>>EPA
United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park
NC 27711
EPA-600/9-79-022
June 1979
Research and Development
Proceedings of the
Symposium on
Experimental Models for
Pulmonary Research
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EPA-600/9-79-022
June 1979
Proceedings of the
Symposium on
Experimental Models for
Pulmonary Research
Edited by
Donald E. Gardner
Edward P. C. Hu
Judith A. Graham
Inhalation Toxicology Branch
Environmental Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Foreword
Chronic and acute diseases are among the major causes of disability and
death in all age groups. As the organ responsible for gas exchange, the lungs
have a unique proximity to the environment and as such are repeatedly exposed
to potentially hazardous gaseous and particulate airborne pollutants as well
as microorganisms. In order to adequately assess the total impact of environ-
mental pollutants on the health of human lung/ it becomes necessary that
disciplined studies with various animal model systems be available.
During the past several years a number of new sensitive and reproducible
in vivo and in vitro pulmonary research model systems have been developed
for studying the unique features of the respiratory tract/ particularly those
related to host defense mechanisms and pharmacological modulations in the lung.
This symposium has been organized to exchange research ideas and techniques
with experts in the scientific community and to explore the possible appli-
cation of these new model systems and techniques for studying environmental
effects on pulmonary health. The conference was a framework which can insure
that the U.S. Environmental Protection Agency is kept up-to-date on the latest
research developments in these areas.
Donald E. Gardner
Edward P.C. Hu
Judith A. Graham
iii
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Contents
Foreword iii
Acknowledgments ix
Program Committee xi
SESSION I: PERMEABILITY OF RESPIRATORY EPITHELIUM
Chemical Modulation of Airway Epithelial Permeability
R. C. Boucher 3
Discussion 20
Chemical Modulation of Alveolar Epithelial Permeability
J. T. Gatzy and M. J. Stutts 23
Discussion 40
SESSION II: RESPIRATORY TRACT AND HOST DEFENSE
Neutral Proteases of Human Polymorphonuclear Granulocytes:
Putative Mediators of Pulmonary Damage
J. K. Spitznagel, M. C* Modrzakowski, K. B. Pryzwansky,
and E. K. MacRae 45
Discussion 60
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Bronchus-associated Lymphoid Tissue and the Source
of Immunoglobulin-containing Cells in the.Mucosa
J. Bienenstock 61
The Role of Polyclonai Cell Activation in the Initiation
of Immune Complex-mediated Pulmonary Injury Following
Antigen Inhalation
B. J. Shenker, T. N. Mann, and W. F. Willoughby 69
Discussion 86
Lymphatic Removal of Fluids and Particles in the
Mammalian Lung
L. V. Leak 89
Discussion 115
SESSION Ills RESPIRATORY TRACT CILIA
Structural Basis of Ciliary Movement
P. Satir 119
Discussion 130
Injury of Respiratory Epithelium
A. M. Collier 133
Discussion 142
SESSION IV: MODEL SYSTEMS OF RESPIRATORY INFECTIOUS DISEASES
Interaction Between Environmental Pollutants
and Respiratory Infections
R. Ehrlich 145
Discussion 162
Experimental Infection of the Respiratory Tract
with Mycoplasma pneumoniae
E. P. C. fti, J. M. Kirtz, D. E. Gardner, and D. A. Powell 165
Discussion 177
Experimental Models for Study of Common Respiratory
Viruses
W. A. Clyde, Jr. 131
vl
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SESSION V: REGULATION OF MUCUS SECRETION AND
CELLULAR DIFFERENTIATION
Mucous Membrane of Respiratory Epithelivm
L. M. Reid and R. Jones 193
Discussion 207
New Methods Used To Investigate the Control
of Mucus Secretion and Ion Transport in Airways
B. Davis and J. A. Nadel 209
Discussion . 225
Mucus Glycoprotein Secretion by Tracheal Explanta:
Effects of Pollutants
J* A. Last and T. Kaizu 227
Discussion 239
Mucus and Surfactant Synthesis and Secretion by
Cultured Hamster Respiratory Cells
J* B. Baseman, N. S. Hayes, W. E. Goldman, and
A. M. Collier 241
Discussion 256
Retinoid Metabolism and Mode of Action
L. M. De Luca, W. Sasak, S. Adamo, P. V. Bhat,
I. Akalovsky, C. S. Silverman-Jones, and N. Maestri 259
Discussion 270
SESSION VI: PHARMACOLOGICAL MODULATION
The Lung Mast Cell: Its Physiology and Potential
Relevance to Defense of the Lung
S. I. Wasserman 273
Discussion 295
Angiotensin Converting Enzyme:
I. New Strategies for Assay
J. W. Ryan, A. Chung/ and U. S. Ryan 297
Angiotensin Converting Enzyme:
II. Pulmonary Endothelial Cells in Culture
U. S. Ryan and J. W. Ryan 307
Discussion 321
vii
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Divergent Effects of Prostaglandins on the Pulmonary
Vascular Bed
P. J. Kadowitz, E. w. Spannhake, and A. L. Hyman 323
Discussion 338
Environmental Influences on Uptake of Serotonin
and Other Amines
A. B. Fisher, E. J. Block, and G. G. Pietra 339
Discussion 353
SESSION VII: SUMMARY AND FUTURE RESEARCH NEEDS
Summary and Future Research Needs
P. A. Bromberg and D. B. Menzel 357
vni
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Acknowledgments
We would like to thank Wendy A. Martin/ Peter A. Murphy/ and Daria T,
Krogulski of Kappa Systems, Inc., for their advice, efforts, and cooperation
in coordinating this symposian and in editing the proceedings.
IX
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Program Committee
Daniel B. Menzel, Ph.D.
Department of Pharmacology
and Physiology
Duke University
Durham, NC 27710
Donald E. Gardner, Ph.D*
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
Judith A. Graham
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
David L. Coffin, D.V.M.
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
Paul Nettesheim, M.D.
National Institute of Environmental
Health Sciences
Research Triangle Park, NC 27711
Philip A. Bromberg, M.D.
Department of Medicine
University of North Carolina
School of Medicine
Chapel Hill, NC 27514
Edward P.C. Hu, Ph.D.
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
Jack Bend, Ph.D.
National Institute of Environmental
Health Sciences
Research Triangle Park, NC 27711
Mirzda Peterson, Ph.D.
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
M. Thomas Wagner, Ph.D.
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
xi
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Session I:
Permeability of Respiratory Epithelium
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Chemical Modulation of Airway Epithelial
Permeability
R. C. Boucher
University of North Carolina
Chapel Hill, North Carolina
Epithelia generally exhibit two functions in organ homeostasis: they
serve as a protective barrier, and they often possess specialized mechanisms
of transport. As the control of ion and mucus secretion by the airway mucosa
will be discussed later in this symposium, this presentation will focus on the
role of airway epithelium as a barrier to passive translocation of large polar
solutes, both as inhaled materials and resident macromolecules, across the
airway. Previous work on the "barrier function* of the airway mucosa, pre-
dominantly morphologic, will be reviewed/ and more recent approaches to the
study of airway epithelial function that combine physiologic measures of air-
way permeability and morphology will be presented.
Until the late 1960s, it had been held that the respiratory epithelium
was impermeant to protein macromolecules. Morphologic studies reported by
Schneeberger-Keeley and Karnovsky, employing horseradish peroxidase (HRP), a
glycoprotein of ^40,000 daltons, as an electron microscopic tracer, suggested
that in the mammalian alveolus macromolecular flow across the epithelium was
completely restricted at the level of the tight junctions adjoining alveolar
pneumocytes (22). However, physiologic studies initially reported by Bensch
et al. suggested radiolabeled albumin crossed the alveolar epithelium at
sites that were not defined (1). These findings were confirmed by others (25).
Little information on the movement of macromolecules across airway mucosa
was available until 1973 when Richardson et al. reported on attempts to local-
ize within the airway wall inhaled protein antigens that elicited acute
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allergic bronchoconstriction in sensitized guinea pigs (21). These authors
were unable to detect antigen, in this case HRP, below the epithelial tight
junctions at times coincident with physiologic airway constriction. A
hypothesis advanced for these results suggested that the rapid onset of bron-
choconstriction (^30 sec after initiation of challenge) involved antigen con-
tact with mast cells residing in the airway lumen. Subsequent transmission
electron microscopic (TEM) studies by Richardson et al. suggested that enzy-
matically intact HBP and ferritin do cross the guinea pig tracheal epithelium
from airway lumen to interstitium (20). They concluded that this movement
occurs via pinocytotic rather than paracellular routes, required at least 30
to 60 minutes for transfer, and no preferential rates of movement of HBP
(40,000 d) over ferritin (450,000 d) could be shown. Inoue and Hogg in
subsequent studies concentrated on defining the ultrastructure of the epi-
thelial tight junctions (13). Freeze fracture studies from guinea pig tra-
chea revealed a junctional compartment that is relatively deep—^0.5 microns—
and composed of an average of 7 strands, interconnected to form compartments.
Studies of respiratory epithelial permeability to macromolecules have
been hampered by several technical problems. Satisfactory probe macromole-
cules for use in estimating the permeability of relatively tight or imper-
meable epithelia have been lacking. Radioactive label from externally labeled
probes often elutes or is cleaved from the parent compound. Because of
the restrictive sieving characteristics of respiratory epithelia, flows of
the lower molecular weight-free label or labeled fragments across the barrier
will dominate and yield erroneously high permeability coefficients. In the
worst cases, free label can bind to resident macromolecules, e.g., albumin,
making evaluation of the nature of the permeant radioactivity by common tech-
niques, e.g., TCA precipitation, impossible. Such considerations have
similarly hindered combined morphologic-physiologic studies of macromole-
cular translocation in respiratory epithelia. Techniques such as autoradio-
graphy have been relatively disappointing for these purposes because there
is little evidence that radioactivity faithfully reflects location of
probe molecules.
These considerations led to the development of sensitive techniques to
monitor the movement of the EM tracer horseradish peroxidase across rela-
tively impermeable barriers. Because of the need to use small amounts and
low concentrations of this material on mucosal surfaces, an assay sensitive
to nanogram/ml quantities of HRP in plasma was required. As the presence
of circulating endogenous peroxidases rendered the enzymatic determination
of HRP in plasma at low concentrations unreliable, a solid phase radioimmuno-
assay (RIA) that detects HRP in plasma to concentrations of 10 ng/ml was
developed. Following application of small volumes of HRP onto the airway
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500,
ABSORPTION OF HRP FROM G P TRACHEA ± S6
t
HRP
15 20 35
TIME mm
30
40
Figure 1. Plasma accumulation of HKP after intratracheal instilla-
tion of 1 mg HRP. Ordinate: fractional plasma absorption of instilled
HRP; abscissa: time.
mucosal surfaces, rate constants of HRP transfer into blood can be estimated
and routes of HKP movement across the epithelium examined morphologically
with the transmission electron microscope.
Early studies showed that after instillation of 1 mg of purified HRP into
guinea pig trachea a small fraction of the instilled HRP was measurable in
plasma 10 minutes later (0.08% instilled dose) and that the rate of HRP ac-
cumulation in plasma was relatively constant from fO to 40 minutes (Fig. t).
To assess effects of an inspired agent on tracheal permeability, the follow-
ing protocol was used: After control blood samples were obtained, 1 mg of
purified HKP in 0.2 ml phosphate buffered saline (PBS) was instilled onto the
tracheal surface through a tracheostomy tube. Blood was sampled for HRP by
RIA at 10, 15, and 20 minutes, with the rate of accumulation in plasma over
this interval calculated by the least squares method. After the 20-minute
measurement, an inhalation exposure to" a test agent was administered and post-
challenge rates of HRP accumulation in plasma determined from 20- to 40-minute
measures of plasma HRP. The significance of aerosol-induced rate changes was
estimated by comparing these rates for individual animals within each group
with paired t-tests. At various points within this time frame animals were
sacrificed and HRP localized in mucosal tissue on transmission electron
micrographs. In addition, respiratory parameters, tidal volume/ dynamic
compliance, and pulmonary resistance, as well as arterial blood gases and
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blood pressure, were measured with the animal in a pressure-sensitive ple-
thysmograph to provide an index of more traditional physiologic effects of
the inspired agent.
We have recently reported data assessing the sensitivity and validity
of this methodology for measuring changes in protein transport in the tra-
cheobronchial mucosa (7). In control studies, it was found that sham chal-
lenge with air did not change HRP plasma accumulation rates, and electron
micrographs at 23 and 40 minutes after HRP instillation showed HRP to be
localized primarily atop the mucous barrier/ suggesting that the instillation
technique did not induce mucosal trauma or direct toxic effects. In contrast/
diethyl ether exposure, which Richardson et al. had shown morphologically to
HRP penetration into guinea pig tracheal epithelium, led to a three-fold in-
crease in rate of plasma HRP accumulation. Electron micrographs confirmed
Richardson's observations that HKP was present in the proximal intercellular
spaces, a finding that could result from a loss of the barrier function of
the tight junctions. A similar association between increased flow of HRP
from lumen to blood in vivo and loss of the barrier function of the tight
junction has been reported in the gut (19). Gel filtration of plasma from
control and exposure animals showed the HRP measured in plasma by RIA to be
intact 40,000 d protein with no smaller molecular weight fragments measurable.
More recently, in collaboration with Ms. Joy Johnson in Montreal and
Dr. Hogg, these techniques were used to pursue earlier morphologic obser-
vations of Simani et al. that inhalation of cigarette smoke increases HRP
penetrance into guinea pig epithelium (14, 23). In these studies, guinea pigs/
20 minutes after intratracheal instillation of HRP, were assigned to one of
five exposure groups: 1) a control group exposed to 20 puffs of air; 2) a
group exposed to 5 puffs of whole cigarette smoke; 3) a group exposed to 20
puffs of whole smoke; 4) a group exposed to 100 puffs of whole smoke; and
5) a group exposed to 100 puffs of smoke that had been passed through a
standard Cambridge filter pad, effectively removing the particulate phase
of smoke and thus exposing the animal to the "vapour" phase of smoke. Staoke
or air was delivered to the animals by a specially designed smoking machine
that, from standard flue cure cigarettes, delivered 20-ml puffs of smoke into
a length of tubing attached to the inspiratory port of the tracheostomy tube.
The results for control, post-exposure rates, and p values for these groups
are shown in Table 1. For the control group the mean rate of HRP accumu-
lation after air exposure, while moderately lower, did not differ statisti-
cally from the control (10-20 minutes) period. Five puffs of whole smoke have
relatively little effect on rates of HRP accumulation, while the mean rate of
plasma HRP accumulation after 20 puffs of smoke was moderately increased,
principally due to a variable effect at 40 minutes. It can be seen that 100
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Table 1. Rates of Plasma HRp Accumulation for Control Periods and After
Exposure to Graded Doses of Cigarette Smoke
(5% delivered dose/min)
20-40
10-20 20-40 p 10-20
Control (N»14) 0.0053 0.0038 NS 0.7
5 puffs (N-12) 0.0055 0.0053 NS 1.0
20 puffs (N-11) 0.0055 0.0099 0.09 1.8
100 puffs (N-15) 0.0048 0.0122 <0.01 2.5
0.40
0.36
8 0.30
a
S 0-2B
S 0.20
e
1 0.15
£0.10
a.
0.06
0.00
6-.0065 ^^
^^^
^iff;--
b'.OOTi T^X^^ ~
•rx-^^-t 100 Puffs - vapour phase + tS.t
*
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
TIME POST-MRP (mini
Figure 2. Rate of plasma HRP accumulation before (b - .0071 percent
delivered dose min ) and after (b » .0065) exposure to vapour phase of ciga-
rette smoke.
puffs of whole cigarette smoke resulted in about a three-fold rise in the
rate of plasma HRP accumulation over control, an increase significant at the
P < 0.01 level. In addition to standard transmission electron microscopic
studies, freeze fracture studies were performed by Sada Inoue on tracheal epi-
thelium from guinea pigs that inhaled equivalent amounts of smoke* In con-
trast to the normal morphology as revealed by this technique/ beading and
disruptions of the fibrils/ loss of discrete compartments, and free fibrillar
endings were noted after smoke inhalation. While the precise relationship
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between the morphology of the junction as revealed by freeze fracture tech-
niques and probe permeation is controversial, the changes are consistent with
the TEM evidence of damaged tight junctions and the increases in probe (HRP)
flow.
Figure 2 shows the application of these techniques to identify the noxious
components of cigarette smoke* This figure shows that exposure to 100 puffs
of the vapour phase of cigarette smoke—that is, whole smoke passed through a
Cambridge filter—is not associated with increased rates of plasma accumu-
lation of HRP over control. These data suggest that the tars and nicotine in
the particulate phase rather than the acroleines, CN and NO in these vapour
A
phase exposures, are likely to be the active agents. Data consistent with
these observations have recently been obtained, showing that high tar, high
nicotine cigarettes produce significantly greater increases in tracheal per-
meability to HRP than low tar, low nicotine cigarettes.
In summary, this technique has been useful in measuring the changes in-
duced by inhaled agents in tracheal mucosal permeability to an intact macro-
molecule, HRP, and in identifying morphologic sites of mucosal damage.
The protocol, however, has raised questions about the normal routes of
HRP movement across the airway mucosa. As noted earlier, Richardson et al»
suggested HRP moves across guinea pig tracheal mucosa via pinocytosis rela-
tively slowly, i.e., requiring 30-60 minutes. However, in our experiments
HRP was easily measurable in plasma 10 minutes after instillation. Electron
micrographs taken at these early intervals, like those of Richardson and co-
workers, failed to show HRP penetration into the lateral intercellular spaces
or basal areas. A similar discrepancy between the rate of HPP movement across
the pulmonary capillary to R lymph duct, and electron microscopic evidence
of tracer egress from capillary lumen, has recently been reported (2). These
observations imply that, standard EM techniques, not sufficiently sensitive
to detect a small number of HRP molecules traversing the tight junction, are
unable to detect transport via a small number of vesicles emptying into the
lateral or basal areas of the epithelium. Alternatively, localized areas of
transport such as the bronchus associated lymphoid tissue (BALT) (16) areas or
increased cell turnover may be missed by the sampling procedure.
To better describe the movement of macromolecules across airway epithe-
lium, precise measurement of translocated probes and the surface area avail-
able for permeation is necessary. In vitro studies with excised trachea,
mounted in Ussing chambers, as first reported by Olver jit al., have proved
useful for such studies (17). In these experiments, epithelium from the post-
erior membrane of canine trachea is mounted as a flat sheet between two
Lucite half chambers. The bathing solutions, generally Ringer's or Krebs-
Henseleit, are gassed, circulated, and warmed. In most situations, these
solutions are identical on both sides of the membrane, eliminating chemical
8
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Table 2. In Vitro Bioelectric Properties of Excised Canine Trachea
in Ussing Chambers
Bioelectric propertiesa
Present study Olver et al,
PD (mV) 33.5 + 2.9 30.7 jf 2.7
~2) 97.6 +• 11.0 108 + 8.0
G (mScm ) 2.91 + 0.18 3.51
a~'"~~
Mean +_ S.E.
and hydrostatic pressure gradients. Via sensing bridges closely apposed to
each surface of the membrane, transmural potential difference (PD) is recorded
with a voltmeter. The membrane can be short-circuited, that is, the trans-
epithelial PD reduced to zero, by imposing the electromotive force (EMP) of
an external battery across the membrane via a second pair of bridges. The
current through the external circuit short circuit current {!__) is recorded
sc
on an ammeter. Because this tissue behaves like an ohraic resistor, resistance
(R) and its reciprocal, conductance (G), can be calculated from the I and
sc
open circuit PD. For our studies, probe molecules, which differed in size but
too large to enter the intracellular space, were added to the mucosal bathing
solution and the rate of probe appearance in the serosal solution was mon-
itored by tracer counting ( C-mannitol, H-inulin) or RIA (HRP) (4). Per-
meability coefficients for these probes were calculated.
The bioelectric properties of canine trachea measured'in these studies
were similar to those reported by Olver et al. (17) (Table 2). Probe mole-
cule addition, particularly HRP, at a dose of 7.5 mg/ml, did not change bio-
electric properties. Rates of entry into the sink were linear 30 minutes
after the molecules were added to the source.
Prom the data of Marin et al., it can be calculated that the electrical
— 2 ~™^ ~"™" M*S 2 S*M —2
G (3.3 mScm ) approximates the sum of JvT- (2.4 mScm ) and J + (1.4 mScm )—
i.e., 2J^a8S!"Ve - 3.8 mScm2 (15). Further, permeability coefficients for Na*
and Cl can be calculated from these fluxes. The ratios of P_. and P.T to
cj. Na
Pmm —3.2 and 1.6, respectively—approximate the aqueous free diffusion ratios:
ntcin
D -/D » 2.8; D« +/D =» 1.9. Because mannitol is assumed to move through
Cl man Na man
extracellular paths, the similarity in the permeability ratios and the ratio
of the free diffusivities suggest passive Na and Cl movement is unrestricted
and occurs largely via aqueous paracellular routes.
The permeability coefficients of the respiratory mucosa in vitro to
probe solutes, the ratios of P . to P .. , and free diffusion ratios of
proce mannitoJ.
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Table 3. Permeability of Canine Tracheal Epithelium to Solutes
_in Vitro
Canine trachea Free diffusion
P7 -1
Probe solute (x 10 cm sec ) P /P .a D /D
probe mannitol probe mannitol
36 —
Cl
22 4~
Na
C -mannitol
H-inulin
Horseradish
peroxidase
£>
52
b
25
16
4.4
0.1
3.2
1.6
1
0.25
0.006
2.8
1.9
1
0.33
0.076
Calculated from the contemporaneous fluxes of mannitol and the probe
across the same preparation (except for Ma and Cl permeation through dog
trachea).
Calculated from the lumen to serosa flux of Cl and the serosa to lumen
flow of Na reported by Marin and coworkers.
D to D , are shown in Table 3. The comparison of permeability co-
probe mannitol
efficients is one of the methods used by Solomon and others to calculate
D
equivalent pore radii (24). In this analysis P = F(a/r)—, where
2 3 ^x 5
F(a/r) - (1 - a/r) (1 - 2.1 a/r + 2.09 a/r - .95 a/r ) and
a = probe molecule radius; r = pore radius; and AX = path length.
Comparison of the permeability coefficients for probe molecules to a common
reference, i.e.,
px F(Vr) Dx
Pref " F(aref/r) * °ref
eliminates the path length. The results of such an analysis are shown in
Figure 3. As the molecular radius of the probe molecule increases, the
permeability coefficient/ relative to that of mannitol, decreases. The di-
rection of this change is predicted for movement in free solution. However,
the ratios for inulin and, particularly, HKP are smaller than those predicted
for free diffusion, suggesting that the movement of these molecules is re-
stricted and that the barrier behaves like a "sieve." A single equivalent
pore of 75 A appears to fit our data. Although this pore size may seem re-
latively large in light of published comments on the relationship between PD,
conductance, and the "tightness" of epithelia (10), the total pore area can be
calculated to occupy 0.05% or less of total surface area. The presence of
albumin, a macromolecule similar in size to HRP, in canine airway liquid (18)
obtained in vivo from normal dogs suggests that these paths are present under
physiologic conditions.
10
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10.0 r
EXCISED DOG TRACHEA
10
o
I O.I
0.
.01
001
Free Diffusion
70 A
Horserodish
Peroxidase
4 8 12 16 20 24 28 32 36 38
MOLECULAR RADIUS (A)
probe'"mannitol
versus
Figure 3. Semilogarithmic plot of ratio of P . /P
probe n
molecular radius. Solid lines show predicted values for equivalent pore radii
70-80 A.
It should be noted that the electrical conductance (G) reflects current
that is carried by small ions* The magnitude of the conductance may reflect
the total area of the aqueous channels in the barrier, but the magnitude of
restricted diffusion or apparent pore dimensions cannot be predicted directly
from G.
The existence of size-dependent restriction of "sieving" solutes across
tracheal epithelium implies movement through paracellular pores rather than
by pinocytosis. Other evidence supports this hypothesis. The rate of flow
of probes was directly related to tissue conductance, indicating movement of
these solutes by the same paracellular paths as ions. The unidirectional
11
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fluxes of the probes were symmetric, i.e., flow from S+M equaled flow from
M*S, as would be anticipated for a diffusional process. Finally, in colla-
boration with Dr. N. S. Wang of McGill, samples of the tissues were examined
ultrastructurally and vesicles were not detected.
Edge damage, i.e., chamber-induced membrane damage, is a concern in all
experiments where tissue is clamped between two chambers. Although rigorous
analyses of edge effects have not been reported in this tissue, varying edge
to surface area ratios, varying edge compression, and comparison of bioelec-
tric properties obtained with our apparatus with those in the literature,
including "edge free" systems, suggest no substantial edge artifacts in these
experiments. As edge-induced increases in G might be expected to decrease PD
measured in vitro, comparisons of in vitro PD to measurements of PD made
directly in vivo in canine trachea, if similar, would also suggest no sub-
stantial edge artifacts.
As reviewed above, the bioelectric properties of excised canine trachea
can be measured in Ussing-type chambers/ permitting the study of the tissue
under short circuit conditions, i.e., no electrochemical driving force for
solute movement. Under these conditions and with no hydrostatic gradient,
asymmetric unidirectional fluxes of a tracer species (i.e., net flow) are
primary evidence for active transport. Olver and Marin have demonstrated
Cl secretion and a smaller Na absorption across the short circuited canine
trachea (17). As shown in Table 1, the canine trachea exhibits an average
peak PD of '30 mV—lumen negative—and an I of 75-100 uamps/cm . The radio-
sc
tracer studies suggest that the open circuit PD is generated by active ion
transport mechanisms that were identified under short circuit conditions.
Several pharmacologic agents, added to the solutions that bathed canine
trachea in vitro, have been shown to alter bioelectric properties and ion
fluxes.
However, the relevance of the in vitro model to in vivo function, both
for our studies and salt and water metabolism, hinges on the similarities in
the basic properties of the two preparations. The selection of appropriate
bioelectric properties for comparison is not obvious since the time course
of PD in vitro is complex. After mounting/ transepithelial PD transiently
falls but subsequently gradually rises to reach a relatively stable plateau
1-2 hours after mounting. We and others have selected this plateau value as
the value likely to represent the PD of the epithelium in vivo. To verify this
supposition and because Hale et al. have reported an j.n vivo canine tracheal
PD of only -5 mV, we have developed a technique to measure PD directly in vivo
(3, 12).
The protocol is begun by balancing the reference electrode—a 19 gauge
needle containing Ringer-4% agar—and the fluid-filled exploring bridge (PE-
160 tubing) in a common reservoir of Ringer. The calomel electrodes connect-
12
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-50
-25
5 mm/sec
£ -50
-25
0 L- j
.25 mm/sec
Figure 4. In vivo PD (mV) recordings from canine trachea.
ing these bridges to the voltmeter were selected to have an offset potential
of 1 mV for each study. Adult male mongrel dogs, mean weight 18 kg, were
anesthetized with intravenous pentobarbital sodium or amybarbital and intubated.
The reference electrode was placed in a subcutaneous space, shown in pre-
liminary studies to be isoelectric with the serosal surface of the trachea.
The exploring electrode was advanced through the endotracheal tube* Contact
with the tracheal surface was insured by continuous perfusion through the bridge
at 0.1 ml/min with warmed, gassed Ringer solution. The potential sensed by
this bridge was transmitted to the calomel half-cell via Ringer-4% agar
bridge that connected the perfusion reservoir with the cell.
The calomel electrodes were connected to a high impedance voltmeter—
filtered to remove 60 cycle interference—and values displayed digitally and
recorded on a strip chart*
Figure 4 shows representative tracings obtained with the fluid-filled bridge.
It can be seen from the top tracing—with relatively fast paper speed—that
the PD recorded from this dog is stable but shows oscillations, which corres-
pond to respiratory movements. The bottom tracing shows a 6-minute interval,
indicating stability of tracing. Continuous tracings are stable, within 10%,
over periods of at least 30 minutes.
To control for possible effects of liquid junction potentials between
bridge perfusate and tracheal liquid, 3H KC1 bridges were compared to the
Ringer bridges employed in the fluid-filled system. In these experiments
a 3M KCl-4% agar-filled 19 gauge needle was inserted into a subcutaneous
13
-------
Table 4. Comparison of Canine Tracheal PD in Vivo and in Vitro
Tracheal transepithelial biopotential
In vivo In vitro
PD (mV) 29.1 + 3.0 33.5 + 2.9
a
Mean + S.E.
space and a PE-160 electrode filled with 3H KCl-4% agar, balanced as dis-
cussed previously, was used as the exploring electrode. The values obtained
with each technique were identical over a range of PDs. The correspondence
of PD with the two systems suggests the effects of liquid junction are neg-
ligible.
For comparing measurements obtained with this system directly to in vitro
measurements, excised canine trachea was mounted in Ussing chambers and PD
recorded by standard techniques. Then the chamber was detached from the in
vitro apparatus, the reference 19 gauge needle electrode inserted into the
solution bathing the serosal surface of the membrane, fluid evacuated from
the mucosal side, and PD recorded directly by introducing the fluid-filled
exploring electrode onto the mucosal membrane surface. The potentials measur-
ed with in vitro and in vivo bridges in this situation were identical (n*=6).
These data suggest that the filtering techniques used to process the In vivo
signals did not contribute to the measured potentials and indicate a close
correspondence between in vitro and in vivo measured PD in the canine trachea.
Further, we have compared PDs measured with the fluid-filled bridge in
other organs with those reported in the literature. Canine gastric PDs from
three dogs were measured under fluoroscopic control. The mean PD, -54 mV, is
similar to that reported by Dennis and others (9). Esophageal PDs in these
dogs were -20 mV, with a regional PD profile similar to that reported in
rabbit and man by Powell and coworkers (26).
PD was measured at three random sites in the trachea and mean value cal-
culated. Table 4 shows the mean PD + S.E. from 27 separate dogs using the
FF technique and, for comparison, the mean peak in vitro PD + S.E. from the
study previously cited from our laboratory. Mean tracheal PD is -29 mV, a
value indistingishable from peak j.n vitro values.
The fluid-filled bridge has made it possible to assess the pharmacologic
modulation of PD in vivo by direct addition of agents to the bridge perfusate,
ensuring application of drug directly at the site of PD measurement. Drug-
containing Ringer is flushed through the bridge perfusion system pump run in
reverse for 1.5 minutes. During perfusion the time required to clear the
drug-free fluid provides a control period. Drug effects were compared to
14
-------
o-
^20^
O
Q.
10
-L-
T 1
TC"1^"^
OUABAIN
IO-3M
--J— J
4 — "
o.
-— r---^
h- _j
[ 1
1 1 T -<
•— • CONTROL
o—o OUABAIN
£±SE
_L
-101 23456
TIME (min)
Figure 5. PD response to ouabain addition to mucosal surface of canine
trachea.
Table 5. Comparison of _in Vivo and in Vitro PD Response in Canine
Trachea to Mucosal Applications of Pharmacologic Agents
Profile of PD response
In vivo APD (mV)
In vitro .1PD (mV)
Ouabain
(10 M)
Amphotericin
(10 M)
Acetylcholine
-33.5%
^77.0%
-28.0%
+58.0%
(5 X 10 M)
Atropine
(10 M)
Histamine
(10 M)
0
0
-10.0%
0
0
+5.0%
Percentage control.
those of a group of 10 dogs with Ringer perfusion only. An example of the
effect of ouabain, 10~ M, on tracheal PD in four dogs is plotted as a function
of time in Figure 5. Ouabain produces a slow reduction in PD. Both the
magnitude of the change and time course resemble those reported for the in
vitro preparation.
15
-------
As the majority of the reports on pharmacologic pertubation of tracheal
bioelectric properties have focused on only the effects of serosal drug ad-
dition, an appropriate comparison of in vivo with in vitro drug effects re-
quired a parallel set of experiments with excised trachea. The pattern of in
vivo response is compared with the in vitro response to mucosal drug additions
in Table 54 It is obvious that the pattern of response to mucosal drug appli-
cation in both preparations is similar.
Hence, 1) the absence of significant liquid junction potentials/ 2) the
similarity between PD as measured with in vivo and in vitro bridges on ex-
cised epithelium, 3) the similarity between PDs measured in other organs with
the fluid-filled electrodes and values in the literature, and 4) the similar-
ity in pattern of response to drug applications in vivo and in vitro indicate
that the in vivo tracheal PD can be measured accurately and reliably with
this technique. Further, the magnitude of that PD measured by this technique
implies that the low values reported by others may be in error. The technique
should allow direct in vivo studies of the genesis and regulation of the airway
biopotential in both animal and man. In regard to the interests of this sym-
posium, effects of environmental pollutants on airway potentials in vivo
are feasible.
There are several likely pathophysiologic implications of airway hyper-
permeability. A loss of the "barrier" function would facilitate movement of
inhaled materials into the airway wall. Recent studies in conjunction with
Dr. J. Richardson at McGill have shown in the chicken that airway hyperper-
meability, induced by methacholine, enhances antibody response to antigen
instilled into the trachea (unpublished data). This enhanced response was
assumed to result from an increased antigen load. The effects of airway hyper-
permeability on airway reactivity in asthmatics to naturally occurring anti-
gens have not been evaluated. However, the hyperreactivity to inhaled chemical
agonists that is a characteristic of asthmatics may, in part, by mediated by
airway hyperpermeability.
It has been shown in the ascaris-sensitive rhesus monkey and guinea pig
that specific antigen challenge leads to mucosal hyperpermeability to topic-
ally applied small molecular weight polypeptides and HRP (5, 6). Further,
the rhesus demonstrates a period of hyperreactivity to inhaled histamine
for an interval after antigen challenge, even after IL has returned to base-
line. Using radiolabeled histamine as a tracer, it has been observed that
post-challenge airway hyperreactivity is associated with an increased frac-
tional absorption of inhaled histamine into the blood (8). These data are con-
sistent with the hypothesis that airway epithelial hyperpermeability, induced
by allergic bronchoconstriction, contributes to airway hyperreactivity by
increasing flows of inhaled bronchoactive agents to effector sites in the
airway wall. However, until experiments can be performed that alter airway
16
-------
permeability in the absence of changes in bronchomotor tone, the association
of airway hyperpenneability and hyperreactivity cannot be taken as causal.
The recent demonstration, in collaboration with Dr. Richardson, that viral
infection in chickens increases airway permeability to HBP suggests that this
mechanism may contribute to the increased airway reactivity with viral infec-
tions in humans.
A hyperpermeable mucosal surface might be expected to impair mucus clear-
ance by two mechanisms. First, if active secretion of chloride secretion
into the airway lumen is important to hydration of the airway, epithelial
hyperpenneability, by increasing the passive "leak" of the chloride toward
the interstitium, dissipating the osmotic gradient that drives water toward
the lumen, would be expected to result in dehydration of the airway* Second,
increased flows of macromolecules, e.g., albumin, through a more permeable
epithelium down the chemical gradient into the airway lumen could occur, as
has been reported for albumin in asthmatics (11). Albumin binds to mucus
glycoproteins, increases cross-linking, and may thereby alter visco-elastic
properties and diminsh mucociliary clearance.
SUMMARY
The epithelium lining the airway appears to function as an effective,
though not absolute, barrier to the movement of large hydrophilic substances
between airway lumen and interstitium. The principal path of translocation
for these substances across the airway mucosa appears to be paracellular.
Experiments on the guinea pig are consistent with the notion that a variety
of inhaled noxious agents, e.g., ether and cigarette smoke, increase epithe-
lial permeability to HRP by increasing the flow of this probe through para-
cellular paths, probably by damaging the epithelial tight junctions. In vivo
tracheal PD in dogs agrees closely with in vitro values and suggests the use
of this technique in rapid screening for epithelial damage.
17
-------
REFERENCES
1. Bensch KG, Dominguez F, Liebow AA: Absorption of intact protein mole-
cules across the pulmonary air-tissue barrier. Science 157:1204-1206,
1967
2. Bienenstock J, Rudzik 0, Clancy RL, et al: Bronchial lymphoid tissue.
In: The Immunoglobulin A System (Mestecky J, Lawton AR III, eds).
New York and London, Plenum Press, 1974, pp 47-56
3. Boucher RC, Bromberg PA, Gatzy JT: Measurement of the in vivo electrical
potential difference in canine trachea with a fluid filled bridge.
Clin Res 26:802A, 1978
4. Boucher RC, Gatzy JT: Trachea! epithelial permeability to non-electro-
lytes. Physiologist 21:11, 1978 (abstract)
5. Boucher RC, Pare PD, Gilmore N, Moroz LA, Hogg JC: Airway mucosal per-
meability in the ascaris suum-sensitive rhesus monkey. J Allergy Clin
Immunol 60:134-141, 1977
6. Boucher RC, Pare PD, Hogg JC: Relationship between bronchial hyper-
reactivity and hyperpermeability to histamine in ascaris sensitive
monkeys. Clin Res, Dec 1977
7. Boucher RC, Ranga V, Pare PD, Inoue S, Moroz LA, Hogg JC: Effect of
histamine and methacholine on guinea pig tracheal permeability to HRP.
J Appl Physiol: Respirat Environ Exercise Physiol 45:939-948, 1978
8. Boucher RC, Ranga V, Pare PD, Moroz LA, Hogg JC: The effect of allergic
bronchoconstriction on respiratory mucosal permeability. Physiologist
20:11, 1977
9. Dennis WH: Potential difference across the pyloric antrum. Am J Physiol
197:19-21, 1959
10. Diamond JM: Channels in epithelial cell membranes and junctions. Fted
Proc 37:2639-2644, 1978
11. Dunnill MS: The pathology of asthma, with special reference to changes
in the bronchial mucosa. J Clin Pathol 13:27-33, 1960
12. Hale X, Sandier L, Niewoehner D: Sensitivity of the in vivo trans-
mucosal potential difference to beta-adrenergic agents. Clin Res 26:
447A, 1978
13. Inoue S, Hogg JC: A freeze-etch study of the tracheal epithelium of
normal guinea pigs with particular reference to intercellular junctions.
J Oltrastruct Res 61:89-99, 1977
14. Johnson J, Boucher RC, Inoue S, Moroz LA, Hogg JC: The effect of graded
doses of whole cigarette smoke on respiratory mucosal permeability.
Am Rev Resp Dis 117:244, 1978 (abstract)
18
-------
15. Marin MG, Davis B, Nadel JA: Effect of acetylcholine on Cl" and Na+
fluxes across dog tracheal epithelium in vitro. Am J Physio1 231:1546-
1549, 1976
16. Michel RP, Inoue S, Hogg JC: Pulmonary capillary permeability to HRP
in dogs: a physiologic and morphologic study. J Appl Physiol 42:13-
22, 1977
17. Olver RE, Davis B, Marin MG, Nadel JA: Active transport of Ha and Cl
across the canine tracheal epithelium in vitro. Am Rev Resp Dis 122:
811, 1975
18. Pennington JE, Reynolds HY: Concentrations of gentamicin and carbenicillin
in bronchial secretions. J Infect Dis 128:63-68, 1973
19. Rhodes RS, Karnovsky MJ: Loss of macromolecular barrier function asso-
ciated with surgical trauma to the intestine. Lab Invest 25:220-229, 1971
20. Richardson J, Bouchard T, Ferguson CC: Uptake and transport of exogenous
proteins by respiratory epithelium. Lab Invest 35:307-314, 1976
21. Richardson JB, Hogg JC, Bouchard T, Hall DL: Localization of antigen
in experimental bronchoconstriction in guinea pigs. J Allergy Clin
Immunol 52:172-181, 1973
22. Schneeberger-Keeley EE, Karnovsky MJ: The ultrastructural basis of
alveolar-capillary membrane permeability to peroxidase used as a tracer.
J Cell Biol 37:781-793, 1968
23. Simani AS, Inoue S, Hogg JC: Penetration of the respiratory epithelium
of guinea pigs following exposure to cigarette smoke. Lab Invest 31:75-
81, 1974
24. Solomon AK: Characterization of biological membranes by equivalent pores*
J Gen Physiol 51:3355-3645, 1968
25. Theodore J, Robin ED, Gaudio R, Acevedo S: Transalveolar transport of
large polar solutes (sucrose, inulin, and dextran). J Appl Physiol 129:
989-996, 1975
26. Turner KS, Powell DW, Carney CN, Orlando RC, Bozymski EM: Transmural
electrical potential difference in the mammalian esophageal in vivo.
Gastroenterology 75:286-291, 1978
19
-------
DISCUSSION
PARTICIPANT: Is there any difficulty that you can foresee in testing alter-
ations of permeability in the jja vitro preparation? In other words, couldn't
you simply put ether into the J.n vitro preparation, and try to open up tight
junctions, as you've mentioned, and see whether you get effects in conductivity
or pore radius or whatever?
DR. BOUCHER: I think that kind of study may have a fruitful future. We're
actually beginning to do cigarette smoke in a parallel fashion in vitro* I
think the point is that you can't just measure conductance, if you're interest-
ed in what's happening as far as equivalent pore radius. But I think our data
with respect to the in vitro preparations, are relatively encouraging. The
potentials that we measured, and I think they were measured in San Francisco,
are quite close to what we measure in vivo.
DR. DAVIS: I'm glad that you do find the same; it seems that our last five
years of work haven't been in vain. I do have one comment and one question.
Originally, we measured j.n vivo versus _in vitro potentials, but we did it a
little bit differently. We took a segment of the trachea from a dog and coat-
ed just the top half of it with fluid. Then we planted the lower half of the
trachea from the same dog in the chamber. We found a correlation between the
two, but we haven't published the results. We now, in fact, have a chronic
dog with emphysema and from the fluid-filled pipe we measure PD, and from
day to day in a chronic dog, that seems to be a fairly standard contact.
DR. BOUCHER: Do you get 30 millivolts?
DR. DAVIS: In the chronic, it's not as high as that. It's around 12 or 15
millivolts. My question is whether you are using our original preparation?
Do you dissect off the trachealis muscle, and have you done histological
studies of that? One of our worries always was that sometimes there are glands
there, and those glands run right up to where the muscle is. Then, there is
the possibility that would cut through one of those glands and open up one of
the gland ducts as a pore through the epithelium.
DR. BOUCHER: There may be large leaks but we have not found them despite
large numbers of klstologic studies. We actually had a more troublesome worry
20
-------
in regard that there were no pores infected. Essentially we did slides; we
did not see a mucosal lens, although we are sure that they are probably
there. There may be an occasional large leak that we're not picking up under
this technique. Again, this is purely a descriptive way of handling the data*
We chose a particular analysis, because it had certain theoretical advantages,
but Stein also used a similar model. The problem that we ran into is this:
(Dr. Boucher refers to a slide.)
This is a piece of tissue from the Ussing chamber, and all these black shapes
probably represent bronchosepticus. They are all gram-negative rods, and
we also found polymorphonuclear and lymphoid cells in this tissue.
DR. DAVIS: Can you tell us a bit more about that tissue? Have you done a
study on that tissue?
DR. BOUCHER: This tissue had approximately 34 millivolts, a pore radius
exactly similar with the others, and a conductance of less than 2. Most of
our conductances run a little more than 2 2.2. We then got a group of un-
infected female beagles. I don't have a picture of their epithelia, but they
were clean; there were none of these black agents in it* The mucosal cul-
tures also were negative/ and the equivalent pore radii were identical. The
bioelectric properties, as well/ were identical. Infected tissue is a real
problem. However, controlling epitheliun in uninfected dogs did not change our
data at all.
DR. DAVIS: Can I also establish that's not what you normally see in your
preparation? I mean, your normal preparation doesn't look like that* That's
not representative of every tissue you put in the chamber/ is it?
DR. BOUCHER: It's representative of most male mongrel dogs, as far as the
infection* Our dogs are all infected* I suspect that most are* We looked at
straight H and E sections from these preparations, and they looked fine; you
don't see it with such techniques.
DR. DAVIS: We've done EM studies. I'll be showing some pictures; I don't
have many pictures of canine trachea with me, but I have some of cat trachea.
DR. BOUCHER: To us this finding was amazing.
21
-------
PARTICIPANT: In the first part of your studies/ where you studied in vivo,
how do you know that the passage is in the tracheal or the bronchial epithelivm,
rather than in the lower airways?
DR. BOUCHER: We instilled a very small volume of material—about 200 micro-
liters—into the trachea, and with two techniques we tried to assess the
125
deposition. We radiolabeled some HRP with I, then cut serial sections and
counted. We found no tracer in the alveolar surfaces. Perhaps more directly,
we fixed the trachea and performed the peroxidase reaction directly in the
airways. We could only find HRP in the trachea and bronchi. Finally, we
evaluated tissue, both from bronchi and the trachea by ultrastructural
techniques. We had such an unrewarding experience with the bronchi, i.e.,
finding positive HRP on the mucosal surface, that we gave that up and just
examined tracheal specimens. I'm fairly confident that these data really re-
flect HRP permeation in the central airway.
DR. SPITZNAGEL: What is the basement membrane in a surface situation? What
do you see when you look at the basement membrane underneath the epithelium?
DR. BOUCHER: It looks relatively intact. It's interesting to look at the
symmetry itself of peroxidase from both the mucosal side and the serosal
side. Peroxidase from the mucosal side is not seen on transmission electron
micrographs, but when you add it to the serosal side, you see peroxidase
accumulating in proximal intercellular spaces between the cells. You do not
see it at all in the basement membrane, even though the concentrations in
that situation are 7.5 milligrams per 1. The supposition is that the basement
membrane isn't a significant barrier, at least as assessed by this relatively
gross technique in this preparation.
DR. COLLIER: On that last slide that you showed, the bacteria in the inter-
cellular spaces are also granules within the epitheliun itself.
DR. BOUCHER: Yes, there were some early changes in the epitheliun. It is not
normal. I believe I said it looked like a viral infection as noted in other
epithelia and as seen in hunans. I discussed it with our veterinarians in
the School of Veterinary Medicine* Apparently all our dogs are infected with
a parainfluenza virus and the super-infected with gram-negative organisms.
22
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Chemical Modulation of Alveolar
Epithelial Permeability
J. T. Gatzy and M. J. Stutts
University of North Carolina
Chapel Hill, North Carolina
The respiratory epithelium is the first continuous cellular barrier en-
countered by environmental agents in inspired air and is, therefore, a po-
tential site of early toxicity. In contrast to Dr. Boucher's contribution,
this paper will focus on the functions of the epithelium that lines the gas
exchange rather than the conducting surface of the lung. The fluid that
covers the surface of the alveolar epithelium plays an important role in
inflation of the lungs during fetal development (30) and in the maintenance
of reduced surface tension and macrophage function in the adult lung (37).
The volume and composition of the fluid are regulated by the permeability
and active solute transfer processes of the epithelial monolayer.
Direct measurement of fluid outflow from the trachea (17) and dilution
of an impermeant solute that was placed in the alveolar lumen (23) demon-
strated that lung liquid is continuously secreted by the lung of the fetal
sheep. The driving force for fluid production is the active secretion of Cl
and K by the epithelium (25). Secretion ceases and fluid is reabsorbed at
birth, a phenomenon that can be simulated by the parenteral administration
of epinephrine and blocked by a 0-adrenergic antagonist (40). The barrier
separating blood and lymph from the fluid that normally fills the lumen of
the fetal lung exhibits many properties of a "tight" epithelium. Lipophylic
solutes permeate at a rate roughly proportional to their lipid solubility.
In contrast, the translocation of hydrophilic molecules is restricted by
molecular size. Conventional pore analysis indicates that the permeation of
small nonelectrolytes is compatible with aqueous channels with an equivalent
23
-------
radius of 0.55 nm (24). A similar analysis of solute flow between blood and
lymph suggests pores of 15 nm in the capillary endothelium (24). Hence, the
epithelium presents the major impediment to solute movement between blood and
the lumen.
Relatively little is known about the composition of the thin fluid layer
at the air-epithelial interface in the adult lung. Several lines of evidence
indicate that surface active material is secreted into the alveolar lumen by
type II pneumocytes (16, 18). Fluid from the surface of alveoli of rats has
been sampled by micropipet and found to be "protein free" (27). When a Ringer
solution is added to the airspace of dogs and rabbits, the pattern of passive
solute permeation between blood and the lumen resembles, in most cases, that
of the epithelium of the fetal lung (35, 36, 41). Similar conclusions can be
reached from studies of the osmotic effectiveness of small, hydrophilic
solutes added to the solution that perfused excised dog lungs (34). The
introduction of a salt solution into the airspace greatly increased the
osmotic activity of solutes in the vascular space. These results suggested
that the epithelial barrier between the new fluid compartment and the blood
was far less permeable than the endothelial barrier between blood and the
lung interstitium. Other studies have shown that the apparent radius of pores
in the epithelium can be greatly increased by excessive inflation (2).
Several conditions and toxic agents damage the barrier between blood and
the airspace and induce pulmonary edema. In all cases, the electrolyte com-
position of the accumulated liquid resembles that of extracellular fluid, but
the protein concentration is determined by the method of edema formation.
For example, increased left ventricular pressure in dogs results in the for-
mation of edema fluid with the protein concentration of the interstitium (39),
and a bulk flow of fluid across the epithelium through a few large holes (3).
Alloxan induces the flow of fluid with the protein concentration of the plasma
through many smaller holes in the endothelial-epithelial barrier (22, 38).
Both methods of edema formation induce peribronchial and perivascular cuffs
(29). These studies illustrate a major drawback of fluid-filled lung pre-
parations. The movement of solutes across the alveolar epithelium cannot be
distinguished from parallel flow across the epithelium of the airways. Since
the surface area of the alveoli exceeds that of the airways by at least se-
veral hundred fold, the contribution of the nonalveolar epithelium to results
from whole lung is often dismissed. However, recent evidence suggests that
equivalent pores in the epithelium of the upper airways are much larger than
those of the alveolar epithelium so that most large hydrophilic molecule
permeation may follow this path (1).
24
-------
METHODS, RESULTS, AND DISCUSSION
We have attempted to elucidate the permeability of the alveolar epithe-
lium and the mode of action of toxic agents which attack this barrier in two
ways. The first approach proposes the excised rat trachea as a model for the
airway epithelium. When the permeability and transport functions of this
barrier are "subtracted" from properties of perfused fluid-filled rat lung,
the characteristics of the alveolar epithelium should be obtained.
Perfused, Fluid-Filled Left Lobe of the Rat Lung
A complete evaluation of the mode of solute translocation and edema for-
mation in fluid-filled lung is hampered by the overwhelming contribution of
fluid in the airspace to the electrolyte and water content of the tissue.
We minimized this problem by preparing lung slices from a perfused lobe (32).
The left lobe of the rat lung was excised and perfused through the pulmonary
vasculature (arterial pressure » 15 cm H.O) with Krebs Ringer bicarbonate
solution (KBR) that contained 6% colloid (1% bovine serum albumin and 5%
Ficoll). The outflow of perfusion fluid from the pulmonary vein ranged from
1.5 to 3 ml/min and was collected in a fraction collector. The lobe was
filled through the main bronchus with KBR that contained radiolabeled high
molecular weight dextran. After one hour, the lobe was drained and slices,
0.5 to 1 mm thick, were prepared with a Stadie Riggs tissue slicer and blot-
ted on KBR moistened filter paper.
Table 1 summarizes the composition of slices from O.-filled and KBR-
filled lobes. The conditions of each experiment were designed to assess the
effects of fluid filling, perfusion, perfusion with reduced colloid and
agents that interfere with the cell's ability to regulate its internal com-
position. Compared with slices from excised gas-filled lungs, tissue from
perfused, fluid-filled lungs contained 30% more water, a volume that corre-
sponds closely to the volume of dextran distribution* This volume represents
luminal solution that was trapped in the slice and was not affected by the
magnitude of lobe filling volume over a range of 4 to 10 ml/kg body weight
(
-------
Table 1. Electrolytes and Water of Slices
from the Left Lobe of the Rat Lung
Conditions n
V f
HO dextran
(1/kg dry wt)
+ + -
Na K Cl
(meq/kg dry wt)
Mean (+SE)
O -Filled
Unperfused
KBR3-Filled
Unperfused 3
KBR perfused ' 7
Low colloid
perfused 8
Ouabain
perfused 3
Metabolic
inhibitor
perfused 4
15 4.5(+0.1)g
7.K+0.3)
6.K+0.2)
458(+13}g 339(+6) 404( + 12)9'
600(+32)
639(+17)
334(+11)
505(+21)
595(+14)
783(+84)h 349(+14) 725(+63)h
788(+16)h 209(+32)h 617(+43)
6.5(+0.4) 2.6(+0.8) 809(+14)h 163(+16)h 640(+50)
iCrebs bicarbonate Ringer solution.
Krebs bicarbonate Ringer solution with 1% bovine serum albumin (BSA)
and 5% Ficoll (w/v).
KBR with 1% Ficoll or bovine serum albumin.
'iCBR with 1% BSA, 5% Ficoll, and 10 M/l ouabain.
eKBR with 1% BSA, 5% Ficoll, 10~3 M/l iodoacetamide, and 10~3 M/l NaCl.
f 14
Volume of distribution of C-carboxyl dextran that was added with KBR
in the airspace.
gO -filled value significantly different
-------
-9
the rate of dextran appearance in the venous outflow was usually below 10
cm/sec, a value lower than the coefficient for albumin flow across perfused,
fluid-filled dog lung (15).
The usefulness of the slice protocol is illustrated by the experiment
shown in Table 2. The addition of amphotericin B to the fluid in the airspace
of the perfused lobe nearly doubled the concentration of K that was recovered
in the fluid after one hour (38). Since water and electrolytes and the volume
of recovered fluid did not change, the increase in luminal K cannot be
attributed to a loss of tissue K • The electrical potential difference between
the airspace and perfusion fluid seldom exceeded 6 mV (lumen negative) so that
the transepithelial K gradient does not reflect a passive distribution. We
conclude that amphotericin induces the active secretion of K into the lumen,
an effect which has been reported for epithelia of colon (4), frog skin (21),
toad urinary bladder (9), and bullfrog lung (11). Further, the increase in K
in the lumen of the rat lung could be reduced by adding ouabain to the perfusion
fluid, suggesting the participation of Na -K ATPase in K transport.
Excised Rat Trachea In Vitro
The contribution of the airway epithelium to the respiratory lining was
estimated from functions of the excised rat trachea (31). To make use of
nearly all of the small tracheal surface the entire cylinder was excised and
tied to the arms of an inverted "y"-tube. The tracheal cylinder was suspend-
ed horizontally in an outer bath of KBR at 37°C. Bubbles of 5% CO -95% 0_
and a gas lift in the tubing recirculated KBR through the lumen of the
trachea. Transtracheal electric p.d. was monitored between KBR-agar bridges
near the midpoint of the tracheal cylinder and in the outer bathing solution.
The bridges were connected through calomel cells to a high impedance volt-
meter. Current from a d.c. voltage source was passed between an axial
silver-silver chloride wire in the lumen and an outer silver-silver chloride
foil that surrounded most of the trachea. Cysteine (1 mM/1) was included in
the bathing solutions to complex any silver that might have been released
from the electrodes. Unidirectional fluxes of radioactive solutes were deter-
mined by adding the tracer to one bathing solution and determining the rate
of radiolabel appearance in the other bath.
Under open circuit conditions 14 tracheas exhibited a transmural elec-
tric p.d. of 9.3 (+ 1.2 SE) mV, lumen (mucosa) negative, and a d.c. conduc-
2
tance of 11.0 (+ 1.2) mS/cm . These bioelectric properties remained constant
~ 22
for at least two hours, unidirectional Na fluxes across the short-circuited
trachea (transmural voltage clamped at zero) were asymmetric and indicative of
+ 2
an active reabsorption (net mucosal to aerosal flow) of 1.7 yeq Na /cm , hr.
Under *-he same conditions, Cl fluxes revealed an active secretion into the
27
-------
10
CO
KBR
KBR + 10~5 M/l
Table 2. Effect of Amphotericin B on the Composition
of Perfused Lung and the Fluid Added to the Airspace
Recovered
A-rspace fluid Tissue
airspace fluid
n H20 Na+ K+ Cl~ Volume (K+)
(1/kd dry wt) (meg/kg dry wt) %_added
Mean (+SE)
59.3(±1D 350(±6) 559(±10) 82(±6)
amphotericin 3 5.7(±0.1) 657(±14) 369(±19) 580(±10) 7K+5) 179 (±5)
Significantly greater (P<.05) than the value for KBR without amphotericin.
-------
lumen of 1.9 ueq/cm , hr. Furthermore, the sum of the net Na and Cl~ move-
ments accounted for 95% of the current required for short-circuiting. Cl~
secretion and Na reabsorption have been reported for excised, short-circuited
42 14
dog trachea (26). K and C mannitol unidirectional fluxes were symmetric
and, therefore, consistent with passive transfer.
Figure 1 depicts the relationship between the passive flow of each
solute and conductance. The linear relationship between mannitol and con-
ductance and the intercept near zero suggest that most of the current carry-
ing electrolytes and mannitol traverse the same path. A comparison of
points on the least square lines at any common conductance describes the
relationship between the permeability coefficients (P) for the .solutes. The
ratio of p + or P_+ to P ... approximates the ratio of the free dif-
Na K mannitol
fusion coefficients for each solute pair (see Table 4). These results are
compatible with an equivalent pore radius in excess of 4 nm. This estimate
and the implication that mannitol, a solute restricted to the extracellular
compartment, and the electrolytes are moving through the same channel suggest
that this path is paracellular. Cl~ movement relative to mannitol flow was
less than the free diffusion ratio and might be explained if the paracellular
path is lined with fixed negative charges.
The addition of amphotericin to the luminal bathing solution (final
concentration » 10 M/l) increased unidirectional fluxes for all solutes
from two- to fivefold. Specifically, net secretion of K could not be de-
tected.
Determination of Alveolar Epithelial Na Permeability and K Secretion
If the trachea is accepted as a model for the remainder of the airways,
then permeability and electrolyte transport by the gas exchange epithelium
can be deduced from concordant measurements in trachea and the perfused,
fluid-filled lobe (Table 3). The unidirectional flow of Na* into the lumen
of trachea exceeded flow in the same direction in the lobe by two orders of
magnitude. Since the lobe is lined by both alveolar and airway epithelia
with a surface area ratio of about 700 to 1, the passive Na permeability of
the alveolar epithelium should be slightly less than that of the lobe. By •
the same reasoning the increase in Na permeability induced by luminal am-
photericin was considerably smaller in the lobe than in trachea, suggesting
an even smaller increase in the alveolar epithelium.
K secretion into the lumen of the lobe was induced by amphotericin but
could not be detected in trachea. This does not exclude the possibility that
K secretion of the magnitude measured in lobe might be present in trachea.
29
-------
20
c\j
u
10
u
c
o
u
0
/Mannitol
00
0
100
200
Permeability Coefficient X 10 cm/sec
Figure 1. The relationship between d.c. conductance and steady-state,
unidirectional solute flow across excised rat trachea in vitro. Permeability
coefficients were calculated from the unidirectional passive flux of tracer
(P =• radiolabel flux per unit area/radiolabel concentration in the source).
Each symbol [0 - K , • - Na (serosal to mucosa),Q- Cl (mucosa to serosa),
and A-mannitol] represents data from one trachea.
points by the method of least squares.
Lines are fitted to the
However, it is clear that a K secretion of about 50 ueq/cm , hr would have to
be induced by amphotericin in the airways of the lobe to account for entire
change in luminal K concentration. Since this secretion could be easily
measured in the trachea it is reasonable to conclude that most of the K is
secreted by the alveolar epithelium.
Excised Bullfrog Lung
An alternative approach to the characterization of alveolar epithelial
permeability and ion transport is to exploit species with large area:: of
30
-------
Table 3. Cation Flow Across Excised Rat Trachea and
the Perfused Left Lobe of the Rat Lung
Flow Lobe Trachea
2
(yeg/cm , hr)
Na (passive)
Untreated
Amphotericin B
K (net secretion)
Untreated
Amphotericin B
0.029b
0.042b
0.0013d
0.0090d
2.7°
15. 5°
<0.056
<0.056
a 2
Estimated surface area - 1400 cm .
Calculated from the rate of change in the amount of Na in airspace
fluid, a KBR with Na replaced by choline.
£
Serosal to mucosal flux.
^he rate of change in the amount of K in the KBR in the airspace.
Serosal to mucosal flux - mucosal to serosal flux.
intact alveolar surface that can be separated easily from the airways. Each
lobe of the lungs of frogs and toads is a single/ large alveolus more than a
centimeter in diameter that is connected by a short airway to the trachea
(23). The luminal surface of the lobe is lined by a continuous epithelial
monolayer. Most of this layer is comprised of type I and type II pneumocytes
(20). A few ciliated cells cover the tips of a fibrous trabecular network in
the interstitium which also contains blood vessels, axons, and muscle bundles.
The interstitium separates the epithelium from a continuous pleural covering.
The alveolar sac can be opened and mounted as a planar sheet between
Ussing chambers. With identical Ringer solutions bathing both sides of the
lung, a transmural bioelectric p.d. of nearly 20 mV (lumen negative) and d.c.
conductance of 1.4 mS/cm were measured (5). Direct and indirect studies
indicate that the epithelium is the site of the biopotential and the major
resistance to transmural ion flow (5, 8).
The passive permeability of the bullfrog lung to electrolytes is similar
to that reported for the fetal and fluid-filled adult mammalian lung (7).
In addition, the bullfrog lung, like the fetal lung, actively secretes Cl
and other halides into the lumen. Cl transport equals the short-circuit
current and can be inhibited by metabolic inhibitors or Br".
Studies that were designed to establish the dimension(s) of an equiva-
lent pore(s) have been equivocal. Table 4 summarizes the permeability co-
31
-------
Table 4. Solute Permeability of Excised Bullfrog Lung
CO
10
Solute
36ci
22Na
C-mannitol
57- D c
Co-B.,,.
3 • -12 •
H-methoxy
iniilin
(unfiltered)
Radius
(run)
0.19
0.29
0.4
7.2
14
n
5
5
29
3
3
P x 107a
(cm/sec)
mean
15.2(+2.6)
9.5(+0.8)
2.7(+0.2)
0.08(+0.03)
. ~~
1.2(+0;. 1)
P /P
solute mannitol
(+SE)
6.9(+0.7)
5.4(+1.0)
1
0.05(+0.01)
0.72
solute mannitol
2.87
1.87
1
0.50
0.32
H-methbxy
inulin 14
(gel filtered)
C-methoxy
inulin 14
(gel filtered)
0.40
0.25
0.12
0.06
0.32
0.32
Permeability coefficient for the unidirectional flux of tracer from the luminal to pleural
bathing solution.
Ratio of the free diffusion coefficients.
C
Cyanocobalamin.
Chromatographed on'a Sephadex G-100 column. About 25% of the total radioactivity was
selected from the peak and an equal number of fractions on either side of the peak.
-------
efficients for the passive flux of solutes of different size across the
short-circuited lung. In contrast to the canine trachea, the coefficients
for Na and Cl movements relative to that of mannitol are greater than the
free diffusion ratio. These results suggest that mannitol movement and the
flow of the larger cyanocobalamin and imilin molecules are restricted, i.e./
the apparent area for large molecule diffusion through aqueous channels is
less than that for small molecules. However, the permeability coefficient
measured for inulin depends on the "purity" of the tracer species. Com-
mercial tritiated methoxy-inulin contains small fragments which permeate the
lung rapidly, resulting in a ratio of inulin to mannitol flows which is more
than twice the ratio predicted for free diffusion. Partial purification
of inulin by fractionation on polyacrylamide gel results in a tracer species
that is restricted, but the possibility of minor contamination by small
fragments cannot be excluded. The contribution of this contamination to the
apparent inulin flux is magnified when restriction is severe* This uncer-
tainty leads to the calculation of several pore radii for the lung (Fig. 2).
Whereas the flows of Na , Cl~, and cyanocobalamin relative to that of man-
nitol describe a pore radius of 1 to 2 nm, the most slowly permeating inulin
species appears to require a radius of 3.5 nm. Despite these reservations,
most of the data favor the notion that the passive flow of hydrophilic mole-
cules across the bullfrog lung involves pores that resemble those of fetal
or adult mammalian lung rather than the transmural channels in the tracheal
mucosa.
The potential value of pore size estimates can be illustrated by experi-
ments that assess the effects of pH on bullfrog lung permeability. Exposure
of the mucosal (luminal) or serosal (pleural) surface of the lung to un-
buffered Ringer solutions that contain HNO , H So , or HC1 induce identical
effects. Bioelectric properties do not change until the pH of the bathing
solution falls below three. Then conductance increases several fold. Volume
flow in response to an osmotic gradient rises* Table 5 illustrates the ef-
fects of exposing either surface of the lung to HC1. The relatively low pH
of the other bathing solution reflects the permeation of protons through the
lung during the three-hour experiments. In both experiments an increase in
conductance was paralleled by a dramatic increase in the mannitol and Cl
permeability coefficients. The ratio of the coefficients (?„,-/P .. .)
Cl mannitol
approached the value for diffusion in free solution. This fall can be ex-
plained by the expansion of existing channels to a radius greater than 4 nm
and/or the creation of new large pores.
Whereas the effects of mineral acids on lung permeability are not sub-
tle, a number of chemical agents induce selective changes in functions of the
bullfrog alveolar epithelium. The replacement of NaCl in the mucosal bath-
ing solution by NaNO inhibits short-circuit current by 40% but does not
33
-------
10
I .1
o
£
•01-
mannitol
•
unrestricted
3-5 nM
•. inulin
5 10
Probe Molecule Radius (nM)
Figure 2. Estimation of equivalent pore radius from the permeability
coefficients for solute flow. The relationship between the permeability
coefficient (P) and pore radius is given by:
P --Si
(cm/sec) Ax
(28)
where D » the diffusivity in aqueous solution (cm /sec); Ax » the path length
for diffusion (cm); P(a/r) = the function that describes steric and fric-
tional interactions between solute and pore, specifically [1-(a/r] [1-2.1
3 5
(a/r) + 2.09(a/r) - 0.95(a/r) ] in which a « solute radius and r » pore
radius. By dividing the permeability coefficients for other probe molecules
by the coefficient for a reference solute, mannitol, Ax is eliminated:
P D , P( a /r)
probe probe probe
^•^^™^^^^™—• SB ^•••^^^^••^•^^^W^" ^^^HH4fe«^H^«iMaM»«M^Ma^MIBB»
P D Ff a /v\
mannitol mannitol mannitol
The lines join predicted ratios of the permeability coefficients for each
solute-mannitol pair. The ratios were calculated from assumed pore radii
and literature values for a and D*
34
-------
01
Table 5. Effect of Bathing Solution pH on the Solute
Permeability of Excised Bullfrog Lung
Bath pH
Luminal Pleural
8.3 8.3
2.5 6.3
6.3 2.5
Solute n
36cr
5
14
C-mannitol
36cr
2
14
C-mannitol
36cr
2
C-mannitol
7b
Ga Px10
(mS/cm ) (cm/sec)
15.2°
0.9
2.7°
669°
6.7
228°
614d
7.4
179d
P D
mannitol mannitol
6.9 2.9
1 1
2.9 2.9
1 1
3.4 2.9
1 1
d.c. conductance.
bpermeability coefficient for the contemporaneous flux of radio-mannitol and Cl
across the same lung lobe.
°Lumen to pleural flux.
'pleural to lumen flux.
-------
affect conductance. Bioelectric properties are not affected by serosal
exposure to NaNO_ (8). The mode of NO. action is not known, but unpublished
flux experiments demonstrate that Cl flow toward the lumen and the trans-
mural movement of Na in either direction are unaffected.
Amphotericin B in the mucosal solution induces not only the secretion
of K into the lumen but also a paracellular K selective pathway (11), an
effect which has also been described for toad urinary bladder (7).
High concentrations of HgCl_ in the mucosal solution (10~ to 10 M/l)
lead to an increase in conductance and general ion permeability and inhibi-
tion of active Cl secretion (8). However, the flow of water in response to
a transmural osmotic gradient is not affected (12). When exposure to Hg is
followed after one minute by the addition of an excess of a sulfhydryl agent,
such as dimercaprol, there is a sustained 40% increase in short-circuit cur-
rent and in the secretion of Cl , but neither conductance nor unidirectional
fluxes of Na are affected significantly (12).
Finally, the beating of cilia on the cells that cover the trabecula is
inhibited by the addition of CdCl_ to the luminal solution (concentration
-53 z
range * 10 -10 M/l) (10). The metal also decreases the rate of tissue
oxygen consumption but does not affect bioelectric properties or transmural
ion movement. Studies with alveolar epithelial cells that were disaggregat-
ed from the lung surface by perfusion through the pulmonary vasculature with
collagenase show that Cd complexed with bovine serum albumin or hemoglobin
does not alter ciliary motility (13). However, these extracellular proteins
do not reverse the effects of Cd that has already reacted with the cells.
In contrast, Cd exposed cells washed with permeant sulfhydryl agents, such
as mercaptoethanol, dimercaprol, or cysteamine, partially or completely re-
cover oxygen consumption and ciliary motility (14). These observations
suggest that the inhibition of ciliary motility requires the interaction of
Cd with intracellular ligands. Measurements of the Cd binding which persists
after the sulfhydryl agent wash suggest that no more than 30% of the total
Cd ligands participate in the inhibition of ciliary motility.
CONCLUSIONS
Evidence from fluid-filled, perfused rat lung lobes, rat trachea, and
bullfrog lung indicates that the alveolar epithelium restricts the movement
of small hydrophilic molecules. The large epithelial surface of the alveolar
epithelium is shunted by the airway epithelium, a less restrictive barrier.
Chemical agents, such as amphotericin B, can induce quantitatively and, per-
haps, qualitatively different effects in the gas conducting and exchanging
epithelia. Whereas exposure to acids (pH < 3) induces a general increase in
solute and water perreability and in pore radius, other edemagenic agents,
36
-------
such as HgCl and CdCl_, affect ion secretion and ciliary motility without
altering the permeability of the alveolar epithelium.
ACKNOWLEDGMENT
Supported, in part, by U.S. Public Health Research Grant HL 16674.
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39
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DISCUSSION
DR. DAVIS: I have one technical question relating to the work on rat trachea
and your system for measuring short circuit current. As you know, the original
Ussing technique depends on the presence of a uniform electrical field across
the tissue* Your approach may have been described before. I thought about how
you measure short circuit current; it1s always worried me that you can't meet
those conditions by having a wire down the middle or something around the
outside. Could you say something about that?
DR. GATZY: Certainly. The requirement is that the field should be uniform.
This will result when an axial wire is placed in the center of the tracheal
cylinder and current is passed between this wire and an outer cylindrical
foil. As long as the wire and foil are of uniform composition and plated
uniformly, you ought to get a uniform current. That's what we tried to do.
This is not the first time that this technique has been used. Clarkson and
Toole used this approach with the intestine. The intestine was mounted
vertically and the rather long piece of intestine required large silver elec-
trodes. So the technique has been used before with some success. There may
be a problem in positioning the electrode inside the very small lumen of the
trachea.
DR. DAVIS: That may, in fact, be a part of the problem with your residual
conductance.
DR. GATZY: We have thought of that and have attempted to look into the
question. By moving the electrodes around to the geometric extremes, it looks
like the maximum error is of the order of 15 percent, but when you add this
change in driving force to flow in both directions, it's a possibility.
DR. DAVIS: It's encouraging that you got matching results. You know that
John Mangos has done similar kind of work using rat trachea. That's published
in the Cystic Fibrosis Abstracts.
DR. GATZY: There is a problem with measurements of net flow as defined by
dilution of inulin in the lumen because inulin permeates through the tissue.
Those results must be scrutinized carefully to determine whether there really
is net addition of volume to the lumen.
40
-------
Under short-circuit conditions we observed sodium reabsorption and chloride
secretion/ much like the flows across the dog trachea except that these flows
seem to be more evenly balanced* As you know, opening the circuit tends to
counter the flows measured under short-circuit conditions* I would predict
little or no net ion flow across rat trachea under open circuit conditions.
DR. BELL: How was albumin on the alveolar surface determined?
OR. GATZY: As I understand Reifenrath and Zimmerman's work, protein that was
recovered in fluid from the alveolar surface was not correlated with the volume
of collected fluid and was considered to result from "contamination." So, under
the normal resting situation, at least in the rat, they consider protein to
be absent from the alveolar fluid. How it gets there after treatment with the
edemagenic agents is still an open question. Protein could leak through the
junctions of the alveolar epitheliun, or through the airway epithelium and then
run down into the alveoli. There seems to be no evidence to clearly distinguish
between these two possibilities.
DR. BELL: Do you believe that under normal, conditions in the lung you would
not find a protein like albumin on the alveolar surface?
DR. GATZY: The only study that sampled alveolar surface fluid directly dismisses
this possibility.
41
-------
Session II:
Respiratory Tract and Host Defense
-------
Neutral Proteases of Human Polymorphonuclear
Granulocytes: Putative Mediators of Pulmonary
Damage
J. K. Spitznagel, M. C. Modrzakowski,
K. B. Pryzwansky, and E. K. MacRae
University of North Carolina
Chapel Hill, North Carolina
Neutrophil polymorphonuclear (PMN) granulocytes and their neutral
elastases are among today's leading candidates for principal mediators of
chronic obstructive pulmonary disease due to panacinar emphysema (9). The
concept of these cells and their enzymes as mediators of lung damage fits
well with the established anatomical and functional importance of elastic
fibers in alveolar septa and respiratory bronchioles. Figure 1 shows the
respiratory bronchiole leading into the pulmonary alveoli. Figure 2 shows
the numerous elastic fibers woven through the alveolar and bronchiolar walls.
This concept also fits with reports that in emphysematous lung elastic fibers
have been damaged. Elastase itself affords an element of specificity that
enhances the chance that it is important in this picture of damage since
purified collagenases and trypsin seem unable to induce experimental
emphysema. As things stand, the most successful animal models of human
emphysema depend on intratracheal installation of an elastolytic protease
such as papain or elastase from polymorphs. intravenous injections will
work but require much higher doses of enzyme. In hamsters 0.2-0.5 mg/100 g
of elastase by intratracheal installation initiates irreversible lung
disease. The exact role of the granulocytes, other than their service as
ready sources and vehicles for elastase, is still uncertain. However, there
appear to be two phases for their participation in lung damage. There is
recruitment of the cells and there is exocytosis (degranulation) of the
elastase. Recruitment is likely to depend on deposit in respiratory
45
-------
Figure 1. Normal bronchiole. H and E stained histological section.
Note respiratory bronchiole (left) and alveoli. Courtesy of Dr. E. MacRae,
Department of Anatomy, University of North Carolina.
bronchioles and alveoli of foreign particulate matter with cytotoxic
materials absorbed onto their surfaces (10). Exocytosis of elastase and
access of the enzyme to elastin fibers may depend on the direct effects of
cytotoxic chemicals that damage the PMN (2) and the counter-inhibitory effects
of these chemicals acting on protease inhibitors from plasma (8). Exocytosis
of proteases may also be induced with chemicals absorbed to particles
phagocytized by the polymorphs.
In view of the rather limited knowledge of the circumstances of local
involvement of PMN in the lung, I have chosen not to discuss them but to
discuss some pertinent aspects of the cell biology of PMN neutral proteases
about which many details are available. I shall try to relate these in a
speculative way to the events that may be taking place in the lung. In
particular, I shall discuss the kinds of proteases associated with PMN, the
way in which they are packaged, the ways in which they come to be released
from PMN, and some aspects of their action on connective tissue.
Human PMN have three neutral proteases that have been studied in
46
-------
Figure 2. Lung. Orcein (elastin) stain. Note elastin fibers about
alveoli. Dots are nuclei. Courtesy of Dr. E. MacRae.
considerable detail, an elastolytic enzyme, a chymotrypsin-like cathepsin
G, and a collagenase (1, 3, 11). There are, in addition, much less extensively
studied enzymes—a plasminogen activator (4) and proteinase 3 (P.,) that has
hardly been characterized at all (1). The elastase and the cathepsin G are
serine esterases active against a variety of large molecules such as casein,
but the important thing about elastase is its unique capacity to cleave the
helical peptide of elastin. Being serine esterases, these enzymes are
irreversibly inhibited by chloromethyl ketone derivatives of synthetic peptides
and by sulfonyl fluorides. The elastase is highly reactive with peptide de-
rivatives that have alanyl-alanyl-prolyl-valyl sequences. Cathepsin G is highly
reactive with peptides containing phenylalanine (11). The enzymes are readily
demonstrated with polyacrylamide gel electrophoresis. They are also inhibited
by naturally occurring protease inhibitors found in plasma, a.-antitrypsin and
a -macroglobulin and a -antichymotrypsin (6). The collagenase is not an esterase,
it is unaffected by the chloromethyl ketone inhibitors. Since it is a metallo-
enzyme it is inhibited by metal chelators such as ethylenediaminetetraacetic
47
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Figure 3. Human, normal, resting neutrophil. A, azurophil granules;
S, specific granules; M, vestigial mitochondria; G, Golgi apparatus; C,
rentriole. Fixation 0.5%
-------
granule apparatus of a resting human PMN. Note that the granules appear
heterogeneous, that is, there are many different sizes and electron densities.
In spite of the confusing varieties, there are only two biochemically
distinct kinds, the specifics or secondary granules and the azurophils or
primary granules. Both granule types are formed while polymorphs are still in
the marrow. Azurophils are formed in the promyelocytes and specifics are
formed in the myelocytes (13). The azurophils tend to be large, about 0.3
micrometers in diameter. The specifics tend to be smaller, about 0.1
micrometers in diameter (15). The important point is that the elastase and
cathepsin G are unequivocally carried in azurophil granules (1, 3). Collagenase,
however, is in the specifics (1 and observations/ this laboratory). We know
this as a result of cell fractionation experiments. Thus purified PMN can
be broken up in a homogenizer, their granules released, and the nuclei
containing the anti-proteases removed. When this mixture of granules is
centrifuged through a sucrose density gradient, several bands containing
different subcellular particles form in the gradient. Figure 4 shows a 60 ml
centrifuge containing such a sucrose density gradient. The subcellular
particles have been centrifuged to density equilibrium. The specific
granules are seen clearly as band H^. The azurophil granules are seen as two
bands marked III and III- (15). Pumped out of the centrifuge tubes and
analyzed, these bands of granules are associated uniquely with certain
constituents. Table 1 summarizes a few of the constituents of the major
granules. The azurophils contain elastase, cathepsin G and myeloperoxidase
(MPO). They also contain acid hydrolases—phosphatase, g-glucuronidase, and
lysozyme. The MPO, besides being strongly antibacterial, serves as a unique
marker for azurophils (15). Most of these enzymes are very cationic proteins.
The ones in the table are ordered according to their degree of cationicity.
The cationic property may promote their binding to connective tissues. The
specific granules have lysozyme and, in addition, they have apo-lactoferrin,
an 80,000 dalton, iron binding, intracellular analogue of transferrin. It
forms a specific marker to trace the fate of these granules (15). Specific
granules also contain a collagenase that is latent as we shall see. Figure
5 shows the results of biochemical and immunochemical analysis of the
fractions. The relative distribution of the enzymes is shown in relation to
the gradient in which the particles are depicted as moving from left to
right. Enzyme peaks are to be compared with the positions of the various
granule populations marked by daggers and by the MPO in the azurophils and
the lactoferrin in the specifics. The elastase is distributed exactly as
are the azurophil granules. Not shown here but with exactly the same
distribution in the azurophil granules is cathepsin G (11). From this
we conclude that both elastase and cathepsin G are azurophil granule enzymes.
It can also be seen that the collagenase is in the specific granules.
49
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Figure 4. Human neutrophil polymorphonuclear granulocyte granules
separated on sucrose density gradient
granules
I, cell membranes; II
The II band comprises the specific
III and III- comprise two subclasses of azurophil granules.
3 i-
mitochondria.
Curiously, the collagenase is doubly latent, that is, it cannot be detected in
intact granules. Moreover, it cannot be detected when the specific granule
membranes have been removed unless it is activated by a proteolytic enzyme.
There is an expected latency in all granule enzymes. It is due to the
membranes that surround the granule enzymes, rendering them inaccessible to
substrate until they are treated to remove the membrane barrier as was
50
-------
;*"
5 *
2j *
s
i !
q ••
L4^
Figure 5. Histogram showing activities of collagenase (white histogram
latent/ black histogram collagenase with trypsin activation), lactoferrin
(specific granule marker), myeloperoxidase (azurophil granule marker),
elastase, and amounts of protein from sucrose density gradient similar to
Figure 4.
Table 1. Some Constituents of the Major Granules of Neutrophil
Polymorphonuclear Granulocytes
Azurophil (primary) granules
Specific (secondary) granules
Acid Phosphatase
Acid 8 Glucuronidase
Myeloperoxidase
Elastase
Lysozyme
Cathepsin G
Lysozyme
Lactoferrin
Collagenolytic Enzyme
done here to measure the MPO, elastase, and cathepsin G. Latency could not
be eliminated so simply with the collagenase. The small white histogram
shows the small activity detected with only membrane removal. The larger
black histogram shows the activity and distribution of collagenase with
51
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membrane removed and latent enzyme activated by another protease. Trypsin
was the first protease we found that could activate the collagenase.
The most effective collagenase activator turned out to be the highly
purified cathepsin G of azurophil granules. Elastase of azurophil granules
was less effective than cathepsin G or trypsin. This was shown with the
digestion of salt soluble guinea pig skin collagen and was measured with
14
release from the collagen of biologically incorporated C-labeled glycine
and proline. The most active enzyme was cathepsin G. Trypsin activity was
intermediate and elastase was least active.
Now, why so much discussion of PMN collagenase if elastase and elastin
are the most important factors in lung damage leading to emphysema? Actually
there is also evidence that lung collagen is damaged as well, especially in
experimental emphysema (8). Hence a mediator of that damage should be
sought. For that reason I shall compare effects of collagenase and elastase
on collagen and discuss further the relationships between latency of
collagenase and the fate of cathepsin G, its activator. Figure 6 is from
sodium dodecyl sulfate polyacrylamide gel analysis of digestion fragments
cleaved from collagen by highly purified samples of these enzymes.
Collagenase cleaves the helical peptides and gives rise to the characteristic
3/4 and 1/4 length fragments seen in gels 3, 4, 5, and 6. The presence of
other cleavage products suggests that other enzymes capable of cleaving the
peptides are present as well. The amount of fragments increases with time
of digestion. The other gels are from collagen and enzyme controls. The
cathepsin G activator is needed to activate the collagenase. Some reports
suggest that, unlike elastase, cathepsin G is not secreted from PMN (14).
If this is so, the collagenase may be relatively unimportant. Could elastase
cleave collagen? It has been believed unable to attack native collagen,
but Figure 7 shows that purified elastase monomerizes native collagen in the
presence of PMN collagenase. It could do this by cleaving the N terminal
peptides that connect the helical peptides into dimers, the g chains, and
trimers, the Y chains. Here it is seen that with increasing time of digestion
the polymers disappear, no small fragments are seen but only a chain monomers
remain. Quantitatively it also seems that the ot chains eventually, with time,
are cleaved to very small peptides as seen in the last gel. The other gels
are collagen and enzyme controls. These observations are in agreement with
Starkey et al. (16). The relative contributions of the two enzymes in our
system remain to be defined.
N
The crucial question, of course, is how these proteinases happen to
emerge from the PMN and become available to attach to elastic and collagen
fibers as purified elastase has been made to do in animal models (9). Do PMN
die and autolyze leaking enzyme over the connective tissue? Evidently that
is unnecessary. Phagocytosis of particles as small as 1 micrometer in
52
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Figure 6. Cleavage products of guinea pig skin salt soluble collagen
reacted with specific granule collagenase. The collagenase in this instance
was activated by cathepsin G which does not attack native collagen. The
cathepsin G was highly purified. The collagenase was used as specific
granule extract. Far left, collagen control; second from left, collagen plus
granule activated granule protein 0 time; gels 3, 4, 5, 6 - 0.5, 1, 2, 4
hours, respectively, with activated enzyme; far right, gel granule enzyme
control. Note progressive appearance of « (3/4) and a (1/4) fragments.
A B
Other cleavage products not identified appeared with progression of time.
diameter, given appropriate coatings of immunoglobulins, for example, can
cause secretion of elastase from human PMN. Phagocytosis of bacteria,
yeast cell walls, and other particles can do this as well. This leakage is
further aggravated by attempts of PMN to phagocytize surfaces they cannot
surround with membrane. Henson has shown this for other PMN granule enzymes
and named it frustrated phagocytosis (7).
We have been especially interested in how (and also why) PMN secrete
granule enzymes during phagocytosis. It seems curious that cells so
important in anti-infectious immunity as PMN should have survived with such
untidy eating habits. Figure 8 is from an immunocytochemical study on
53
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GP-
I
- -DP
Figure 7. Cleavage fragments of collagen digested with azurophil
granule elastase. Elastase cleavage of salt soluble guinea pig skin collagen,
Control collagen on left (A). B, elastase (gp) plus collagen at time 0.
C, D and E, time course of digestion 0.5, 1 and 2 hours incubation. Note
disappearance of all but a chains.
phagocytosis of Escherichia coli with human PMN. The PMN were allowed to
phagocytize _E. coli, washed in saline, and fixed in 1% parafonnaldehyde. The
paraformaldehyde fixative leaves the cell membranes impermeable by antibody.
The fixed cells were reacted with antibody against myeloperoxidase conjugated
to fluorescein. Any fluorescein bound to the cells indicates antibody
specifically bound to myeloperoxidase that has emerged onto the PMN surface.
Normally MPO is buried behind two membranes, the cytoplasmic membrane and
the granule membrane. Therefore, it cannot be stained in resting cells.
Here the antigen MPO is on the outer surface of the cell. A communication
has been established from granule matrix to cell surface. Antibody to
lactoferrin was conjugated to rhodamine and used to stain the same cells.
The results in the far right panel show that a little lactoferrin also found
its way to the cell surface. The nuclear staining is nonspecific and due to
methyl green which fluoresces dark red in ultraviolet light, it is used for
orientation.
54
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Figure 8. Human polymorph phagocytizing Escherichia coli 5 minutes
after bacteria were added to cell monolayer. Left is a phase contrast view
showing bacteria just within a space visible between nuclear lobes. Middle,
the fluorescein conjugate of anti-human myeloperoxidase shows that
myeloperoxidase has emerged onto the external surface of the cell membrane.
Right, double staining with a rhodamine conjugate of anti-human lactoferrin
shows that a small amount of lactoferrin is also on the cell surface. The
same cell is seen in all three panels. It was fixed with 1% paraformaldehyde.
This fixative leaves the cell membrane impermeable to the immunoglobulins of
the fluorescent conjugates. Thus only antigen on the outside of the cell
surface is stained.
These immunocytochemical results suggest that the mouth of the nascent
phagolysosome was open, wide open, to the fluid phase and that the granules
joined or fused their membranes with the phagolysosome membrane. To investi-
gate this further we have, with Dr. Edith MacRae, used scanning electron
microscopy to evaluate events on the phagocytizing cell surface. Figure 9
shows that indeed the phagolysosome where you see E. coli about to enter does
gape widely, offering ample opportunity for exocytosis of the enzymes from
the phagolysosome.
55
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Figure 9. Human polymorph phagocytizing Escherichia coli 5 seconds
after bacteria were added to the cell monolayer. Note the gap between the
lips of the nascent phagolysosome and the bacterial envelope.
Findings from our laboratory and from the literature lead us to
conclude that the PMN have potent enzymes capable of digesting both elastic
fibers and collagen fibers. These enzymes do leave the cells, but whether
they react with the connective tissues and alter them remains to be shown.
It is also clear that phagocytosis and stimulation with several soluble
mediators and chemicals may also induce release (2). Smoke particles with
toxic chemicals absorbed to their surfaces are reported to be chemotactic
and to stimulate phagocytosis (10). F^en in the normal mode of antimicrobial
phagocytosis granule enzymes are exocytosed onto the PMN surface and they
can dissolve in the fluid phase bathing the cells. The azurophil granule
contents containing elastase may conceivably within suitable localized
spaces evade inhibition by
-------
now known to depolymerize collagen (see above). This depolymerization
would be expected to weaken the collagen (16).
If proteolytic enzymes generally tend to be exocytosed by phagocytizing
PMN, why do they not cause damage at all times? For example, why doesn't the
PMN response in pneumococcal pneumonia autolyze the lung? It is probably be-
cause biological control is available, perhaps through the antiproteases of
plasma (6), perhaps through mechanisms yet to be discovered. Most important
among known mechanisms with respect to PMN neutral proteases are plasma a..-
antitrypsin and a -macroglobulin. But there are, in addition, plasma a1~
antichymotrypsin, plasma B -anticollagenase, and plasma antileukoprotease.
That a -antitrypsin may indeed afford critical control and prevent proteolytic
damage to lungs is suggested by the studies on the relationships between
homozygous a -antitrypsin deficiency and pulmonary emphysema. Finally, it
should be noted there are antiproteases in bronchial secretions as well as in
plasma.
Experimental results recently reported from Janoffs laboratory (8)
suggest that cigarette smoke concentrate interferes with antiprotease activity
due to pure human serum a.-antitrypsin, challenged with pure human PMN
elastase and porcine pancreatic elastase. Moreover, Galdston e_t al. (5) have
reported that pack years of smoking, elastase-like esterolytic activity of
polymorphs plus trypsin inhibitory activity of plasma seem to account for 68%
of variability in pulmonary function tests observed in MZ and ZZ a.-anti-
trypsin phenotypes with chronic obstructive pulmonary disease. One must con-
clude that the evidence that proteolytic enzymes could be factors in pulmonary
damage is highly suggestive; whether these enzymes are from PMN remains to be
seen.
SUMMARY
The principal need is for direct, rigorous demonstration that PMN,
charged with elastase, are recruited to the strategic part of pulmonary structure
in sufficient numbers, and with appropriate kinetics to account for low grade
but progressive long-term damage to connective tissue, especially to elastin
and possibly to collagen. Moreover, it will be necessary to show the nature
and existence of conditions that lead to exocytosis of the proteases from
polymorphs, that lead to the failure of biological controls due to anti-
proteases, and that promote degradative enzyme interactions with connective
tissue fibers.
ACKNOWLEDGMENT
Supported by a grant from the USPHS AI 02430.
57
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REFERENCES
1. Baggiolini M, Bretz U, DeWald B: Subcellular localization of granulocyte
enzymes. In: Neutral Proteases of Human Polymorphonuclear Leukocytes
(Havemann K, Janoff-A, eds). Munich, Urban & Schwarzenberg, 1977, p 7
2. Blue N, Janoff A: Possible mechanisms of emphysema in cigarette smokers.
Am Rev Resp Dis 117:317, 1978
3. DeWald B, Rindler-Lugwig R, Bretz U, Baggiolini M: Subcellular
localization and heterogeneity of neutral proteases in neutrophilic
polymorphonuclear leukocytes. J Exp Med 141:709, 1975
4. Granelli-Piperno A, Vassali JD, Reich E: Secretion of plasminogen acti-
vator by human polymorphonuclear leukocytes. J Exp Med 146:1693, 1977
5. Galdston M, Melnick EL, Goldring RM, Levytska V, Curasi CA, Davis AL:
Interactions of neutrophil elastase, serum trypsin inhibitory activity
and smoking history as risk factors for chronic obstructive pulmonary
disease, in patients with MM, MZ, and ZZ phenotypes for alpha antitrypsin.
Am Rev Resp Dis 116:837, 1977
6. Heimberger N: Proteinase inhibitors of human plasma--their properties and
control functions. In: Proteases and Biological Control (Reich E,
Rifkin DB, Shaw E, eds). Cold Spring Harbor Conferences on Cell Prolifera-
tion. New York, Cold Spring Harbor Laboratory, 1975
7. Henson PM: The immunologic release of constituents from neutrophil
leukocytes mechanisms of release during phagocytosis and adherence to
nonphagocytosable surfaces. J Immunol 107:1547, 1971
8. Janoff A, Carp H: Possible mechanisms of emphysema in smokers.
Cigarette smoke condensate suppresses protease inhibitors in vitro. Am Rev
Resp Dis 116:65, 1977
9. Karlinsky JB, Snider GL: Animal models of emphysema. Am Rev Resp Dis
117:1109, 1978
10. Kilburn KH, McKenzie W: Leukocyte recruitment to airways by cigarette
smoke and particle phase in contrast to cytotoxicity of vapor. Science
189:634, 1975
11. Ohlsson K, Olsson I, Spitznagel JK: Localization of chymotrypsin-like
cationic protein, collagenase, and elastase in azurophil granules of
human neutrophilic polymorphonuclear leukocytes. Hoppe-Seyler1s Z
Physiol Chera 358:361, 1977
12. Powers JC, Gupton BF, Lively MO, Nishimo N, Whitly RJ: Synthetic in-
hibitors of granulocyte elastase and cathepsin G. In: Neutral Proteases
of Human Polymorphonuclear Leukocytes (Havemann K, Janoff A, eds).
Munich, Urban and Schwarzenberg, 1977
58
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13. Pryzwansky KB, Hausch PG, Spitznagel JK, Herion JC: Immunocytochemcial
distribution between primary and secondary granule formation in develop-
ing human neutrophils correlation with Romanowsky stains. Blood 53:179,
1979
14. Schmidt W: Differential release of elastase and chymotrypsin from
polymorphonuclear leukocytes. In: Neutral Proteases of Human Poly-
morphonuclear Leukocytes (Havemann K, Janoff A, eds). Munich, Urban &
Schwarzenberg, 1977
15. Spitznagel JK, Dalldorf FG, Leffell MS, Folds JD, Welsh IRH, Cooney MH,
Martin LE: Character of azurophil and specific granules purified from
human polymorphonuclear leukocytes. Lab Invest 30:774, 1974
16. Starkey PM, Barrett AJ, Burleigh MC: The degradation of articular collagen
by neutral proteinases. Biochim Biophys Acta 483:386, 1977
59
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DISCUSSION
DR. JAKAB: Polys are a kind of a kamikaze cycle, and their half-life is
relatively short in the blood. When they get into the lung/ is it neces-
sary for them to be stimulated for the release of the enzymes or could it
be that they just die and release their enzymes and result in the damage?
DR. SPITZNAGEL: It is surprising, but when you look at sections of normal
lung, there are few polymorphonuclear leukocytes. This is certainly not
true, for example, in the mucosa of the gut wall. That is full of poly-
morphonuclear leukocytes at all times, and you can even see them working
out into crevices in the mucosa of the gut. There are not the large numbers
of macrophages that Joe was talking about earlier with reference to lung.
Polys require some specific stimulus to bring them to the lung and to keep
them there. Apparently, microbial cells being in large numbers, as in pneumonia
will do this, and Kilburn has shown that instillation of cigarette smoke
particles will also attract polymorphs to lung. Unless direct quantitative
studies can show that polys are arriving in lung tissue and perishing very
rapidly over a relatively long period of time, it is difficult to imagine polys
as the major source of destructive enzyme.
60
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Bronchus-associated Lymphoid Tissue and the
Source of Immunoglobulin-containing Cells
in the Mucosa
J. Bienenstock
McMaster University
Hamilton, Ontario
Although the lungs are not usually regarded as primary lymphoid organs, it
may be of interest to recall that in 1958 Humphrey and co-workers (1, 11)
showed that following intravenous hyper immunization with pneumococcal antigens,
the lung tissue was the predominant source of specific antibodies, surpassing
even the bone marrow, spleen, and lymph nodes. One explanation for these
findings may have been that the lungs contained a substantial population of
immunocompetent cells present by either direct immigration from the circulation
or by local proliferation of a precursor population. Also, it appears from
this work that the lungs can respond well to antigens introduced parenterally.
A few years ago we observed the presence of follicular lymphoid aggregates
in the mucosa of the rabbit bronchial tract with a peculiar epithelial relation-
ship (4, 5). These lymphoid follicles can, with practice, be discerned as dense
white patches under the dissecting microscope or upon treatment of the respiratory
tract with 2% acetic acid. The distribution of this lymphoid tissue is random
along the length of the bronchial tract as far as the small bronchioles and
seems to be concentrated around bifurcations. The tissue is present in all
mammals examined so far with the possible exception of the Golden hamster.
Chickens also possess considerable amounts of this tissue which projects into
the lumens of the large bronchi. Morphologically, this bronchus-associated
lymphoid tissue (SALT) was remarkably similar to the Peyer's patches of the
gut. Interestingly, Klein in 1875 (13) wrote, "...these lymphoid follicles of
the bronchial walls are, therefore, in every respect analogous to the lymph
follicles found in other mucous membranes, e.g. tonsils and in the intestine."
61
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The epithelium which overlies the SALT follicles is unlike that found
elsewhere in the bronchus and is constructed of flattened, irregular shaped
cells. Often the SALT epithelium is heavily infiltrated with lymphocytes from
the underlying follicles (3). This lymphoepithelium is reminiscent of the
follicle-associated epithelium overlying Peyer's patches in the gut (6) and is
deficient in glandular tissue and goblet cells as well. In addition, cilia are
irregularly distributed and ultrastructural examination reveals the presence
of microvilli. Rare plasma cells are seen scattered throughout the lymphoid
follicles and occasionally plasma cells are found in the overlying lymphoepithe-
lium (20). The follicles contain the high-walled endothelium associated with
postcapillary venules, and this type of endothelium appears to be more prominent
following antigen challenge (20).
The lymphoepithelium seems to act as a sampler of the luminal antigen much
as does the follicle-associated epithelium of the Peyer's patches and also the
bursa of Fabricius (6). Horseradish peroxidase introduced into the lumen of the
rabbit tracheobronchial tree is selectively taken up by the lymphoepithelium
overlying the SALT follicles (26). A fairly extensive proliferation of cells
in the follicles occurs after antigen challenge, particularly with Bacillus
Calmette-Guerin (20), and following in vivo injections of tritiated thymidine,
heavily labeled cells are seen 24 hours later in a crescentric manner capping
the luminal aspects of the follicles (5). However, the SALT seems to develop
in the virtual absence of antigen. When fetal pulmonary tissue was transplanted
to ectopic subcutaneous sites in syngeneic adult mice, HALT follicles still
developed but appeared immature (5, 15). The fact that germ-free animals
possess a BAIT mass considerably less than that seen in conventional animals
strongly suggests that antigen promoted complete SALT development (4).
The predominant types of cells present in BALT, as judged by surface mem-
brane antigens/ are neither T nor B cells (24). In young adult rabbits, 18.4%
of BALT cells bore a thymic antigen whereas in Peyer's patches and intestinal
lamina propria, this figure was 16.6% and 11.1%, respectively. Approximately
40% of BALT cells bore surface immunoglobulin and 17.2% of these had surface
IgA, a value intermediate between that of Peyer's patches (29.9%) and gut lamina
propria (7.3%). The finding of BALT intermediacy between the Peyer's patches
and the intestinal lamina propria provides some clues as to the functions of
the BALT.
Some evidence indicates that the BALT is functionally similar to the Peyer's
patches. It has been demonstrated (25) that cells derived from the BALT had
virtually the same capacity as Peyer's patch cells to repopulate the spleen of
irradiated allogeneic recipients with IgA-containing cells. Of greater signifi-
cance, cells derived from the BALT were nearly as effective as Peyer's patch
cells in repopulating the lamina propria of the bronchus and gut with IgA plasma
cells.
62
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These findings demonstrated that cells from one mucosal site could move and
perhaps differentiate at another mucosal site, and supported our original hy-
pothesis, in 1973, that the HALT might be part of a more universal mucosal
lymphoid system (4).
In 1974, we proposed that there may be a common mucosal immunologic system
(2). Montgomery (16) has shown that rabbits orally immunized with DNP-pneumo-
cocci have a selective appearance of specific IgA antibody in the milk in the
apparent absence of such antibody in the circulation. Similar feeding experi-
ments have been done by Hanson and co-workers in humans using a non-pathogenic _E.
coli (9). The subjects which had received JJ. coli subsequently developed a rise
of specific IgA antibody against JS. coli in the milk and antigen-specific IgA
antibody-containing cells appeared in their colostrum. Lamm and co-workers (22)
have shown that dividing cells from the mesenteric lymph node have a tendency
to selectively localize in the mammary glands when adoptively transferred into
syngeneic mice in late gestation. This selective migration was enhanced greatly
by lactation and could be induced in nonpregnant animals by the suitable in-
jection of sex hormones. Thus, the antibody and cells in milk specific for
enteric antigens probably result from the migration of cells from gut to breast
tissue. We have done similar experiments with dividing cells from the bronchial
lymph nodes and mesenteric lymph nodes and have shown that these cells have a
tendency to home to mucosal tissues including the breast, bronchus, bowel, and
cervix where they make predominantly IgA (14). Thus, it seems likely that a
common mucosal system may well exist, at least for those mucosal sites where
cells are potentially primed or sensitized and have a tendency to traffic to
another mucosal site.
One question which has not been completely answered is what the role of
antigen might be in lymphocyte traffic between mucosal surfaces. This traffic
appears to be independent of antigen although the presence of antigen, to which
the cells have been primed, clearly causes a greater number of IgA cells to
appear in the lamina propria of the bowel mucosa (18). It may be postulated
that antigen might cause cells to divide locally or even retain cells once they
have immigrated into the mucosal lamina propria. In this event, if cells were
not exposed to the sensitizing antigen they might leave (presumably by lym-
phatics) , die, or, alternatively, find their way via the mucosal epithelium
into the luminal spaces. The cytokinetic experiments referred to earlier
found heavily labeled cells within the lymphoepithelium of the HALT 24 hours
after the in vivo injection of tritiated thymidine (5). We have chosen to
interpret this finding as partial support for the suggestion that these cells
may be on their way out into the lumen although no direct evidence for this
exists.
63
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Careful examination of preparations of BALT led to the appreciation that
there were a number of cells present which had metachromatic granules in the
cytoplasm giving the appearance of primitive mast cells (3). Since some of
these cells may well have emanated from the bronchial lamina propria, a sys-
tematic attempt was initiated to look for them in BALT tissue. We did indeed
find, with special stains and ultrastructural techniques, the presence of
basophil-like cells, particularly in the peripheral zones of the BALT follicles.
These cells were also seen above the basement membrane in the epithelium ad-
jacent to these follicles and resembled the basophiloid cells which have been
described by Patterson in several animal species in the bronchial washings (17)
Collan (7) has described granular lymphocytes in the rat intestinal epithelivn
The relationship of these cells to mast cells is unclear, but we have also ob-
served such granular lymphocyte-like cells in preparations from the gut lamina
propria and have identified these primarily in the epithelium in rabbits (23).
Recently, Guy-Grand ^t al. (10) have made a number of observations about these
cells in the small intestine and have concluded that the cells in the gut epithe-
lium appear to arise from a special population of T cells present in the Peyer's
patches. It is tempting to speculate that a similar Peyer's patch (or BALT?)
derivation applies to the basophiloid cells in bronchial epithelium, Meta-
chromatic, granule-containing cells are also found in Peyer's patches. These
cells, which lie in the spaces formed between epithelial cells, are potentially
in direct contact with antigen which traverses the mucosal epithelium and is
then exocytosed into the lateral interepithelial spaces. Much more work needs
to be done in this area to establish the relationship of epithelial basophiloid
cells to T cells or mast cells and to explore the functional activity of this
cell type.
There is some evidence for T blast traffic between mucosal surfaces of a
similar nature to that described above for B blasts, particularly precursors
of IgA (2t). Relatively little is known about T blast traffic to the respira-
tory tract and it is an open question whether such a common mucosal system
exists for T cells. Waldman and Henney (27) first showed that following intra-
tracheal immunization, cells washed out from the lungs could proliferate and
release migration inhibition factor (MIP) on exposure to specific antigen. it
is unclear whether the cells from which this lymphokine was derived were T cell
or B cells, especially since both types of cells will produce MIF. in any
event, local immunization has been shown to produce a local cell-mediated
specific immune reaction. Immunization protocols have been devised mostly in
experimental animal models to prove that local immunization results in local
antibody and local cell-mediated immune reactions; such reactions are more con-
sistent and of a higher intensity than when antigen is introduced parenterally
It must be remembered when looking at these results that the lung, from the
standpoint of mucosal immune responses, is divided into at least two compart-
64
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ments, that which is mucosal and that which is peripheral and very similar to
the blood in terms of the antibodies contained therein (12). If one draws an
analogy with the bowel, it appears, especially from the work of Pierce and
colleagues (18, 19), that parenteral immunization followed by local challenge
leads to a higher accumulation of IgA antibody-producing cells in the intestine
than with any other immunization protocol. It follows, therefore, that anti-
body and concomitant protection against a particular organism might best be
achieved locally in the respiratory tract if parenteral immunization occurred
first and was then followed by local presentation of antigen. The best way to
prime for complete protection of the lungs against subsequent infection is not
well understood*
Local immunization of the lungs may lead to local IgE antibody formation
which could, of course, be deleterious. Gerbrandy and Bienenstock (8) have
shown that following intratracheal immunization potential IgE-forming cells
could be demonstrated in the draining mediastinal lymph nodes in greater numbers
than following peripheral immunization. Intraperitoneal immunization also pro-
duced the same results, but this observation is confusing since it has been
known for many years that intraperitoneal immunization is a good method of
priming for the IgE response and antigens introduced into the peritoneal cavity
will drain through the diaphragm into the mediastinal nodes* Obviously until
the potential hazards of producing a local IgE type of immune response versus
a protective immune response at a local mucosal surface are better understood,
immunization protocols will perforce not be able to maximize and mobilize the
appropriate immune response at the appropriate mucosal site.
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globulin _in vitro. A study of the synthetic ability of various tissues
from rabbits immunized by different methods. Biochem J 68:252-261, 1958
2. Bienenstock J: The physiology of the local immune response and the
gastrointestinal tract. In: Progress in Immunology II (Brent L,
Holborow J, eds). Amsterdam, North-Holland Publishing Co, 1974, 4:197-207
3. Bienenstock J, Johnston N: A morphologic study of rabbit bronchial
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5. Bienenstock J, Johnston N, Perey DYE: Bronchial lymphoid tissue. II.
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6. Bockman DE, Cooper MD: Pinocytosis by epithelium associated with lymphoid
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7. Collan Y: Characteristics of non-epithelial cells in the epitheliun of
normal rat ileum. Scand J Gastroent 7 (Suppl 18):5-66, 1972
8* Gerbrandy JLF, Bienenstock J: Kinetics and localization of IgE tetanus
antibody response in mice immunized by the intratracheal, intraperitoneal
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9. Goldblum RM, Ahlstedt S, Carlsson B, Hanson L, Lidin-Janson G, Sohl-
o
Akerlund A: Antibody-forming cells in human colostrum after oral im-
munization. Nature 257:797-799, 1975
10. Guy-Grand D, Griscelli C, Vassalli T: The mouse gut T-lymphocyte, a novel
type of T cell: Nature/ origin/ and traffic in mice in normal and graft-
versus-host conditions. J Exp Med 148:1661-1677, 1978
14
11. Humphrey JH, Sulitzeanu BD: The use of [ C] amino acids to study sites
and rates of antibody synthesis in living hyperimmune rabbits. Biochem
J 68:146-161, 1958
12. Kaltreider HB, Salmon SE: Immunology of the respiratory tract. Functions
properties of bronchoalveolar lymphocytes obtained from the normal canine
lung. J Clin Invest 52:2211-2217, 1973
13. Klein E: In: The Anatomy of the Lymphatic System. II. The Lung. Londo
Staith, Elder and Co, 1875
14. McDermott M, Bienenstock J: Evidence for a common mucosal immunologic
system. I. Migration of B immunoblasts into intestinal, respiratory and
genital tissues. J Immunol (In press)
15. Milne RW, Bienenstock J, Perey DYE: The influence of antigenic stinmlatio
on the ontogeny of lymphoid aggregates and immunoglobulin-containing cells
in mouse bronchial and intestinal mucosa. J Heticuloendothel Soc 17:
361-369, 1975
16. Montgomery PC, Rosner BR, Conn J: The secretory antibody response anti-
DNP antibodies induced by dinitrophenylated type 3 pneumococcus. J Immunol
Commun 3:143-156, 1974
17. Patterson R, Tomita Y, Oh SH, Susko IM, Pruzansky JJ: Respiratory mast
cells and basophiloid cells. Clin Exp Immunol 16:223-234, 1974
18. Pierce NP, Gowans JL: Cellular kinetics of the intestinal immune response
to cholera toxoid in rats. J Exp Med 142:1550-1563, 1975
19. Pierce NF, Sack RB, Sircar BK: Immunity to experimental cholera, m.
Enhanced duration of protection after sequential parental-oral administra-
tion of toxoid to dogs. J Infect Dis 135:888-896, 1977
20. Racz P, Tenner-Racz K, Myrvik QN, Painter LK: Functional architecture of
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21. Rose ML, Parrott DMV, Bruce RG: Migration of lymphoblasts to the small
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of mesenteric lymphoblasts and mesenteric T lymphoblasts in syngeneic mice.
Immunology 31:723-730, 1976
22. Roux ME, McWilliams M, Phillips-Quagliata JM, Weisz-Carrington P, Lamm ME:
Origin of IgA-secreting plasma cells in the mammary gland. J Exp Med 146:
1311-1322, 1977
23. Rudzik 0, Bienenstock J: Isolation and characteristics of gut mucosal
lymphocytes. Lab Invest 30:260-266, 1974
24. Rudzik 0, Clancy RL, Perey DYE, Bienenstock J, Singal DP: The distribution
of a rabbit thymic antigen and membrane immunoglobulins in lymphoid tissue,
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25. Rudzik 0, Clancy RL, Perey DYE, Day RP, Bienenstock J: Repopulation with
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26. Tenner-Racz K, Racz P, Myrvik QN, Ockers JR, Geister R: Uptake and trans-
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134:482-494, 1971
67
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The Role of Polyclonal Cell Activation in the
Initiation of Immune Complex-mediated
Pulmonary Injury Following Antigen Inhalation
B. J. Shenker, T. N. Mann, and W. F. Willoughby
Johns Hopkins University
Baltimore, Maryland
Certain forms of environmental lung disease provoked by inhaled sub-
stances are thought to result from immunologically mediated inflammatory
injury to the pulmonary parenchyma; such diseases have been termed "allergic
alveolitis" or, alternatively, "hypersensitivity pneumonitis." To better
understand the pathogenesis of such injury, animal models have been studied
in which protein antigens, polyclonal cell activators derived from plants,
or other organic materials have been administered in aerosol form to normal
or immune animals.
Inhalation of bovine serum albumin (BSA), a protein antigen, causes
little, if any, injury in USA-immunized rabbits, presumably because it does
not have access to humoral antibody (18). However, we have observed that
inhalation challenge with mixtures of antigen plus concanavalin A (Con A),
a polyclonal cell activator, produced a severe necrotizing pneumonitis in
immune recipients, in association with localized deposits of immune complexes
of inhaled antigen, host antibody, and complement (19, 20). We now present
evidence that this enhancement of immune complex formation in the lung by in-
haled Con A occurs as a direct consequence of Con A's ability to stimulate
lymphocytes iii vivo. To do this, we have employed purified cholera toxin
(CT), a cAMP agonist which inhibits leukocyte stimulation by Con A in vitro.
First, we have compared three different regimens of in vivo CT adminis-
tration for inhibition of Con A-induced stimulation of peripheral lympho-
cytes in vitro, and also for inhibition of Con A-induced inflammation in the
69
-------
skin. We then demonstrate that CT administration selectively inhibits cell-
mediated, delayed hypersensitivity skin reactions but not the antibody-medi-
ated vasculitis associated with the Arthus reaction. Finally, we show that
when we block cell-mediated reactivity to Con A in vivo, we also block the
ability of Cbn A to trigger immune complex reactions in the lung between in-
haled antigen and humoral antibody. Lastly, these inhibitory effects of CT
can be correlated with elevations in lymphocyte cAMP concentrations associ-
ated with CT administration.
MATERIALS AND METHODS
New Zealand White male rabbits used in this study weighed 2-3 kg and
were purchased from Hare (Hare, N.Y.). The rabbits were housed in laminar
flow rooms supplied with filtered air and given both rabbit chow and water
ad lib. All animals were immunized with BSA (Sigma, St. Louis, Mo.) incor-
porated into an alum precipitate as previously described (18). Each rabbit
received a total of 50 mg BSA, 10 mg of protein initially, followed by 20
mg each on days 7 and 21, injected bilaterally in four equal parts at dorsal
and gluteal sites.
Aerosols containing BSA and Con A were administered as previously de-
scribed (18). Briefly, aerosols were generated in a DeVilbiss model 35A
ultrasonic nebulizer containing a solution of 100 mg BSA and 100 mg Con A
in 20 ml of PBS. Each aerosol was vented into a Plexiglas chamber into which
two rabbits' heads could be inserted simultaneously. Each aerosol adminis-
tration required approximately 60-90 minutes to complete, and was adminis-
tered to each animal on 3 consecutive days. All animals were then sacrificed
24 hours after the last aerosol by an intravenous overdose of nembutal.
Concanavalin A
Con A was prepared from Jack bean meal (Difco, Detroit, Mich.) according
to the method of Agrawal and Goldstein (1), modified as previously described
(20). The purified Con A gave a single precipitin band by immunodiffusion
in gel against rabbit anti-Jack bean meal antibody, and a single symmetrical
peak by velocity sedimentation in a 10-40% sucrose density gradient.
Cholera Toxin
CT was purchased from Schwarz/Mann (Orangeburg, N.Y.). It was charac-
terized by the supplier as giving a single band in disc electrophoresis. One
milligram of lyophilized CT was initially resuspended in 1 ml HO, then di-
luted in saline to a final concentration of 100 yg/ml or 50 ug/ml. Prior to
injection, the diluted CT was filtered through a 0.22 Millipore filter
70
-------
(Bedford, Mass.)- In search for optimal conditions of inhibition, several
different regimens of CT administration were employed in initial studies.
In some experiments (protocol A), rabbits were injected via the marginal ear
vein with 40 wg of CT/kg, 24 hours prior to the first aerosol, followed 48
hours later with a second intravenous injection of 20 jjg/kg. In others
(protocol B), daily injections of equal amounts were given on four consecu-
tive days, beginning 24 hours prior to the first aerosol; the total daily
dosage in the latter protocol was 20 ug/kg body weight or approximately 160
pg/animal. Finally, a third group of rabbits (protocol C) was given 40 yg
of CT/kg intraperitoneally, followed 48 hours later with a second injection
of 20 Ug/kg.
The amounts of CT employed in these studies were derived empirically
in preliminary experiments. It is apparent from such studies that the amounts
of CT required for suppression of immune effector reactions in vivo are near
lethal values. Although no rabbit died in the experiments presented here
(nor in most subsequent experiments carried out to date), we have on occasion
seen animals receiving similar amounts of CT die within 24-48 hours of the
first injection. At autopsy, the only obvious pathological finding in these
animals was the occasional presence of fluid in the pleural cavities.
Lymphocyte Cultures
Rabbits were bled from the central artery of the ear and the peripheral
lymphocytes isolated by the method of Boyum (2). The cells were washed twice
with Hanks' balanced salt solution (HBSS) (Microbiological Associates,
Rockville, Md.) and, if necessary, any remaining red blood cells lysed with
0.84% ammonium chloride. The lymphocytes were diluted in culture medium to
give a suspension of 2 x 10 cells/ml. The culture medium employed consisted
of RPMI 1640 (Microbiological Associates) containing 10 mM HEPES buffer
(Sigma, St. Louis, Mo.), 10% heat inactivated pooled normal rabbit servm,
2 mM glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin (Micro-
biological Associates).
Equal volumes (0.1 ml each) of diluted cells (2 x 10 cells) and of
medium containing Con A were added to each well of microculture plates
(Costar Plastics, Cambridge, Mass.). After incubation for 52-54 hours at
37°C in humidified air containing 5% CO , 0.05 ml of media containing 0.5
jiCi of H-thymidine (specific activity - 5.0 Ci/mM, Amersham/Searle,
Arlington Heights, HI.) was added to each well. After 72 hours of total
incubation time, the cells were harvested on Reeve Angel glass fiber filters
(Whatman Inc., Clifton, N.J.) with an automatic cell harvester (Model 24V,
Brandel, Rockville, Md.). The filters were air dried and then placed in
plastic scintillation vials (Minivials, RPI, Rochester, N.Y.). Five milli-
71
-------
liters of scintillation fluid (4 g Cmniscint to 1 L toluene, RPI) were added
to each vial and the samples counted in a Beckman LS 250 or LS 7000 liquid
scintillation spectrophotometer. The degree of lymphocyte stimulation is
expressed as the net cpm (total cpm minus cpm of unstimulated control cul-
tures) • Data are reported as the mean of triplicate cultures -*• standard
error of the mean.
Cyclic Nucleotide Determinations
Lymphocytes were isolated from arterial blood as described above and
0.5 ml aliquots containing 2 x 10 cells in HBSS were placed in 16 x 125 mm
Pyrex test tubes, frozen rapidly in alcohol and dry ice, and then stored at
-60°C. For assay, the cells were thawed slowly, 3 ml of ice-cold acetone
added, and the cell suspension then allowed to stand for 15 minutes in an
ice bath. The cell suspensions were then heated in a 60-70°C water bath
for 3 minutes followed by centrifugation at 900 x jj for 15 minutes at 2-5°C.
The supernatants were transferred to Pyrex culture tubes and lyophilized.
Radio immuneassays (RIA) for cAMP and cGMP were carried out under identical
conditions, as previously described (10), using commercial reagents (Schwarz/
Mann, Orangeburg, N.Y.). Briefly, lyophilized samples were resuspended
in 1.0 ml of cold (2-5°C) 0.05 M sodium acetate buffer (pH 6.2) and 0.3 ml
transferred to each of two 12 x 75 mm polypropylene culture tubes (one for
cAMP and one for cGMP), followed by the addition of 0.1 ml of radioactive
cyclic nucleotide [2-0-succinyl cyclic AMP (or cGMP) tyrosine methyl ester
1 25
( I)] and Of 1 ml of rabbit IgG anti-CAMP or anti-cGMP to each tube. After
mixing, all RIA tubes were held at 4*C for 16-20 hours, followed by addition
of 2.5 ml of 60% saturated ammonium sulfate to each tube* Thirty minutes
later, the tubes were centrifuged at 900 x 3 for 30 minutes at 2-5*C. The
supernatants were decanted and any excess fluid removed with a cotton-tipped
applicator. The precipitate radioactivity was measured in a Beckman 310
125
gamma spectrophotometer; the efficiency of I activity measurements was
determined to be 75%. For each assay, standard curves were constructed
using known amounts of cyclic nucleotides (ranging from 0.05 to 10.0 pmoles/
tube). These curves were then used to relate the amount of cyclic nucleo-
tide in unknown samples as a function of precipitated radioactivity.
Skin Tests
Rabbits were skin tested 20-24 hours prior to sacrifice. All skin teats
were done on the animal's left side, which had been previously shaved.
Arthus skin tests were induced by intradermal injection of 0.05 ml of BSA
dissolved in PBS at a final concentration of 1 mg protein/ml. Cell-mediated
inflammatory skin reactions were induced by intradermal injection of 0.05 ml
72
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of Con A (200 ug Con A/ml PBS). In some instances, where negative reactions
were anticipated, sterile india ink (1%) was added to the Con A and BSA skin
test solutions. The india ink provided a marker for the positive identifi-
cation of non-inflammatory skin test sites; by itself, the ink caused no in-
flammatory response. After sacrifice, each test site was excised and fixed
in formalin for subsequent processing for histologic examinations.
Histology
All tissue was fixed in formalin and paraffin-embedded tissue sections
stained with hematoxylin and eosin. Lungs were inflated with isotonic buf-
fered dormalin at a hydrostatic pressure of 10 can HO. Sections were taken
from all lobes; each section extended from the hilar region out to the peri-
phery. Injury was judged and graded on the extent or degree of inflammatory
change/, and also in terms of the presence or absence of parenchymal necrosis.
RESULTS
Effects of CT on Peripheral Blood Lymphocytes (PEL)
The effects of CT in vivo were examined using three alternative adminis-
trative protocols. In protocol A, a total of 60 yg CT/kg of body weight
was injected intravenously, two-thirds being given at time 0 and the remain-
ing one-third at 48 hours; in protocol B, a total of 80 ug CT/kg of body
weight was injected intravenously, given daily, starting at time 0, as four
equally divided doses; in protocol C, a total of 60 ug CT/kg body weight was
injected intraperitoneally, two-thirds at time 0 and the remaining one-third
at 48 hours. Peripheral blood lymphocytes were isolated from bleedings ob-
tained at daily intervals, and tested for their ability to incorporate H-
TdR when stimulated with Con A in vitro.
All animals given CT intravenously showed a drop in PBL responsiveness
within 24 hours following the initial injection (Fig. 1). Animals treated
with CT via protocols A or B (intravenously) showed over a 90% inhibition
in proliferative responses to Con A, and remained unresponsive throughout the
remainder of the experiment. On the other hand, administration of CT via the
intraperitoneal route was notably less effective/ in terms of both the
rapidity of onset as well as the extent of inhibition observed.
Effects of CT Administration on Con A-induced Cutaneous Inflammation
The ability of Con A to activate T-lymphocytes (and basophils) has been
demonstrated in vivo as well as in vitro; intradermal injections of Con A
induce a diffuse, local inflammatory reaction, predominantly mononuclear in
73
-------
60ug CT/kg IV • • Protocol A
80ug CT/kg IV a-- -a Protocol B
60ug CT/Kg IP A A Protocol C
TIME FOLLOWING INITIAL CT INJECTION (hours)
Figure 1. Effects of CT administration in vivo on responsiveness of
peripheral blood lymphocytes (PEL) to stimulation in vitro by optimal con-
centrations of Con A. Three different protocols (A, B, and C) for CT adminis-
tration have been employed and their effects compared. Results are plotted
as percent of H-TdR incorporation observed prior to CT administrations
(time = 0), as observed in PBL obtained 24, 48, 72, and 96 hours following
the initial CT injection. Each point represents the mean value observed/
using triplicate assays, in each of three animals; vertical bars express the
standard error.
character, and resembling the classic tuberculin-type, delayed hypersensi-
tivity reaction (8, 9, 13). Therefore, we examined the ability of CT to
inhibit Con A-induced cutaneous inflammation, as might be expected if CT
inhibited leukocyte activation in vivo as well as in vitro.
Ten micrograms of Con A were injected at duplicate sites in rabbits
receiving CT according to protocols A (3 animals), B (3 animals), or c (3
animals), and also into non-CT treated controls (3 animals). Both schedules
of intravenous CT administration (A and B) effectively inhibited the Con A-
74
-------
Ttv-vr •>*••
\U**.9ttti .. -
Figure 2. Local inflammatory response to intradermal injections of Con
A in the rabbit: a) Diffuse inflammatory infiltrate observed in the colla-
genous dermis of a normal rabbit 24 hours following injection of 10 pg Con A
(X155); b) Absence of any inflammatory reaction 24 hours following injec-
tion of 10 ug Con A in a CT-treated rabbit (protocol A). The black particulate
material present in the center of the field is sterile india ink which had been
admixed with the Con A to positively identify the injection site (X155).
75
-------
Table 1. Effects of CT Administration on Con A-induced Cutaneous
Inflammation
Skin test grade*
Rabbit No. CT administration protocols (duplicate sites)
293 (A) 40 ug/kg IV, day 0, + ++
294 20 ug/kg, 48 hr later + 0
295 + 0
296 (B) 20 ug/kg IV, daily 0 0
297 for 4 days + 0
298 0 0
299 (C) 40 ug/kg IP, day 0,
300 20 ug/kg, 48 hr later
288
249
251 None
252
Intensity of inflammation graded from 0 (no inflammation) to -f-f-n-
(extensive inflammation).
induced inflammatory response (Fig. 2), whereas intraperitoneal administration
gave little, if any, inhibition (Table 1). These results paralleled those
obtained in the preceding experiment, again indicating that, whereas intra-
venous administration of CT effectively blocked in vivo the lymphocyte's
responsiveness to Con A, intraperitoneal administration was decidedly less
effective. Based on these observations, protocol A was employed in subse-
quent experiments.
Differential Effects of CT Administration on Delayed Hypersensitivity and
Immune Complex Reactions in the Skin
The direct active Arthus reaction, induced by injecting antigen intrader-
mall y into an actively immunized animal, consists in part of a necrotizing
vasculitis mediated by immune complexes of antigen, antibody and complement,
localized within the vessel wall, and, in part, of a diffuse cutaneous leuko-
cytic infiltrate (similar to that produced by Con A) considered to represent
a delayed-type hypersensitivity response (3). Importantly, the vasculitis
does not require participation by sensitized T-lymphocytes, but instead is
mediated by immune complexes, together with homocytotropic antibodies which
76
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Table 2. Differential Effects of CT Administration on Delayed
Hypersensitivity and Immune Complex Reactions in the Skina
Arthus reaction
(50 ug SSA)
Total
g CT/kg Diffuse Delayed hypersensitivity
Rabbit No.
133
134
138
140
body weight
0
0
0
0
Vasculitis inflammation
++++ ++++
++++ ++
1 J ^^L ^ i^Li
~^r « ^r ~ ~ ~ ~
•M-f + +
( 10 yg Con A)
++++
++-M-
•H-+H-
4--W-
135
136
137
139
141
60
60
60
60
60
++4*+ 0
++++ 0
•H-++ 0
+++-H- 0
•M-++ 0
++
•»•+
•H-
0
•f
alntensity of inflammation graded from 0 (no inflammation) to +++++
(extensive inflammation).
participate in initiation of complex formation, and neutrophils which respond
to the complement-mediated chemotactic stimulus (3). Thus, it can be readily
produced in animals immunized passively with antibody alone.
Using both the BSA-induced Arthus reaction (in BSA-immunized rabbits)
as well as the Con A skin test, we were thus able to compare the effects of
CT administration upon an immune complex-mediated vasculitis with its effects
upon delayed-type hypersensitivity reactions.
As in the previous experiment, intravenous administration of CT inhibited
the inflammatory response to Con A (Table 2). Of particular interest, however,
was its effect upon the active Arthus reaction, where it inhibited the diffuse
inflammatory or delayed hypersensitivity component of the Arthus, but not the
immune complex-mediated vasculitis (Fig. 3). Thus, these findings argue
eloquently for a selective inhibitory effect by CT upon effector lymphocytes
of the delayed-type hypersensitivity reaction, while leaving simple immune
complex-mediated reactions intact.
Effects of CT upon the Immunologic Pulmonary Injury Associated with
Inhalation of Antigen Plus a Polyclonal Cell Activator
We have recently reported that polyclonal cell activators such as Con A
77
-------
Figure 3. Direct active Arthus reaction to BSA injected intradermally
into a BSA-immunized rabbit, a) Arthus reaction in a control animal. Notice
both the necrotizing vasculitis (mediated by immune complexes containing BSA,
anti-BSA antibody, and complement) and the diffuse inflammatory infiltrate
(representing a delayed hypersensitivity reaction to BSA) (X155). b) Direct
active Arthus reaction in a rabbit treated with CT. An intense necrotizing
vasculitis is still seen, but the diffuse inflammatory component is notably
reduced (X155).
78
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and phytohemagglutinin induce a diffuse interstitial pneumonitis when in-
haled in aerosol form (19). More importantly, they also exert a pronounced
enhancing effect upon immune complex injury involving inhaled antigen, hu-
moral antibody, and complement; such injury is severe and is characterized
by foci of frank parenchymal necrosis (20). We have proposed that this en-
hancement may be mediated, at least in part, through polyclonal activation
of leukocytes within the lung by Con A, leading in turn to the interstitial
inflammation typically observed following inhalation of Con A (20). In
particular, Con A is known to cause T-lymphocytes to release vascular per-
meability factors (12) in vitro, and to cause mast cells and basophils to
release vasoactive amines in vitro (11); inhaled Con A could presumably be
having the same effect on these cells in vivo. In any event, inhaled Con A
might alter the cellular barriers which normally isolate intra-alveolar
antigen from humoral antibody and complement. If this hypothesis is true,
then administration of CT should block both Con A-induced interstitial pul-
monary inflammation and immune complex-mediated parenchymal necrosis, even
though, as seen in the preceding experiment, CT has no apparent effect on
simple immune complex-mediated ueactions such as the Arthus vasculitis.
The BSA-immunized rabbits were divided into two groups of five each?
one group was given CT intravenously, according to protocol A; the other
group did not receive CT but served as a control. All animals were challenged
with Con A/BSA aerosols at 24, 48, and 72 hours following the initial CT
injection, and were sacrificed at 96 hours. Histologic examinations of lung
tissues revealed that both the extent and severity of inflammation were
greatly reduced in the CT-treated animals as compared to the controls (Fig.
4). Of particular importance was the striking reduction in the incidence of
immune complex-type necrosis, with such lesions being observed in only two
lobes from a single animal receiving CT, whereas such lesions were present in
a total of 14 lobes of the control group, with all animals being affected
(Table 3) .
Jn vitro studies have shown CT to act through adenylate cyclase to in-
crease cellular cAMP levels. In order to look for a similar effect iri vivo,
we obtained daily bleedings from the five rabbits receiving CT via schedule
A, as well as from the five controls, harvested the lymphocytes and measured
their cAMP levels by radioimmunoassay (Table 3). The average cAMP concen-
tration in cells obtained following the initial CT injection was 18.6 + 2.4
pmoles/10 cells, significantly greater than that in cells from control
animals (7.0 + 1.2 pmoles/10 cells, P = 0.002).
In some cases, different experimental observations were carried out
simultaneously in the same animals, thereby providing a form of internal con-
trol and underscoring the mutual relevance of the different phenomena. For
79
-------
$cw^32$rr£5
k_ l-J-V ' V-^sX*. k.
.S~^'Tv£ ^EFct^
P^f • i>^ JA^>^^
t"»*^"kS; !v;^7TlStV'
fe-5^p^g!g ^
xi- • . •» f S*\ ^ %-..--s > V. >^ •
Figure 4. Pulmonary injury following administration of BSA/Con A
aerosols to BSA-immunized rabbits. A) Severe interstitial pneumonitis
with areas of frank parenchymal necrosis in a control animal not given CT
(X60). b) Inhibition of pulmonary injury in a CT-treated animal. A small
focus of interstitial inflammation is seen in the right upper corner (X60).
80
-------
00
Table 3. CT-induced Suppression of Immunologic Pulmonary Injury3 in BSA-immunized
Rabbits Challenged with BSA/Con A Aerosol
Rabbit No.
131
133
134
138
140
135
136
137
139
141
ug CT/kg
body wt .
0
0
0
0
0
60
60
60
60
60
MeL
0
0
0
+
0
|++++j
+
0
RUL RML
f+T+T| I7++T
o j+Ti
+ ++++
I+++I [+++++ I
Em
0 ++
0 +
++ ++++
0 +
0 ++
Lung injury
RLL, RLL LUL
Ti p
+ 0 f+++1
++ + +
+ o Lt+ttJ
(++j ++ j+^+^+l
0 0 J++++J
00 +
++00
++ o r++++ 1
0 +0
0 0 +++
LLL,
n
++
0
0
0
0
ND
+
+
0
Lymphocyte cAMP
LLL (pmoles/10 cells)
0
0
0 7.0 + 1.2
0
0
0
0 18.6 + 2.4
0 p = 0.002
0
Lesions graded from 0 (no injury) to +++++ (severe injury).
ND = not examined.
Lobes containing one or more foci of
parenchymal necrosis are enclosed in boxes.
MeL = mediastinal lobe; RUL = right upper lobe; RML = right middle lobe; RLL = hilar portion of
right lower lobe; RLL = peripheral portion of right lower lobe; LUL = left upper lobe; LLL, = hilar
portion of left lower lobe; LLL = peripheral portion of left lower lobe.
-------
example, the selective action of CT upon cell-mediated versus antibody-
mediated reactions in the skin, its effects on Con A/BSA aerosol challenge,
and on cAMP levels in peripheral blood lymphocytes, were examined simulta-
neously in animals 131-141 (see Tables 2 and 3). Thus, observations that CT
treatment was able to block the immune complex-mediated parenchymal necrosis
but not the Arthus vasculitis, were made simultaneously in individual CT-
treated animals*
DISCUSSION
We have previously proposed that inhaled polyclonal cell activators
enhance local immune complex injury, in an indirect fashion, through their
ability to stimulate pulmonary lymphocytes. These activated lymphocytes
could then "trigger" immune complex formation between intra-alveolar antigen
and humoral antibody by releasing lymphokine mediators or, possibly, through
direct cell-cell cytotoxic reactions. This hypothesis has been critically
tested in this study by examining whether selective inhibition of lymphocyte
responses to Con A in vivo in fact blocked not only cell-mediated interstitial
lung injury, but immune complex-mediated parenchymal necrosis as well.
To achieve such a selective inhibition in vivo, we elected to use CT
for several reasons. First, the ability of CT to inhibit lymphocyte effector
functions in vitro through its actions as a cAMP agonist is now well docu-
mented (15, 16). Moreover, we favored CT over other known cAMP agonists
since it binds irreversibly to cell membranes, and its cAMP stimulating
effects are notably prolonged relative to those of the beta adrenergic stimu-
lators (14). In addition, Warren and colleagues (17) had previously reported
that CT, when administered intravenously to mice, inhibited cell-mediated
inflammatory responses to schistosome egg antigen, in association with a
doubling of spleen cell cAMP content.
As a first step, we demonstrated that CT administration could inhibit
peripheral blood lymphocytes. Lymphocytes harvested from CT-treated animals
were shown to have impaired proliferative responses when treated with optimal
concentrations of Con A in vitro, and the local in vivo inflammatory response
to intradermal injections of Con A was also suppressed. Second, we showed
that the effect of CT on immune effector responses was selective, by demon-
strating that it inhibited both the cell-mediated response to Con A and the
classic delayed-type hypersensitivity response to the protein antigen, BSA,
but that it failed to inhibit the cutaneous Arthus vasculitis mediated by
antigen, antibody, and complement. The latter is particularly noteworthy,
since there is good evidence that this vasculitis is triggered by an anaphy-
lactic reaction involving the release of vasoactive amines (and possibly
platelet activating factor) from mast cells and basophils sensitized with
82
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homocytotropic antibody (3). Thus, the inability of CT to block the Arthus
vasculitis also argues against any significant inhibition of basophil or
mast cells by CT in these studies.
The present demonstration that CT administration blocks both the inter-
stitial pneumonitis [associated with injury by Con A alone (20)] and the
immune complex-associated necrotizing injury supports the hypothesis that
cell-mediated lung injury acts to enhance or facilitate immune complex for-
mation in the lung. Stimulation of T-lymphocytes appears to be the critical
initiating event for the following reasons: 1) Con A is known to directly
stimulate T cells (but not B cells) (6); 2) T-lymphocytes are normally pre-
sent in the lung (4); 3) Con A has little effect upon macrophages (5); and
4) we have now demonstrated that inhibition of immune complex lung disease
by CT is associated with inhibition of T-lymphocyte functions, in the ab-
sence of demonstrable effects on the immune complex-mediated, basophil/mast
cell-initiated Arthus vasculitis.
It should be noted that in some instances the cutaneous response to
Con A and also the production of interstitial pneumonitis were only partially
blocked by CT, yet inhibition of immune complex injury was striking. Thus,
initiation of immune complex injury seems dependent upon the extent of cell-
mediated injury produced. Similarly, this finding may also explain the re-
lative inability of BSA alone to initiate immune complex injury (although cell-
mediated responses to BSA were observed by the direct active Arthus skin test)
since only a small percentage of bronchoalveolar T-lymphocytes would theo-
retically specifically recognize BSA antigen. In contrast, all or most T-
lymphocytes should respond to polyclonal activation by Con A except, of course,
in animals treated with CT.
Demonstrations of significant increases in cAMP in peripheral lymphocytes
from CT-treated animals indicate that CT is exerting its inhibitory effects
in vivo through stimulation of intracellular "second messengers." This find-
ing parallels those changes observed when such cells are treated with CT in
vitro (15, 16), and the 2.7-fold increase observed here agrees well with the
value of 2.1 that Warren and colleagues observed in mouse spleen cells follow-
ing a single injection of CT (17).
In conclusion, these studies call attention to the possible importance
of polyclonal cell activators which, when present in the environment, may
play a critical role in initiating environmental lung disease. Such poly-
clonal activators include not only a vast array of plant proteins, including
lectins such as that employed in this study, but also endotoxins, enzymes,
and even simple inorganic chemicals (7). in addition, this study addition-
ally emphasizes the importance of regulation, by intracellular mediators such as
cAMP, of immune reactive cells, and in particular their ability to respond in
83
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the production of immune injury. Consideration of such factors may prove
useful in future clinical studies of environmental lung disease, and may
facilitate further definition of risk factors both at the individual as
well as environmental level.
ACKNOWLEDGMENTS
This work was supported in part by Grants HL 19819-03 and 1 T32 AI07147-
01 from the National Institutes of Health, and by a fellowship from the
American Lung Association.
REFERENCES
1. Agrawal BBL, Goldstein IJ: Protein-carbohydrate interaction. VI.
Isolation of concanavalin A by specific absorption on cross-linked Dextran
gels. Biochim Biophys Acta 147:262-271, 1967
2. Boyum A: Separation of leukocytes from blood and bone marrow. Scand
J Clin Lab Invest (Suppl) 21:97, 1968
3. Cochrane CG, Janoff A: The Arthus reaction. In: The Inflammatory
Process, Vol. 111 (Zweifach BW, Grant L, McCluskey RT, eds). New York,
Academic Press, 1974, pp 85-162
4. Daniele RP, Altose MD, Rowlands DT: Mmunocompetent cells from the lower
respiratory tract of normal human lungs. J Clin Invest 56:986, 1975
5. Edelson PJ, Conn ZA: Effects of concanavalin A on mouse peritoneal
macrophages. I. Stimulation of endocytic activity and inhibition of
phagolysosome formations. J Exp Med 140:1364, 1974
6. Moller G, Andersson J, Polilit H, Sjoberg O: Quantitation of the number
of mitogen molecules activating DNA synthesis in T and B lymphocytes.
CLin Exp Immunol 13:89, 1973
7. Novogrodsky A: A chemical approach for the study of lymphocyte acti-
vators. In: Mitogens in Immunobiology (Oppenheim JJ, Rosenstreich DL,
eds). New York, Academic Press, 1976, pp 43-56
8. Schatz M, Patterson R, Sommers HM, Harris KE: Mitogen-induced lympho-
cytic and cutaneous reactions in dogs. Int Arch Allergy Appl Immun
54:121-128, 1977
9. Schwartz HJ, Catanzaro PJ, Leon MA: An analysis of the effects of skin
reactive factors released from lymphoid cells by concanavalin A in vivo.
Am J Pathol 63:445, 1971 '
10. Shenker BJ, Gray I: Cyclic nucleotide metabolism during lymphocyte
transformation. I. Changes in cAMP and cGMP concentration and enzyme
activity associated with cyclic nucleotide metabolism. Cell Immunol
(In press)
84
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11. Siraganian RP, Siraganian PA: Mechanism of action of Con A on human
basophils. J Immunol 114:885, 1975
12. Sobel AT/ Branellec AI, Blanc CJ, Lag rue GA: Physicochemical charac-
terizations of a vascular permeability factor produced by Oon A-stimulated
human lymphocytes. J Immunol 119:1230, 1977
13. Stadecker MJ, Leskowitz S: fine cutaneous basophil response to mitogens.
J Immunol 113:496, 1974
14. Strom TB, Carpenter CB: Regulation of alloinununity by cyclic nucleotides.
In: Immunopharmacology (Hadden JW, Oof fey PG, Spreafico F, eds) .
New York, Plenum Medical Book Co, 1977, pp 47-59
15. Strom TB, Carpenter CB, Garovoy MR, Austen KF, Merril JP, Kaliner M:
The modulating influence of cyclic nucleotides upon lymphocyte-mediated
cytotoxicity. J Exp Med 138:381, 1973
16. Vischer TL, LoSpalluto JJ: The differential effect of cholera toxin on
the lymphocyte stimulation induced by various mitogens. Immunology
29:275-282, 1975
17. Warren KS, Mahmoud AAF, Boros DL, Rail TW, Mandel M, Carpenter CCJ:
In vivo suppression by cholera toxin of cell-mediated and foreign body
inflammatory responses. J Immunol 112:996-1007, 1974
18. Willoughby JB, Willoughby WF: In vivo responses to inhaled proteins.
I. Quantitative analysis of antigen uptake, fate, and immunogenicity
in a rabbit model system. J Immunol 119:2137-2147, 1977
19. Willoughby WF, Barbaras JE, Vfaeelis R: Immunologic mechanisms in experi-
mental hypersensitivity pneumonitis. Chest 69 (Suppl):290-291, 1976
20. Willoughby WF, Willoughby JB, Cantrell B, Wheelis R: In vivo responses
to inhaled proteins. II. An animal model of allergic alveolitis.
Lab Invest (In press)
85
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DISCUSSION
DR. NETTESHEIM: I have two questions* Would you say a word about how you
explain the difference between the intra-tracheal delivery and the inhalation
delivery and the presence in the serum? Is that/ perhaps, related to the
amount of BSA delivered to the lung, or is there something else? Whatever
happened to the experimental asthma model in guinea pigs/ where you immunize
with BSA, and then with inhalation of the same antigen; it was very severe
asthma. HOW would that type of reaction fit in with what you have described
here?
DR. WILLOUGHBY: It's very difficult to achieve an exact comparison between
the two. We get 100 milligrams of BSA, of which 4 milligrams goes in the
rabbit. In our instillation, we instill 5 or 10 milligrams. Perhaps the
single most important aspect is that when you instill it, it all goes into
one small area. We know by counting different lobes in our preparation,
it1s very profusely distributed throughout the lung. We feel that both are
meaningful models. Instillation certainly relates to aspiration, I would thinJc
inhalation perhaps more to the environmental dust situation* Your second
question is: The asthma models are airway models, and they're classically
carried out in the guinea pig. We're looking at chronic disease. The rabbit
works best, because it doesn't have a receptor, and you don't kill the animal.
You can kill the guinea pig, unless you work it just right.
DR. WASSERMAN: Do your animals have an immediate hypersensitivity skin test
of IgE antibody, BSA? You didn't show us electron micrographs of the Con A-
induced lesion alone. Does that look like the interstitial process that you
see in Con A plus antigen, or is it somewhat different?
DR. WILLOUGHBY: As far as the IgE response, it was a very tiny response, we
have not actually looked by PCA. However, most people feel that Arthus
requires microprecipitable antibody for its initiation. By the very fact that
we can get an Arthus reaction suggests that with BSA there must be some homo-
cytotropic antibody. And more importantly, since the cholera toxin didn't block
it, the cholera toxin must not be having a major effect on mast cells, although
it's neutral in vitro. Your second question was about the EM. These are
preliminary photographs, and we haven't done the others. It's a reasonable
thing to look at, and we probably will.
86
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OR. BASEMAN: How do you account for the half-life of BSA in your model?
DR. WILLOUGHBY: First of all, in the instillation, these animals are
not immunized* Secondly, you're looking at a dynamic thing* When you look
at the half-life, you inject i.d., and you follow the half-life as it dis-
appears, but it's disappearing in one route, and it's being absorbed from the
gut at the same time. We do not see a normal first order decay of the BSA or
BSA label in the circulation for several reasons* First and foremost, it's
not intact BSA, but has been extensively degraded* Secondly, unlike in most
half-life studies, labeled protein, such as BSA, one injected over a short
period of time into the bloodstream* In the experiments being considered
here, two processes are involved. One is the half-life of the material in
circulation, and the second is continuing absorption or reappearance or
appearance of new material into the circulation. What we're looking at is
a result of two different processes, that is, the appearance of the label in
the serum as well as its disappearance. Hence, one does not see a simple first
order decay in concentrations. Third, most of these half-life studies are
carried over a period of weeks to months* And I don't know exactly what the
half-life of the albumin is in the noniramunized animal, but I suspect it's
anywhere from two weeks to four weeks. However, the studies that we're showing
are just carried out over a matter of 24 hours. Hence, even if we did classic
half-life studies, the line would appear very flat, and one would not see a
notable decrease due to catabolism alone*
87
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Lymphatic Removal of Fluids and Particles
in the Mammalian Lung
L. V. Leak
Howard University
Washington, D.C.
Physiologists have long realized the importance of the lymphatic vascular
system in the removal of fluids, proteins, particulate components, and cells
that are not reabsorbed at the venular limb of the blood vascular system (20).
Likewise, information from a number of studies has established that the
lymphatic system serves primarily as a transport system designed to maintain
homeostasis of the interstitial environment by draining excess fluids and
proteins from the interstitium for its return to the blood stream (1, 5, 6,
9, 20, 31). The importance of the lymphatic system lies in the fact that even
normal blood capillaries are permeable to macromolecules in proportion to
their molecular size and that these molecules, particularly proteins, disrupt
the normal Starling pattern of exchange between capillaries and tissues. If
left undisturbed, this would lead to a considerable accumulation of fluid
within the interstitial spaces, leading to edema and altering the hemodynamics
of tissue fluids (20). By cannulating the large lymphatic trunks which drain
various regions, much information has been obtained regarding the overall con-
tribution of lymphatics, not only in the maintenance of fluid homeostasis for
the various tissues throughout the body (4, 5, 7, 21), but also in regard to
its role in the removal of hormones (12), enzymes (3, 8, 19, 26), lipoproteins
(31), as well as cells (10, 24).
Although we now have some ideas regarding the overall contribution of
the lymphatics to the maintenance of fluid homeostasis in various tissues of
the body, the precise topographical organization and ultrastructure of this
drainage vascular system within the lung still remain unclear.
89
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Notwithstanding the voluminous literature on the anatomy and pathology of
pulmonary lymphatics, there have been few definitive studies at the ultrastruc—
tural level which consider: (a) their precise topographical arrangement and
the ultrastructural basis of interstitial fluid and particulate removal,
(b) the structural basis for lymph formation in the lung and the subsequent
propulsion of lymph in a unidirectional stream toward collecting vessels, and
(c) their participation in pulmonary defense mechanisms and involvement in th*
genesis and dissemination of various respiratory disease processes includinq
the metastasis of lung cancer.
Just as there is a rich plexus of lymphatic vessels distributed throughout
other areas of the body in which there is an abundant amount of connective
tissue, the same is true for the lungs (23). In our efforts to gain a new
perspective on the old problem of the structural organization of pulmonary
lymphatics and their role in the removal of excess interstitial components
we have employed improved techniques of tissue preservation and combined
transmission and scanning electron microscopy to study the topography and
ultrastructure of pulmonary lymphatic vessels.
MATERIALS AND METHODS
The lungs used in the study were from rats, mice, dogs, and guinea pigs.
However, most of the illustrations in this presentation are from the lungs of
rats. In an attempt to determine the fine structure of pulmonary lymphatica
and their precise distribution within the pulmonary interstitial areas and
their close proximity to the alveoli, use was made of combined intratracheaj.
and vascular perfusion methods of fixation.
Surgical Procedure
For control animals, rats were anesthetized with an intravenous inject!
of sodium pentobarbital. The heart was exposed through a median sternotomy
incision and heparin was injected intravenously. Saline containing heparin
and procaine were perfused through the lung by inserting a number 260 poly-
ethylene tubing intio the pulmonary artery through the right ventricle. The
lungs immediately branched as the saline displaced the blood in the pulmonarv
blood vessels. Aftjer 2-5 minutes, the saline was replaced with a fixative
consisting of a glutaraldehyde-formalin mixture (11) or 2.5% glutaraldehyde
in phosphate buffer. At the same time as fixative was being perfused into
the blood vessels a number 29 gauge needle was inserted into the trachea and
the airway perfused with fixative. After the lungs were perfused with fixa-
tives for 15-30 minutes, the hardened lungs were resected and placed in
fixatives for an additional two hours. The lungs were cut into small pieces
and subsequently processed for transmission and scanning electron microscoov
90
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Experiments with Colloidal Ferritin and Carbon
To follow the movement of intratracheally instilled particles in the
lungs of rats, suspensions of colloidal ferritin (.r 80 A diameter) or
dolloidal carbon (/> 350 A diameter) were administered via the trachea. After
periods of 5, 15, and 30 minutes and 1, 3, 6, and 24 hours, the lungs were
perfused as discussed above and small pieces processed for transmission
electron microscopy.
Dextrail Experiments
Dextrans of varying molecular weights (60,000 to 300,000) were injected
via the saphenous veins of young adult rats. At 5, 15, 30, and 60 minutes,
the lungs were perfused with saline followed by glutaraldehyde-formalin mixture
in phosphate buffer at pH 7.4 and at 0°C (27).
Preparation of Lungs for Scanning Electron Microscopy
Following perfusion fixation, lungs were cut into small sections and
postfixed in 1% OsO at 5°C for one hour, rinsed, and dehydrated in a graded
series of alcohols to 100% and gradually infiltrated with Freon 113. Hie
specimens were then processed by the critical point drying method (2, 25) using
Freon 13 and a Bomar critical point drying machine. After drying, the speci-
mens were mounted on stainless steel studs with silver conducting paint and
subsequently coated with a thin layer of carbon followed by a coating of gold
palladium in a Hummer coating machine. Specimens were observed in an ETEC-Auto-
Scan scanning electron microscope.
RESULTS AND DISCUSSION
Topography and Ultrastructure of Pulmonary Lymphatics
For a cohesive account of pulmonary lymphatics in relation to their
role in the dynamics of lymph formation and the removal of particles and
cells from the pulmonary interstitium, it is crucial to give a portrait of
the lymphatic vascular system as it relates to the movement of fluids, plasma
proteins, and cells across the blood-interstitial-lymphatic interface and the
air-interstitial-jLymphatic interface in the lung.
In considering the general organization and morphology of pulmonary
lymphatics these vessels can be classified by two categories. There is a
superficial plexus and a deep plexus of vessels which, unlike the accompanying
blood vessels, do not comprise a circulatory system, but a system which sub-
serves the lungs by providing a one-way drainage vascular system for the con-
-------
Figures 1a and b. Radiograph illustrating a rich network of pleural
lymphatics in the lower lobe of human lungs. In Figure 1b the periodic
constrictions along the length of the vessels indicate the location of valves,
(From ref. 30).
92
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stant removal of extravascular fluids, plasma proteins, and cells for their
subsequent return to the blood vascular system.
The superficial plexus is located within the connective tissue layer
of the visceral pleura. The extensive network of lymphatics within this region
is appreciated when these vessels are filled with vital dyes (trypan blue) or
radiopaque substances which outline the vessels, making it possible to observe
an anastomosing network of vessels (Fig. 1) that contain numerous valves point-
ing in all directions (23, 29, 30).
The deep plexus of lymphatic vessels is also referred to as the intra-
pulmonary or parenchymatous lymphatics. It consists of an interconnecting
network of vessels which surround bronchi and pulmonary arteries and veins.
It is also generally agreed that a rich plexus abounds within the interlobular
septa. These vessels are thought to provide interconnections between the deep
and superficial lymphatics as indicated by a flow of injected dyes from the
pleura to the deep parenchymatous lymphatic plexus (13, 30). Although it is
generally agreed that lymphatics abound within the connective tissue sleeves
surrounding pulmonary blood vessels and bronchi, the existence of alveolar
lymphatics or their presence at the air-blood barrier is still unclear.
By using improved micro-injection techniques combined with vascular
perfusion, a rich plexus of lymphatics can be identified within the connective
tissue sleeve which extends to the terminal and respiratory bronchioles.
These lymphatics are also juxtaposed to alveoli and are separated from the
air space only by the alveolar epithelium and a thin layer of adjoining con-
nective tissue. At this level of the bronchial tree, the lymphatics are of
a smaller caliber than those surrounding the large bronchi. The lymphatics in
these regions are extremely thin-walled and are situated at the sites of fluid
formation which, if left to accumulate, would lead to pulmonary edema (13).
The smaller and thin-walled lymphatics (10 pm in diameter) represent the
initial segment of the pulmonary lymphatic vascular system/ i.e., the lymphatic
capillary. Although the continuous layer of endothelial cells is extremely
attenuated, many areas between adjacent cells lack adhesion devices. Therefore,
adjacent cells are easily separated and can readily accommodate the movement
of interstitial fluids and particles into the lymphatic lumen. Therefore,
from a functional standpoint pulmonary lymphatics begin as blind end saccules or
tubes within a thin band of connective tissue at the level of terminal and
respiratory bronchioles.
The unidirectional flow process begins at the tissue-lymph interface with
the uptake of interstitial fluids and plasma proteins by the smallest and more
permeable vessels, the lymphatic capillaries. From these channels, the lymph
is propelled into an extensive system of collecting vessels whose continuity is
frequently interrupted by lymph nodes that serve as a filtering or screening
system. These vessels contain valves which prevent the regurgitation of lymph.
93
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§*•£*.. ~ -72*"
teSfr*'
*««*'.
Figure 2. Light micrograph shows the position of lymphatics (L) in
relation to a terminal bronchiole (TB) and the adjacent alveoli (*). X900,
(From ref. 17).
94
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The collecting vessels usually follow the overall distribution of the arteries
and veins within the lung. Lymph drained from the collecting vessels enters
the main lymphatic vessels which drain toward the hilar regions of the lungs.
Upon superficial examination of respiratory bronchioles and the adjoining
alveoli in paraffin sections, the general organization of the connective tis-
sue is unimpressive. However, on closer inspection of one-micron thick Epon
sections, a plexus of lymphatic vessels can be detected at the light micro-
scopic level (Fig. 2). The content of precipitated lymph along with occasional
lymphocytes or macrophages make it easier to differentiate lymphatics from
blood vessels. Electron micrographs of perfused lung tissue confirm the pres-
ence of precipitate within the lymphatic lumen (Fig. 3). In addition, the
irregularity of its wall and close topographical relationship to the adjoining
alveolar space (saccule) are also appreciated. In such areas the lymphatic
capillary is separated from the air space by the alveolar wall and a thin
band of connective tissue. In some areas blood capillaries may be interposed
between the lymphatic vessel and alveolar saccule.
The Ultrastructure of Pulmonary Lymphatic Capillaries
The endothelial cells of the pulmonary lymphatics, like those in other
tissues, are extremely attenuated over large areas. At this level of organi-
zation, these vessels resemble lymphatic capillaries as no smooth muscle cells
were observed in their walls. In addition, they lack a continuous basal
lamina and there were numerous anchoring filaments extending into the sur-
rounding connective tissue (Fig. 4). The adjacent cell margins overlap
extensively and the intercellular clefts are of variable widths, occasionally
accommodating the passage of lymphocytes from the interstitium (Figs. 5 and 6).
It is likely that plasma proteins as well as other cells also gain access to
the lymphatics in this manner.
Other salient features of the endothelial cells include numerous plasna-
lemmal imaginations and many microfilaments occur throughout the cytoplasm
(Fig. 7). Recent studies of Lauweryns et a_l. (14) demonstrated that the
5-6 nm filaments in pulmonary lymphatic endothelial cells formed the charac-
teristic arrowhead complexes when reacted with heavy meromyosin, suggesting
that these filaments represent actin.
Pulmonary Collecting Lymphatic Vessels
Proximal to the terminal bronchiole, the thickness and complexity of the
surrounding sleeve of connective tissue are gradually increased. Likewise,
the plexus of peribronchiolar and perivascular lymphatics also becomes more
elaborate (Figs. 8 and 9). The lymphatic vessels have a larger diameter and a
thicker wall consisting of a continuous layer of endothelial cells which are
95
-------
; • ;
BC
RB
Figure 3. Survey electron micrograph shows a pulmonary lymphatic
capillary (L) and its relationship with a respiratory bronchiole (RB). A gray
flocculent precipitate (*) fills the lymphatic lumen and is also located in
the connective tissue. The lymphatic capillary is separated from the respira-
tory bronchiole by a blood capillary (BC) and a band of connective tissue (CT)
which contains a thin process from fibroblasts (F) and collagen fibrils (CF).
X7,000.
96
-------
ttBttir >^.
Figure 4. A portion of pulmonary lymphatic capillary illustrating its
close relation to the adjoining connective tissue (CT). Numerous anchoring
filaments (af) extend from the abluminal surface and project into the surround-
ing connective tissue. The endothelium (E) contains plasmalemmal invaginations
and vesicles { v).
97
-------
'•'• >'*% '-'%#•*: *~ "*v/ V'--'V'
^Sf* ^£Ti% ^
- r !*&: 9f& J5$;
'^ .^ ".- _. . J^Lr 4tt.. MB( A '» .- *^'* ' ! Bfl_• '
. .
•**• •-*..;
'
. "i
*.
Figures 5a and b. Electron micrographs illustrate the extensive over-
lapping of adjacent endothelial cells (E) at intercellular junctions (j).
The width of the intercellular cleft (*) is also variable. (a) X56,000;
(b) X56/000.
98
-------
m
Figure 6. Portion of a juxta-alveolar lymphatic capillary (L) whose
lumen is filled with an electron dense precipitate (*). A lymphocyte (ly)
is located within the cleft of an intercellular junction (J). The lymphatic
endothelium (E) is extremely attenuated except in areas occupied by the
nucleus (N). The lymphatic capillary is separated from the alveolar sac by
a thin band of connective tissue (CT). X7,600.
99
-------
Figure 7. Electron micrograph of a segment of lymphatic capillary
endothelium (E) illustrating cytoplasmic filaments (cf), microtubules (mt) ,
and ribosomes (r). X77,700.
100
-------
Figure 8. Lymphatic vessels within the perivascular connective tissue
sleeve (CT) of the larger vessels which still maintain close proximity to the
alveolar sac (AS). X7/350.
101
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Figure 9. The branching of peribronchial and periarterial vessels is
demonstrated in these scanning electron micrographs. In (a) parts of lymphatic
vessels are seen at L1 and L2 which contain a clump of cells (arrow). L3
also contains a clump of cells (arrow) and forms an anastomosis with L4 by
way of a valve (V). Lymphoid tissue (*), similar to that observed in scanning
images of lymph nodes, surrounds the lymphatic vessels. Bronchial (B) and
blood vessel (BV) are as marked. (a) X250; (b) X500. (From ref. 17).
102
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held in close apposition by maculae adherents. The wall of the larger vessel
is distinguished by the periodic occurrence of valves. The valves consist of
leaflets of endothelial cells which project into the lumen as folds (Fig. 10).
The two layers of cells are separated by a thin band of connective tissue
consisting of collagen and elastic fibrils and an occasional fibroblast (Fig.
10). Ihe regular spacing of valves and constriction at the base of each
give a beaded appearance characteristic of the distended lymphatic collecting
vessel. When observed in the scanning electron microscope, the bileaflet
nature is readily apparent in three-dimensional relief (Figs. 9 and 11). It
is evident that the endothelial cells are duplicated as a double layer of
cells encircling the lumen of the vessel. The folds are separated along
the midline to form paired leaflets which appear as two thin cusps. The sur-
face of each leaflet is lined with flattened endothelial cells. The leaflets
project into the lumen at an angle such that their free edges fit together as
a miter joint without fusing with each other (Fig. 11). The valves project
into the lumen in the direction of fluid flow, providing free movement of lymph
toward the larger vessels and lymphatic trunks.
Figure 10. Electron micrograph of collecting vessels illustrating the
appearance of valves (arrows) which arise as a duplication of the endothelium
which folds into the lymphatic lumen (L). (a) X3,000; (b) X8,000. (From
ref. 17).
103
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Figure 11. This scanning electron micrograph illustrates the appearance
of a valve as seen in a cross-section of a lymphatic duct. A pair of leaflets
extend from the wall in a circumferential fashion and project into the lumen
of the vessel at such an angle that their free edges fit together like a miter
joint. X240.
The wall of the collecting vessel is also distinguished by the presence
of smooth muscle cells. Located in the tunica media, the smooth muscle cells
may vary from single cells arranged in an incomplete spiral to several com-
plete layers of smooth muscle cells (Fig. 12). The smooth muscle cells are
connected to each other by communicating (gap) junctions and make contact with
the endothelium by myoendothelial intercellular junctions (Fig. 13).
Trace Experiments
Pulmonary lymphatics also share the distinction of engulfing large
molecules and particulate matter from the surrounding interstitium. Advantage
is taken of this phagocytic property not only to label pulmonary lymphatics
but to ascertain the mechanisms involved in the movement of fluids and large
particulate substances across the air-tissue-lymph interface as well as the
blood-tissue-lymph interface.
In an attempt to monitor the uptake of colloidal particles from the
104
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alveolar spaces, ferritin and colloidal carbon suspensions were instilled
into the trachea of young adult rats. In samples of lungs observed at 15 and
30 minutes after tracer was instilled in the trachea, large amounts of ferritin
and carbon could be seen in the lumen of the bronchial tree (Figs. 14 and 15).
Tracer particles were also observed in vesicles within alveolar macrophages
and squamous cells of the alveolar epithelium (Fig. 16). At time periods of
up to three hours, the tracers were observed within vesicles in macrophages
located in the interstitium and in vesicles within the lymphatic endothelium
(Figs. 17 and 18). For periods of up to six months following intratracheal
Figure 12. Portion of collecting lymphatic with closely associated
smooth muscle cells (SMC), some of which make contact with the endothelial
cells (arrows). The lumen (L) of collecting vessel also contains a dense
precipitate. The endothelial cells (E) contain the usual complement of
organelles including large vesicles (v) with an electron dense substance.
X11 ,000.
105
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Figure 13. The smooth muscle cells (SMC) of the tunica media and the
lymphatic endothelial (E) cells of collecting vessels are closely apposed to
each other to form myoendothelial junctions (arrows). X15,048.
Figure 14. An electron micrograph depicting the presence of ferritin
particles (*) within alveoli. X20,000. (From ref. 17).
106
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injections of colloidal carbon, the tracer was observed as large accumula-
tions within autophagic vacuoles in the lymphatic endothelium as well as in
macrophages located in the lymphatic lumen and in pulmonary lymph nodes.
Although ferritin particles were observed free within the interstitium, this
was not the case with the carbon tracer observed within vesicles in macrophages
and lymphatic endothelium.
Intravascular Injection of Tracer Substances
Although interstitial injections of tracer substances permit the uptake
of tracer to be first removed by the lymphatics before their appearance is
observed in the blood vascular system, the problem still exists for the
development of some trauma due to unphysiological pressure in the vicinity
of the injection site, as in the case of interstitial injection, or the pro-
duction of a mild inflammatory response as indicated by the migration of
macrophages and neutrophils to the site of irritation. This possibility is
overcome by using intravenous inj ections of tracer particles or substances
which can be rendered electron dense for visualization in the electron
Figure 15. Intratracheally injected carbon is shown within alveolar
macrophages. X13,000. (From ref. 17).
107
-------
,•
^k
- '€
I6)b
Figure 16. A portion of alveolar macrophage which contains numerous
vesicles that are filled with ferritin particles (*). X12,400. in Figure
16b, carbon tracer particles (arrow) are observed in a vesicle within the
epithelium lining of the alveolar wall. X15,378.
108
-------
. « • f
^
Figure 17. Ferritin is located within large vesicles (v) at 12 hours
after injection into the trachea. X79,800.
109
-------
,A.
v:&:S**S'
•;• •• •
w
...;.£•,:.'
Figures 18a and b. Carbon particles (C) are also accumulated within
vesicles within the lymphatic endothelium (E). (a) X13/224; (b) X10,944.
110
-------
microscope by chemical means once the tissue has been fixed and processed.
This difficulty can be avoided by using the procedures of Simionescu and
Palade (27) in Which dextran is injected intravenously. Dextrans of varying
molecular weights (60,000 to 300,000) were injected via the tail or saphenous
veins in young adult rats. It is well documented that dextran injected
intravascularly is able to pass across the blood capillary wall and is
removed from the interstitium by lymphatic vessels for return to the systemic
circulation (9). To observe dextran mainly in the interstitium and the
lymphatic vessels, the lungs were perfused with saline to free the blood
vessels of injected dextran solutions, leaving the tracer in the interstitial
areas and within lymphatics. After fixation with a glutaraldehyde-formaldehyde
mixture in phosphate buffer at a pH 7.4 at 0-C (27), dextran particles were
retained in a homogenous distribution in the plasma and interstitium, pre-
sumably as a result of fixation of the surrounding proteins. This procedure
produces an electron dense product whose density is considerably enhanced
by postfixation in osmic acid in a phosphate buffer solution.
Figure 19. Part of lymphatic vessel from animal injected with dextran
which appears as a very electron dense precipitate (*) in the connective
tissue, within vesicles (v) and within the lymphatic lumen (L) at 15 minutes
after an injection of a 10% solution of dextran via the saphenous vein.
X23,000.
111
-------
Examination of pulmonary tissue within 15 minutes after dextran injec-
tions shows the presence of this tracer throughout the interstitium and
within the pulmonary lymphatic vessels (Fig* 19). Dextran gains access to
the lymphatic capillary lumen via vesicles and within the intercellular clefts
of loosely adherent overlapping intercellular junctions* It is of special
interest to note that the uptake of dextran from the surrounding interstitium
by pulmonary lymphatics is very similar to the pattern of movement of peroxi-
dase across the blood-tissue-lymph interface in other tissues of the body (16),
The presence of tracer particles within alveolar macrophages and in
vesicles of squamous cells of the alveolar epithelium at short intervals
(15-30 minutes) and the subsequent appearance of tracer particles in pulmonary
lymphatic vessels provide morphological evidence at the ultrastructural level
suggesting that large particles are able to cross squamous alveolar epithelial
cells within vesicles* Once in the connective tissue compartment, which has a
negative interstitial fluid pressure (22), the particles move toward the
lymphatics and are subsequently removed by these vessels in a fashion similar
to that in other regions of the body. The observation in the present study
and those of other workers (13, 28) suggest that particulate materials are
able to cross the pulmonary epithelium within vesicles to gain entrance into
the pulmonary interstitium. This removal of fluids and proteins from the
pulmonary interstitium is similar to the pattern of lymphatic drainage in
other tissues (15, 16, 18). Therefore, fluids, particles, and cells are re-
moved from the pulmonary interstitium by way of vesicles and the clefts of
intercellular junctions.
ACKNOWLEDGE ENTS
This work was supported in part from funds received from Grants
NHLBI-13901 and NIAID-10639.
REFERENCES
1. Allen L: Lymphatics and lymphoid tissue. Ann Review Physiol 29:197-224
1967
2. Anderson TF: Techniques for the preservation of three dimensional
structures in preparing specimens for the electron microscope* Trans NY
Acad Sci Ser II 13:130-134, 1951
3. Bloomstrand R, Werner B: Alkaline phosphatase activity in human
thoracic duct lymph. Acta Chir Scand 129:177-191, 1965
4. Bollman JL, Cain JC, Grindlay JH: Technique for collection of lymph from
liver, small intestine or thoracic duct of the rat. J Lab Clin Med
33:1349-1352, 1948
5. Courtice FC, Simmonds WT: Physiological significance of lymph drainage
of the serous cavities and lungs. Physiol Rev 34:419-448, 1954
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6. Drinker CK, Hardenberg E: Absorption from the pulmonary alveoli.
J Exp Med 86:7-18, 1947
7. Engeset A, Hoger B, Hesheim A, Kolbenstuedt A: Studies on hunan peripheral
lymph. I. Sampling method. Lymphology 6:1-5, 1973
8. Flock EV, Bollman O: Alkaline phosphatase in the intestinal lymph
of the rat. J Biol Chem 175:439-449, 1948
9. Grotte G: Passage of dextran molecules across the blood-lymph barrier.
Acta Chir Scand [Suppl] 211:8-84, 1956
10. Hall JG, Morris B, Moreno GD, Bessis MC: The ultrastructure and function
of the cells in lymph following antigenic stimulation. J Exp Med 125:99-
109, 1967
11. Karnovsky MJ: A formaldehyde-glutaraldehyde fixature of high osmolarity
for use in electron microscopy. J Cell Biol 27:137A, 1965
12. Lascelles AK, Morris B: The flow and composition of lymph from the mam-
mary gland in merino sheep. Q J Exp Physiol 46:206-215, 1961
13. Lauweryns JM: The blood and lymphatic microcirculation of the lung. Path
Ann 6:365-415, 1971
14. Lauweryns JM, Baert J, Desloecker W: Fine filaments in lymphatic
endothelial cells. J Cell Biol 68:163-167, 1976
15. Leak LV: Electron microscopic observations on lymphatic capillaries and
the structural components of the connective tissue-lymph interface.
Microvasc Res 2:361, 1970
16. Leak LV: The transport of exogenous peroxidase across the blood tissue
lymph interface. J Ultrastruc Res 39:24-42, 1972
17. Leak LV: Lymphatics and their role in the removal of interstitial
fluids and particulate matter. In: Respiratory Defense Mechanisms,
Part II (Brian JD, Proctor DF, Reid, LM, eds). New York, Marcel Dekker,
1977, Chapter 17, p 631
18. Leak LV: Studies on the permeability of lymphatic capillaries. J Cell
Biol 50:300-323, 1971
19. Lewis GP: Intercellular enzymes in local lymph as a measure of cellular
injury. J Physiol (Lond) 191:591-607, 1967
20. Mayerson HS: On lymph and lymphatics. Circulation 28:839-842, 1963
21. Mayerson HS, Patterson RM, McKee A, Lebrie SJ, Mayerson P: Permeability
of lymphatic vessels. Am J Physiol 203:98-106, 1962
22. Meyer BJ, Meyer A, Guyton AC: Interstitial fluid pressure. II. Negative
pressure in the lung. Circ Res 22:263-271, 1968
23. Miller WS: The Lung, 2nd ed. Springfield, 111., Thomas, 1947
24. Morris B: The cells of the lymph and their role in immunological reac-
tions, in: Handbuch der Allgemeinen Pathologic (Messen H, ed). Berlin,
Springer Verlag, 1972, p 405
113
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25. Porter KR, Kelley D, Andrews PM: The preparation of cultured cells and
soft tissue for scanning electron microscopy. In: Proceedings of the
Fifth Annual Stereoscan Scanning Electron Microscope Colloquium. Morton
Grove, 111., Kent Cambridge Scientific Inc., 1972, pp 1-19
26. Roberts JC, Courtice FC: Measurements of protein leakage in the acute
and recovery stages of a thermal injury. Aust J Biol Med Sci 47:421-433
1969
27. Simionescu N, Palade GE: Dextrans and glycogen as particulate tracers
for studying capillary permeability. J Cell Biol 50:616-624, 1971
28. Sorokin SF, Brain JD: Pathways of clearance in mouse lungs exposed
to iron oxide aerosols. Anat Pec 181:581, 1975
29. Staub NC: Pulmonary edema. Physiol Rev 54:678-811, 1974
30. Trapnell DH: The peripheral lymphatics of the lung. Br J Radiol 36:
660-672, 1963
31. Yoffey J, Courtice FC: In: Lymphatics Lymph and the Lymphomyeloid
Complex. London, Academic Press/ 1970
114
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DISCUSSION
DR. BROOY: Do you have any evidence of whether particles that move from
the air space by way of the epithelium and reach the basement membrane? Do
you have any evidence that those particles reach the small lymphatic capill-
aries by the basement membrane pathway, before they get to the interstitivm?
DR. LEAK: The passage of particles from the air space into the pulmonary
lymphatics occurs by (a) endocytosis of particles by the pulmonary epithelium;
(b) movement of vesicles across the epithelium and exocytosis of parti-
cles onto the tissue front across the basal lamina and into the interstitium;
and (c) once in the interstitium particles move toward the lymphatics and enter
the lymphatic capillaries via clefts of loosely adhering intercellular junc-
tions and also via plasmalemmal vessels. In our studies which follow the
movement of ferritin from the air space into the interstitium we have observed
ferritin particles within vesicles of the epithelium, within the interstitiura
and within vesicles of the lymphatic endotheliun and finally within the Ivanen
of the lymphatic vessel. In similar studies with colloidal carbon very few
particles were seen free in the interstitium, but most appeared in macro-
phages within the interstitium •
DR. BRODY: Do you have any evidence that macrophages lift particles and move
from the interstitium to the lymphatic space? You show the particles in the
lymphatic lining cells and in macrophages in the lymphatics, and one would
conclude that they do, in fact, move.
DR. LEAK: We have not been fortunate enough to see macrophages in the process
of passing along the clefts of intercellular junctions as we saw for the
lymphocytes as illustrated in this study*
DR. BRODY: Can you say that the particles may be picked up in the interstitium
by macrophages?
DR. LEAK: Probably so, based on our earlier studies of dermal lymphatics
we did observe interstitially injected particles within macrophages which
subsequently entered dermal lymphatics via the clefts of intercellular junctions*
DR. BRODY: Do you think that's because it happens so rapidly?
115
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DR. LEAK: Possibly, since the uptake of interstitially injected dye by
lymphatic capillaries occurs with surprising rapidity and since lymphatics
are more difficult to locate than blood vessels, it is easy to miss macro-
phages that may be in the process of entering lymphatic capillaries by way of
the intercellular junction.
DR. BROMBERG: You've shown us these tight endothelial junctions in the lym-
phatic capillaries. As you go to bigger lymphatic vessels that have more of a
wall, is there a change in the nature of the intercellular junctions? Is there
something like a tight junction, there?
DR. LEAK: Yes, there are also tight (occluden) junctions in the lymphatic
collecting vessels; however, they do not form a continuous seal or belt
(zonule) that is found in epithelial cells. They appear as discrete junctions
to form a spot-weld effect. There are two major differences between inter-
cellular junctions in lymphatic capillary and lymphatic collecting vessels.
In the lymphatic capillary, the adjacent cells are extensively overlapped,
while very little overlapping occurs between adjacent cells of the collecting
vessels. The extensively overlapping cells of the lymphatic capillaries are
easily separated to form open (patent) intercellular junctions, thus allowing
the rapid movement of fluids and particles from the interstitiun directly into
the lymphatic capillary lumen.
DR. BROMBERG: I was wondering whether those collecting lymphatics actually
might have the ability to concentrate macromolecules that they picked up in
the interstitial area, the lymphatic capillary area, and move along? Is it
possible there are fluid transport mechanisms that, presumably, would require
some tight junctions of the epithelium?
DR. LEAK: We 'have not detected any differences in the electron density of
lymph protein particles within the lumina of lymphatic capillaries and collect-
ing lymphatic vessels. It has been suggested by some investigators that there
is a mechanism for concentrating lymph within the vessels by a constant re-
moval of water and electrolytes as lymph is propelled up stream. There are
however, no data to support this.
DR. DAVIS: Do you have any evidence to suggest how furosemide has its re-
markable effect on pulmonary edema, and does it affect the pulmonary lymphatics?
DR. LEAK: We have not studied the effects of furosemide on pulmonary lymphati
although we have noticed an increased delitation of lymphatics during pulmonarv
edema. Lymphatic capillaries can rapidly dilate because the extensively over-
lapping intercellular junctions are easily separated.
116
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Session III:
Respiratory Tract Cilia
-------
Structural Basis of Ciliary Movement
P. Satir
Albert Einstein College of Medicine
Bronx, New York
All motile somatic cilia, including those of the human respiratory
tract/ are similar in ultrastructure in that they consist of an axoneme
of 9+2 microtubules, surrounded by a specialized extension of the cell
membrane. The nine peripheral microtubules of the axoneme differ from
other cytoplasmic microtubules in that they are doublets, composed of a
complete microtubule, subfiber A, to which is attached an incomplete, shorter
set of protofilaments, subfiber B. Important axonemal interconnections ex-
tend from specific points on each subfiber A. They are: (a) the two rows
of arms, (b) the radially directed spokes, and (c) circumferentially
directed interdoublet links. Together these elements function as the ciliary
motor, powered by ATP hydrolysis.
In the last decade or so, considerable advances in our understanding
of the precise arrangements of these axonemal structures, together with
some important analyses of their biochemistry and function, have enabled us
to put together a reasonable working hypothesis of the coupling of structure
and function within a cilium to produce movement. Most of the basic work
on which this account is largely constructed has been derived from studies
of systems other than the mammalian respiratory tract. Indeed, mammalian
somatic cilia are relatively poorly studied in terms of motile mechanism
and basic beat properties (see ref. 9 for earlier review). Although the
ciliated cells of the tracheal epithelium do not form a continuous sheet and
the exact ciliary stroke of tracheal cilia is not yet understood, it is
119
-------
evident that these cilia are capable of moving mucus sheets in a coordinated
manner to effect respiratory clearance. There is reasonable circumstantial
evidence, which will be summarized here, to conclude that the basic mech-
anism of ciliary motion, like the ultrastructure, is similar in all 9+2
cilia, including human tracheal cilia. It remains to be seen whether the
fundamental biochemical details are identical or whether subtle but vital
differences will crop up, affecting beat form and cellular control of motion.
Additionally, cilia which primarily move mucus may differ from cilia whicn
move water.
Although the basic ultrastructure throughout the length of the axoneme
is identical, for example, in sea urchin sperm tails, Tetrahymena or mussel
gill cilia, and tracheal cilia, differences do occur at either end so that
the origins, overall lengths, and attachments of the individual doublet
microtubules are not the same. For example, a sperm tail may be 50 um long,
a tracheal cilium only one-tenth to one-fifth of that length; one cilium
may taper, another have a blunt, rounded end, and so on. One comparison is
shown in Figure 1. These differences again are thought to modify the actual
form of beat, while the basic motile mechanisms remain reasonably constant.
SLIDING MICROTUBULES IN CILIARY MOTION
Ciliary motion is based on the sliding of the doublet axonemal micro-
tubules. Several major aspects of this sliding microtubule mechanism are
now well established:
(1) The axoneme alone is responsible for ciliary motility. Detergent-
treated models of cilia beat normally in appropriate solutions containing
mM concentrations of Mg and ATP. Detergent treatment destroys the
integrity of the ciliary membrane, but causes no substantial change in the
arrangement of axonemal components. In the intact cell, the ionic environment
of the axoneme and concentrations of ATP and divalent cations are maintained
and regulated by the ciliary membrane. The presence of the membrane permits
cellular control of axonemal function.
(2) The doublet microtubules of the axoneme slide relative to one an-
other without measurable contraction (cf. ref. 9) during bend production in
a ciliary stroke. In a recent test of the geometrical predictions of this
statement, Shingyoji e_t al. (13) pipetted ATP onto local regions of Triton-
extracted sea urchin sperm whose heads had been attached to a micropipette.
This induced local equal and opposite bends of the axoneme, without change
in head or tail position, which conforms exactly to the necessary con-
straints of a sliding system. Further, Triton-treated axonemes exposed
briefly to trypsin, then washed and placed in ATP, do not beat but in-
stead disintegrate by telescoping apart (17). Here sliding of individual
120
-------
Rat
trachea
Clam
gill
9D+2
9S,D
9S + 2
8S,D
Domino
Figure 1. Comparison of ultrastructure of the axoneme (9D+2) and tip
configurations of mammalian respiratory and molluscan gill cilia. The find-
ing of 9S+2 and 8S, D+2 tips in separate cilia is indicative of relative
sliding of doublets. See ref. 8 for details of nomenclature and interpre-
tation. The tracheal cilia end in a rounded tip and the doublet microtubules
are all roughly of equal length; this is not so for the gill cilia, whose
tapering tip results in the domino cross-section. X92,500.
121
-------
•
base
Figure 2. Sliding microtubules in a trypsin-treated ciliary axoneme
to which ATP is added for preparative details. Reproduced with permission
(12). X18,800.
microtubules is directly observed. Figure 2, taken from the work of Satir
and Sale (12), shows an axoneme prepared for electron microscopy after such
sliding has taken place. Sliding in the trypsin-treated axonemes is rela-
tively isotropic i.e., at least seven and possibly all nine doublets are
capable of sliding. It is also unidirectional. If doublets are numbered
1-9 in the conventional way, doublet no. n+1 is always displaced tipward
relative to doublet no. n. Lastly, sliding is such cases is uncoupled from
bend generation and bend propagation and largely unconstrained: final ex-
tension can be many tens of micrometers longer than the original axoneme.
Only one constraint seems to limit sliding in the preparations: some over-
lap of doublets is always necessary, for reasons considered in the next
paragraph.
(3) Active sliding is generated when the arms attached to subfiber A
of each doublet (i.e., doublet n) also bind to subfiber B of the adjacent
doublet (n+1) to push that doublet. The generation of active force in this
system is accompanied by ATP dephosphorylation. Because the arm action is
repetitive and cooperative, we can define an arm cycle, which is discussed
below. Presumably, sliding in trypsin-treated axonemes stops when the num-
ber of active arms is no longer sufficient to move the doublet microtubules.
At that time, evidently, a small amount of overlap between adjacent doublets
still remains (Fig. 2). Individual doublets appear inactive when completely
separated from the axoneme.
(4) Active sliding and bending are two separable systems; the latter
relies on coordination and restriction of sliding, and is trypsin-sensitive.
122
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In the intact (non-trypsin treated) axoneme, microtubule sliding rarely ex-
ceeds 0.3-0.4 um maximum displacement, that is/ only a few percent of total
possible sliding. Further, sliding is not unidirectional. This has been
interpreted to mean that during beat, doublets slide asynchronously such
that the active sliding of doublets no. 1-4 produces the ciliary effective
stroke, while doublets 6-9 are passively moved and, alternatively, active
sliding of doublets no. 6-9 produces the recovery stroke, while doublets
1-4 are passively moved (12).
Much less is known about the conversion of active sliding into bending
and about mechanisms of bend propagation than about sliding itself. The
radial spokes, the interdoublet links, and perhaps the arms themselves
presumably form part of an elaborate mechanical feedback system within the
axoneme that switches sliding on and off appropriately. The spokes, in
particular, interact with central sheath projections in a highly circum-
scribed manner such that as bends develop at the base of a straight axoneme
during a beat, the spokes become tilted in a manner consistent with attach-
ment of the spoke head as a method of constraining further sliding and pro-
ducing bending (22). It seems likely that the spoke-central sheath inter-
action is central to the local conversion of sliding to bending, or at least
to the trypsin-sensitive system which causes the conversion. Spokes, es-
pecially the spoke heads, and interdoublet links appear to be the principal
structures digested by trypsin under conditions where sliding restraints
disappear. Further, mutants of motile Chiamydomonas/ lacking either struc-
tural component of the spoke-central sheath complex, are paralyzed despite
the presence of the intact sliding system (23).
(5) Calcium ion concentration surrounding the axoneme provides a
usual control of ciliary activity. Cilia beat normally in the presence
2+ —7
of axonemal free Ca < 10 M. In many organisms, alteration of axonemal
2+ —6
free Ca to greater than 10 M causes major alteration in ciliary behavior,
including complete cessation of motion (20). However, the trypsin-resistant
2+
sliding of ciliary axonemes seems insensitive to changes in Ca that phy-
siologically account for ciliary arrest (21), so that it seems probable
that the site of Ca control resides in the trypsin-sensitive feedback
system responsible for bend generation and propagation. In organisms where
2+
increasing Ca does not cause complete arrest, it is the mode of bend gen-
eration and propagation that are usually altered, while sliding must
obviously continue.
EVIDENCE FOR THESE MECHANISMS IN RESPIRATORY CILIA
Ciliary malfunction has recently been convincingly documented in man
(cf. review in ref. 1). Immotile cilia in males result in a syndrome,
formerly known as Kartagener's triad, which couples infertility due to
123
-------
nonmotile sperm to respiratory disease (sinusitis, bronchiectasis) due to
nonfunctional respiratory cilia. (The third aspect of the syndrome described
by Kartagener, situs inversus, is not always present and its link to
ciliary function is still obscure.) The diseases responsible for the syn-
drome are congenital and affect females as well as males* Several labora-
tories have now shown ultrastructural defects present in sperm tail
axonemes and somatic cilia in patients whose cilia and sperm are function-
ally nonmotile. In the original cases, the cilia lacked arms (cf» ref. 1).
Sturgess ejt al. (15) described a second variant, where the cilia possess
arms but lack radial spokes. From the complexity of ciliary structure
and assembly, it is likely that other variants will be found, including those
where the ultrastructural defect is not apparent (Herzon, personal communication)
Although the biochemistry remains to be worked out, the mutant classes in humans
resemble those in lower organisms where the rationale for ciliary paralysis has
been defined. Presumably, in armless axonemes the doublets are incapable of
sliding. It would be interesting to know whether the doublets of the spokeless
human axonemes would slide upon appropriate treatment; presumably, these axonemas
are defective in the conversion of sliding into propagated bending. Despite
this impressive circumstantial evidence for mammalian cilia, there is as yet no
direct evidence regarding the mechanism of motion. However, sliding of axonemal
microtubules in mammalian sperm has been demonstrated (6, 16).
In 1967, Spock (14) reported that addition of serum from cystic fibrosis
(CF) homozygotes or heterozygotes to excised rabbit ciliated tracheal epi-
thelium, which was beating in a coordinated manner, resulted in "ciliary
dyskinesia." Although much controversy has been generated regarding the
existence of specific CF factors, and dyskinesia has never been adequately
described in a quantitative manner, ciliary beat clearly looks more or less
coordinated under different conditions. Bogart et a_l. (2) report a CF-like
dyskinesia when the divalent cationophore A23187 is added to the medium
bathing the tracheal epithelium. Can it be that mammalian ciliary axonemes
are also responsive to changes in Ca and that dyskinesia is the equivalent
of ciliary arrest? If this were so, agents such as cigarette smoke, indus-
trial pollutants, and immune complexes, which might alter the cell or ciliary
membrane, could all act to produce dyskinesia via a final common pathway in-
volving increased free axonemal Ca .
DYNEIN ARM CYCLE
The aspect of the mechanism of ciliary motility that is proving most
amenable to analysis at present is the arm cycle that produces microtubule
sliding. The biochemistry of the arms is unsettled in detail, but clear
in general principle. The arms are multi-protein structures whose main
124
-------
functional components are a class of ATPase isoenzymes called dyneins (eft
ref. 5). In Tetrahymena ciliary axonemes, the ATPase of the arms seems to
correspond to that of sea urchin sperm tails, dynein-1 (4). Dynein-1 can be
extracted in a form that has latent ATPase activity and sediments at 21S,
and this may represent the intact arm that is-the moving cross-bridge between
adjacent axonemal doublet microtubules and that generates sliding. Hie
21S particle is apparently composed of three subunits of about 330K daltons
and one each of subunits of 126, 95 and 77K daltons. It is not yet known
whether the inner and outer rows of arms are exactly identical in structure
o r composition.
In negative stain, the subunit construction of the arms is visible (7).
The arms have an overall tripartite construction; in at least one position
they are slightly tilted (ca. 40°) toward the base of the subfiber A to
which they are attached (doublet no. n). In the absence of ATP and the
presence of 2 mM Mg , both inner and outer arms attach to the subfiber B
of doublet n+1, forming bridges between doublets as illustrated in Figure
3a. This is known as the rigor position (3). Such rigor bridges can be
plasticized by the addition of ATP, as in Figure 3b (see also ref. 24).
In trypsin-treated axonemes, presumably held together largely by rigor
arms in the absence of ATP, upon ATP addition, as the arms are plasticized,
the axonemes fall apart, with or without concurrent microtubule sliding (21).
This step is evidently intrinsic to the dynein-microtubule interaction
per se. For example, Takahashi and Tonomura (18) have recombined dynein
arms to doublets from which such arms had previously been extracted. In the
presence of Mg with no ATP/ in a state equivalent to rigor, the arms rebind
to and decorate either subfiber A or subfiber B. Upon addition of ATP, equi-
valent to plasticizing the rigor bridge, the arms detach from subfiber B
exclusively.
It is postulated that sliding of the doublet microtubules is accom-
plished by successive detachment-reattachment of the dynein arms in this
way, each detachment requiring binding of new ATP, which is then hydrolyzed
at some point in the subsequent cycle, providing the energy for eventual
directional force generation. There is little experimentation and no gen-
eral agreement yet on the exact details of the cycle, but one possibility
which combines the known structural information with a plausible, though
highly speculative, enzymology is shown in Figure 4. The enzymology is
simply drawn as a parallel to the work of Taylor and his colleagues (cf.
ref. 19) on the course of actin-myosin kinetics. For cilia, it is known
that vanadate is a specific inhibitor of dynein-ATPase activity and doublet
sliding, but probably not of doublet detachment (Sale, personal communi-
cation) . This may prove to be an important tool in clarifying the point in
the cycle at which ATP is actually hydrolyzed. Eventually, the combination
125
-------
a
Figure 3. (a) Triton-treated axonemes before ATP addition. All doub-
lets are bridged by rigor armst X126,000. (b) After ATP addition. Rigor
arms are plasticized, so that the doublets are no longer completely attached
to one another. X126,000.
of such approaches to the biochemistry, physiology, and morphology of the
dynein arm and other axonemal structures should yield a fundamental under-
standing of the underlying mechanochemical transduction events in ciliary
motion.
126
-------
vj^
D-ATP
I
D-ADP-P,
I
T-D-ADP-P,
A
ADP'P, 0-T
ATP- J
D-ATP
POSTULATED
^•M
^
{^
A"""
r^
r
1C
POS
TU
L4
STANDARD IMAGE
EXTENDED FORM
MECHANOCHEMICAI
TRANSDUCTION
RIGOR IMAGE
FLATTENED FORM
JED
ENZYMOlOGY EM ARM CYCLE
Figure 4. Hypothetical dynein arm cycle (10). From top to bottom successive
stages of one complete cycle are shown. At each level/ the postulated enzymology
is correlated with the postulated arm morphology. The hatched vertical line
along subfiber B of doublet n+1 as diagrammed indicates hypothetical arm attach-
ment site.
ACKNOWLEDGMENTS
This work was supported by a grant from the USPHS (HL 22560). I
thank P. Setzer for help in preparing Figure 1, R. Ramer for bibliographic
and secretarial help, and S. Lebduska for general assistance.
REFERENCES
1. Afzelius B: The role of cilia in man. In: Contractile Systems in Non-
muscle Tissues (Perry SV, et a_l, eds). Amsterdam, Elsevier/North Holland,
1976, pp 275-282
2. Bogart BI, Conod EJ, Conover JH: The biologic activities of cystic
fibrosis serum. I. The effects of cystic fibrosis sera and calcium ionophore
A23187 on rabbit tracheal explants. Pediat Res 11:131-134, 1977
3. Gibbons BH, Gibbons IR: Properties of flagellar "rigor waves" produced by
abrupt removal of adenosine triphosphate from actively swimming sea urchin
sperm. J Cell Biol 63:970-985, 1974
4. Gibbons IR, Fronk E: A latent adenosine triphosphatase form of dynein 1
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5. Gibbon IR, Fronk E, Gibbons BH, Ogawa K: Multiple forms of dynein in sea
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6. Lindemann CB, Gibbons IR: Adenosine triphosphate-induced motility
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7. Sale WS, Satir P: Direction of active sliding of microtubules in
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9. Satir P: The present status of the sliding microtubule hypothesis
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13. Shingyoji C, Murakami A, Takahashi K: Local reactivation of Triton-
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16. Summers KE: ATP-induced sliding of microtubules in bull sperm
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17. Summers KE, Gibbons IR: Adenosine-triphosphate-induced sliding of
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18. Takahashi M, Tonomura Y: Binding of 30S dynein with the B-tubule
of the outer doublet of axonemes from Tetrahymena pyriformis and
adenosine-triphosphate dissociation of the complex. J Biochem
(Tokyo) 84:1339-1356, 1978
19. Taylor EW: Chemistry of muscle contraction. Ann Rev Biochem 41:577-616
1972
20. Walter MF, Satir P: Calcium control of ciliary arrest in mussel gill
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21. Walter MF, Satir P: Calcium does not inhibit active sliding of
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22. Warner FD, Satir P: The structural basis of ciliary bend formation.
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23. Witman GB, Fay R, Plummer J: Chlamydomonaa mutants: Evidence of the
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80:573-588, 1979
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DISCUSSION
DR. DAVIS: Is there any evidence in the mammalian epithelium that there is
a low calcium outside the cell?
DR. SATIR: No, perhaps I didn't make that clear. The calcivm outside the
cell can be quite high/ because presumably of calcivtn pumps in the ciliary
membrane. So, cilia can normally beat in external concentrations of calcium
of several millimolar, even though the internal calcivm concentration must
be often less than 10 M. In one study, Bogart et al. have used the calciun
ionophore. We used the calcium ionophore with gill cilia. They have taken
this protocol over to mammalian cilia and have shown that indeed they can
reproduce the ciliary dyskinesis which they think they see with cystic fibroaia
factors with calcivm ionophore. If that's true/ then that is a rather im-
portant finding.
DR. DAVIS: Is there anything in the cell that would act as a store for calcivm
as we have in other cells? In other internal calciun stores that could in-
teract?
DR. SATIR: There are indeed internal calcium stores, and it's thought that
cilia can, at least in some cases, respond to nervous stimulation, and this
may be in part by releasing internal calciun stores* This still needs to be
worked out in detail. I should also say that probably the calciun stores are
not from mitochondria. There are other calciun stores in the cell. Mito-
chondria operate at a different calciun level.
DR. BROMBERG: Dr. Satir, these little beasties are very long and very active;
they must be using a lot of ATP per unit length. Does it all get up there from
the basal regions of the cell by infective processes/ or do you think diffusion
is fast enough to account for it?
DR. SATIR: I haven't done the calculations myself, but two or three labor-
atories have done calculations on the utilization of ATP versus diffusion.
They calculate that in the sea urchin sperm tail, which is 50 micrometers
long, diffusion is sufficient to supply the appropriate level of ATP. in some
cilia there are adenylate kinase and perhaps other enzymes that can generate
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DR. GATZY: Is it my impression that the ciliary beating is sort of a two-
dimensional process, even though the shafts are or appear to be fairly uniform?
DR. SATIR: No, that's not right. Ciliary beating is three-dimensional.
At least in the gill cilia which I've studied very extensively, one can show
that with scanning electron microscopy and by a variety of other techniques.
The idea is that there is an effective stroke of the ciliun. In the best
studied cases, it's clear that this effective stroke of the cilia lies per-
pendicular to the central microtubules* If you take a line passing between
the central microtubules, this is the plane of the effective stroke of the
cilium. The ciliun then swings out in three dimensions and comes back in a
recovery stroke. In fact, it seems that the hydrodynamics of the ciliun are
such that the cilion is a low Reynolds number system, which means that viscous
forces govern motion rather than inertial forces. One consequence of that
is the form of beat is important in moving material, and it's important to have
a relatively vertical effective stroke and a relatively horizontal recovery
stroke.
DR. GATZY: Then you would expect to see specialized structures in the mem-
brane or in the shafts that would be related to localized changes in calcium,
since they are likely to occur at some point in the cycle everywhere.
DR. SATIR: We don't know whether calcium governs the normal beat cycle. That
is not the case.
DR. GATZY: Or magnesium or whatever?
DR. SATIR: As far as magnesium and ATP are concerned, the cycle looks iso-
tropic, as far as the inside of the axoneme is concerned. In other words, we
could take the isolated axoneme, and put it into ATP and magnesium of the
appropriate concentrations, and it works. If the membrane simply maintains
that concentration around the axoneme, presumably it will work.
DR. GATZY: To get the bending, some areas have to behave passively and some
actively. Is that correct?
DR. SATIR: Yes, that's what I said.
DR. GATZY: There must be some localization.
DR. SATIR: That1s right, and we don't really understand that*
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Injury of Respiratory Epithelium
A. M. Collier
University of North Carolina
Chapel Hill, North Carolina
The respiratory tract of humans provides a large surface area that is
constantly in contact with the environment, The lower respiratory tract is
normally sterile/ emphasizing the remarkable efficiency of the defense
mechanisms of this organ system. At the present time little is known about
the effects produced by inhalation of foreign particulates and infectious
agents on the epithelial lining of the respiratory tract. It is known that
for the respiratory tract to function properly the alveolar macrophages and
elements of the mucociliary escalator must be intact. Once these defense
mechanisms have been altered, a buildup of both viable and nonviable inhaled
substances occurs on the epithelium which may jeopardize the health of the
host.
The anatomic and physiologic complexities of the respiratory tract have
presented problems in interpreting the effects of exogenous agents on this
organ system, since the data base on normal function has not been well de-
lineated. This has led us to establish less complex iia vitro respiratory
organ culture models that can be more easily observed under controlled con-
ditions. In this paper/ studies will be reviewed in which tracheal organ
culture has been used to examine the pathogenic potential for epithelial
injury by Mycoplasma pneumoniae, Bprdetella pertussis/ respiratory syncytial
virus, and parainfluenza virus type 3. The manner in which these human
respiratory pathogens injure respiratory epithelium in tracheal organ culture
will be contrasted.
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TRACHEAL ORGAN CULTURE MODEL
Tracheas removed from most animals and human fetuses are suitable for
study in this model system (3, 4). The organ culture is prepared by removing
the trachea with sterile technique. After a midline incision is made in the
anterior neck, the trachea is exposed and transected at the levels just
below the larynx and above the carina. The trachea is then cut into rings
with a scapel. Each ring contains a cartilage which provides support.
The rings of tissue are placed on areas of cross-hatch scratches in a small
plastic petri dish and enough tissue culture medium added to just cover the
rings. The culture is then maintained at 36°C in 5% CO- and air.
The inner surfaces of the tracheal rings are lined by organized, dif-
ferentiated respiratory epithelium that can be maintained up to six weeks
when the medium is changed every three days. At any time, the dish may
be removed from the incubator and the epithelium examined with an inverted
microscope without opening the petri dish. This permits one to examine
for the presence and vigor of beating of the cilia which serves as a good
indicator of viability of the epithelium and degree of injury produced by
test substances.
One trachea provides 13-15 rings, thus permitting tissue with the
same genetic makeup, for test and control dishes. This model is very
maneuverable, in that rings of tissue may be added or removed from a dish at
any time under sterile conditions. The rings can be grasped on their outer
surface by forceps and moved without touching their epithelial inner surface.
The animal may be exposed to the test substance prior to sacrifice
and removal of the trachea for organ culture, or the tracheal rings may be
prepared and then exposed to the test substance. In our laboratory we have
had more experience with the latter method.
MYCOPLA34A PNEUMONIAE IN HAMSTER TRACHZAL ORGAN CULTURE
Mycoplasma pneumoniae is the most common cause of pneumonia in teenagers
and young adults. This microorganism is the smallest infectious agent capable
of growth on lifeless media. In an attempt to better understand the pathogen-
esis of disease produced by this microorganism, hamster tracheal organ cul-
tures were inoculated with both virulent and attenuated strains of M.
pneumoniae and other human mycoplasma species that produce no known disease
in man (Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale and
Mycoplasma salivarium). The virulent strain of M. pneumoniae produced
ciliostasis by day 3 and the attenuated strain by day 6. The other strains of
mycoplasmas had no effect on ciliary activity, implying that the ciliostatic
property possessed by virulent M. pneumoniae might play a role in the virulence
of this respiratory pathogen (6).
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To be able to quantitate the ciliary beat frequency, a calibrated strobo-
scope was used as the light source for the inverted microscope (4). The
speed of the ciliary beat could be determined from the flash frequency which
gave the illusion of arrested ciliary motion. A series of separate cultures
was established containing tracheal rings from the same animal; tenfold
dilutions of the virulent M. pneumoniae organism pool were employed as ino-
cula. The control rings were placed in sterile broth. Cessation of ciliary
activity was produced within four days by the two greatest inocula, while the
smallest inoculum required seven days to reach this point. This provides
evidence of a dose response effect and suggests that a critical degree of
infection is required for tissue injury to occur*
Individual tracheal rings can be removed from the culture dish at any
time and examined by microscopy techniques. When tracheal rings fixed with
formalin and stained with hematoxylin and eosin were examined, there was
good preservation of the ciliated epithelium in the control rings after 48
hours in organ culture. In contrast, the epithelium of comparable tracheal
rings infected for 48 hours in organ culture with M. pneumoniae demonstrated
nuclear swelling with chromatin margination, cytoplasmic vacuolization, and
loss of cilia from some epithelial cells (4, 6).
In an attempt to localize the M. pneumoniae organisms in relationship
to the injured cells, tracheal rings were removed from the organ culture and
frozen sections prepared. These sections were treated with rabbit M.
pneumoniae antiserum followed by goat anti-rabbit globulin conjugated with
fluorescein isothiocyanate. When the M. pneumoniae infected tissue was
examined with an immunofluorescent microscope, the antigen was found to be
located along the luminal border of the epithelial cells. In tissue that
had been infected for 72 hours, clumps of specific antigen were seen in the
lumen and down into the epithelium on each side of the nonstaining nuclei. '
Control tissue demonstrated no specific fluorescence. Since the M. pneumoniae
were below the resolution limits of the imntunofluorescence microscope, it was
not possible to delineate their exact location with respect to the epithelial
cells by this technique (7).
To answer this question, the tracheal rings were examined by electron
microscopy. Tissue was removed from the organ culture dish, fixed in
glutaraldehyde and osmium tetroxide, embedded in Epon, and thin sections cut.
When tracheal rings infected for 48 hours were examined, densely staining
filamentous organisms were seen down between the cilia. At higher magnifi-
cation the mycoplasma organisms were seen to attach to the epithelial cell
by a specialized tip structure. The tip structure consisted of a dense
central filament surrounded by a lucent space enveloped by an extension of
the organism's unit membrane. The outer membranes of the microorganism and
mammalian cell were in close approximation, but were not fused (4, 6).
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On further examination, M. pneumoniae were frequently seen attached in
the region of the terminal bar where the luminal surfaces of individual
epithelial cells were joined. Organisms were also seen penetrating the
intercellular spaces, and multiple organisms forming microcolonies were seen
in intercellular spaces between epithelial cells (4).
Tracheal rings infected for 72 hours revealed the pathologic changes
of both epithelial cell cytoplasmic vacuolization and loss of cilia. M.
pneumoniae organisms were seen in the intercellular spaces but no organisms
were seen within the epithelial cells. This explained the findings seen with
immunofluorescence and the thicker frozen sections. The antigen was present
in the intercellular spaces (6).
Another technique that we have used to examine the tracheal organ culture
model has been that of scanning electron microscopy (16). This method of
observation permits an in-depth look at the luminal surface of the epithelial
layer. Numerous filamentous M. pneumoniae organisms were seen attached to
the luminal border, and the apical portions of infected ciliated cells were
seen sloughed into the lumen. The phenomenon, called ciliocytophthoria,
was found to be the characteristic pattern of epithelial cell injury pro-
duced by M. pneumoniae infection.
The tracheal organ culture model permits the examination of the bio-
chemical basis of the alterations produced in epithelial cell injury by
infectious agents (10-12). Exposure of hamster tracheal rings to virulent
M. pneumoniae organisms in organ culture leads to alterations in macro-
molecular biosynthesis and metabolic activity of the respiratory epithelial
cells prior to the occurrence of histopathologic changes. These changes
were not seen with avirulent organisms derived from the same parent strain*
Within 24 hours after infection of tracheal rings by virulent M. pneumoniae.
inhibition of host cell ribonucleic acid and protein synthesis was evident.
The addition of erythromycin 24 hours or earlier after infection prevents
the onset of abnormal orotic acid uptake and subsequent cytopathology.
However, 48 hours after infection, rescue of host cells by erythromycin does
not occur and pathological changes become evident.
Our data indicate that the intimate contact between virulent mycoplasmas
and the respiratory epithelium alone does not account for the subsequent
interruption of host cell metabolism, but must be accompanied by continued
multiplication and biochemical function of attached mycoplasmas. The
mechanism of injury mediation is not yet known, but the primary effect of
mycoplasma infection on tracheal organ culture may be at a transcriptional
or translational level.
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MYCOPLASMA PNEUMONIAS IN HUMAN FETAL TRACKEAL ORGAN CULTURE
Human fetal trachea in organ culture has also been used to study the
epithelial changes produced by M. pneumoniae (2, 5). The ability of myco-
plasma species to produce ciliostasis was very similar to that reported
above in hamster tracheal organ culture. Only virulent M. pneumoniae injured
the human tissue. When examined by light microscopy, the epithelial changes
produced by M. pneumoniae followed the attachment of the organisms between
the cilia. This was followed by the apical portion of the cell becoming
eosinophilic and protruding into the lumen with the cilia forming a sunburst
pattern. The ciliated apical portion of the cell was later sloughed into
the lumen/ with the basal portion of the cell being left behind to maintain
the epithelial barrier.
Immunofluorescence studies, as previously described with tissue from
the hamster tracheal organ culture system, showed the M. pneumoniae antigens
to be localized on the epithelial luminal border between the cilia of the
human tissue. The organisms were also seen to invade the submucosal glands.
When the human tracheal tissue was examined by electron microscopy,
the M. pneumoniae organisms were seen to attach to the luminal aspect of
the cell by their specialized tip structure. No evidence of organisms
entering the epithelial cells was seen. The ciliocytophthoria pattern
of epithelial cell injury produced by M. pneumoniae was also observed at the
electron microscopic level. The luminal portion of the ciliated cell first
protruded into the lumen and was followed by a constricting and pinching off
of the apical surface. The injury produced by M. pneumoniae may be due to
parasitization of individual cells, since heavily parasitized cells showed
marked damage of subcellular organelles, and adjacent less heavily parasitized
cells appeared normal.
BORDETELLA PERTUSSIS IN HAMSTER TRACHEAL ORGAN CULTURE
The hamster tracheal organ culture model has been employed to examine
the effects of B. pertussis on respiratory epithelium (8). This human bac-
terial pathogen is the causative agent of whooping cough in children. When
the tracheal rings were infected with phase I B. pertussis and examined
by light microscopy, the bacteria were seen attached to the ciliated cells,
but, unlike M. pneumoniae, there was no attachment to the nonciliated cells.
After attachment, the ciliated cells were injured and later extruded from
the epithelial layer. The extruded cells were seen to contain pyknotic nuclei
with bacteria attached only to their luminal surface. After 72 hours of
infection in organ culture, no ciliated cells remained, and the spaces left
by the extruded ciliated cells in the epithelial layer had been filled in
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by the nonciliated cells. No attachment of B. pertussis to the remaining
nonciliated cells was observed.
Injury of the ciliated epithelial cells was also examined by electron
microscopy. Transmission electron photomicrograph showed ciliated cells
that were heavily infected with J3. pertussis. Some of these ciliated cells
were in the process of being extruded from the epithelial layer. These cells
possessed markedly damaged subcellular organelles while the adjoining cells
appeared normal. At no time were B. pertussis organisms seen within the
epithelial cells.
We have examined comparable tracheal epithelium infected in organ cul-
ture with j3. pertussis by scanning electron microscopy (15). After 48 hours
of infection, multiple extruded cells were seen on the luminal surface.
This technique, at higher magnification, also provided a three-dimensional
view of parasitized ciliated cells being extruded from the epithelial layer.
The ciliated cells that had been extruded had bacteria attached to their
luminal surface and there were marked membrane changes, with some appearing
as hollow shells with their nucleus having been expelled. No B. pertussis
organisms were seen attached to nonciliated cells.
RESPIRATORY SYNCYTIAL VIRUS IN HUMAN PETAL TRACHEAL ORGAN CULTURE
Respiratory syncytial virus is an important cause of bronchiolitis
in young children. The response of the respiratory epithelium in organ cul-
ture to infection with respiratory syncytial virus is different from that of
M. pneumonias and B. pertussis. When the epithelium was examined by light
microscopy, the first changes seen to develop were perinuclear inclusion
bodies in the ciliated cells. This was followed by swelling of the ciliated
cells with protrusion into the lumen and loss of cilia. Respiratory syncytial
virus infection produced fusion of the infected cell membrane with the cell
membranes of adjoining epithelial cells, thus forming a giant cell containing
multiple nuclei (9).
When human tracheal rings are removed from the organ culture, after
infection, and stained with fluorescent antibodies that are specific for the
antigens of respiratory syncytial virus, individual cells along the luminal
border are seen to fluoresce. The respiratory syncytial virus was found to
infect only ciliated cells in the epithelial layer. No specific fluores-
cence was seen in cells of the lamina propria or cartilage layer.
Tissue from organ cultures comparable to that just described was pro-
cessed for electron microscopy and examined. Viral particles were seen bud-
ding from the luminal aspect of the ciliated cells and from the surface of the
cilia. The typical perinuclear inclusion bodies produced by respiratory
syncytial virus infection of epithelial cells were seen to be made up of
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a dense intertwining of RNA cores. These cores were later packaged at the
membrane of the cell where they formed new viral particles.
PARAINFLUENZA VIRUS TYPE 3 IN HAMSTER TRACHEAL ORGAN CULTURE
Another important viral pathogen of children is parainfluenza virus type
3. The epithelial cell injury produced by this virus has been studied in
hamster tracheal organ culture (13). Thirteen days after infection in the
organ culture the formation of giant cells, similar to those seen in the
human epithelial tracheal organ culture with respiratory syncytial virus,
was observed. The giant cells of epithelium infected with parainfluenza
virus type 3 did not lose their cilia. This virus also produced changes in
cells of the lamina propria and cartilage in this system with the formation of
multinucleated giant cells. Immunofluorescence microscopy showed parain-
fluenza virus type 3 specific antigen not only in the epithelial layer but
also in the lamina propria and cartilage cells* When comparable tissue was
examined by electron microscopy/ typical syncytial formation of epithelial
cells was observed. Virus particles were observed budding off the cells in
the epithelial, lamina propria, and cartilage layers of the trachea.
CONCLUSION
In this paper I compared the changes produced in organized, differenti-
ated epithelium maintained in tracheal organ culture by different infectious
agents (M. pneumoniae, B. pertussis, respiratory syncytial virus, and parain-
fluenza virus type 3). This simple system demonstrated that differentiated,
organized epithelium responds differently to individual infectious agents.
This model has been useful in defining the susceptible cell type in the
organized epithelium of pathogens. Only limited use of this model has been
made in studying the effects of pollutants on respiratory epithelium.
Some of these are: 1) Adalis £t a_l. have examined the effects of cadmium
on epithelium in hamster tracheal organ culture (1). 2) Schiff studied the
effects of nitrogen dioxide on influenza virus infection in hamster tracheal
organ culture (17). 3) Mossman et al. reported on the effects of carbon
particles on tracheal epithelium in organ culture (14).
The in vitro tracheal organ culture model should provide a valuable
screening tool to further explore the injury produced by different pollutants
and to establish the lowest levels of pollutants required to produce
epithelial morphological and metabolic injury. The model should also prove
useful to study synergism between infectious agents and pollutants.
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REFERENCES
1. Adalis D, Gardner DE, Miller FJ, Coffin DL: Toxic effects of cadmium
on ciliary activity using a tracheal ring model system. Environ Res
13:111-120, 1977
2. Collier AM: Pathogenesis of Mycoplasma pneumoniae infection as studied
in the human foetal trachea in organ culture, in: A CIBA Foundation
Symposium on Pathogenic Mycoplasmas (Elliot K, ed). London, Elsevier,
1972, pp 307-320
3. Collier AM: Techniques for establishing tracheal organ cultures.
Tissue Culture Manual 2:333-334, 1976
4. Collier AM, Baseman JB: Organ culture techniques with mycoplasmas.
Ann NY Acad Sci 225:277-289, 1973
5. Collier AM, Clyde WA: Relationships between Mycoplasma pneumoniae
and human respiratory epithelium. Infect Imnmn 3:694-701, 1971
6. Collier AM, Clyde WA, Denny FW: Biologic effects of Mycoplasma
pneumoniae and other mycoplasmas from man on hamster tracheal organ
culture. Proc Soc Exptl Biol Med 132:1153-1158, 1969
7. Collier AM, Clyde WA, Denny FW: Mycoplasma pneumoniae in hamster
tracheal organ culture: Immunofluorescent and electron microscopic
studies. Proc Soc Exptl Biol Med 136:569-573, 1971
3. Collier AM, Peterson LP, Baseman JB: Pathogenesis of infection with
Bordetella pertussis in hamster tracheal organ culture, j infect
Dis 136:196-203, 1977
9. Henderson FW, Ha SC, Collier AM: Pathogenesis of respiratory
syncytial virus infection in ferret and fetal human tracheas in organ
culture. Amer Rev Resp Dis 118:29-37, 1978
10. Hu PC, Collier AM, Baseman JB: Alterations in the metabolism of
hamster trachea in organ culture after infection by virulent
Mycoplasma pneumoniae. Infect Immun 11:704-710, 1975
11. Hu PC, Collier AM, Baseman JB: Interaction of virulent Mycoplasma
pneumoniae with hamster tracheal organ cultures. Infect Immun
14:217-224, 1976
12. Hu PC, Collier AM, Baseman JB: Surface parasitism by Mycoplasma
pneumoniae of respiratory epithelium. J Exptl Med 145:1328-1343,
1977
13. Klein JD, Collier AM: Pathogenesis of human parainfluenza type 3
virus infection in hamster tracheal organ culture. Infect Immun
10:883-888, 1974
14. Mossman BT, Adler KB, Craighead JE: Interaction of carbon particles
with tracheal epithelium in organ culture. Environ Res 16:110-122,
1978
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15. Muse KE, Collier AM, Baseman JB: Scanning electron microscopic study
of hamster tracheal organ cultures infected with Bordetella pertussis»
J Infect Dis 136:768-777, 1977
16. Muse KZ, Powell DA, Collier AM: Mycoplasma pneumoniae in hamster
tracheal organ culture studied by scanning electron microscopy.
Infect Immun 13:229-237, 1976
17• Schiff LJ: Effect of nitrogen dioxide on influenza virus infection
in hamster trachea organ culture. Proc Soc Exptl Biol Med 156:546-549,
1977
141
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DISCUSSION
DR* ADALIS: Or. Collier, have you observed changes in the number of ciliated
cells and the location of the nuclei within the ciliated cells during organ
culture?
OR* COLLIER: The ciliated cells do not replicate and we do see a decrease in
this cell type particularly after three weeks in organ culture* I have not
observed changes in the location of the nuclei within the ciliated cells*
142
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Session IV:
Model Systems of
Respiratory Infectious Diseases
-------
Interaction Between Environmental Pollutants
and Respiratory Infections
R. Ehrlich
NT Research Institute
Chicago, Illinois
During this morning's session we have heard several papers discussing
the mechanisms which are active in protecting the respiratory tract against
damage due to inhalation of airborne contaminants. The mechanisms include the
mucociliary system, phagocytic activity of alveolar macrophages, and the
immunity of the respiratory tract* These mechanisms are indeed the same
which play a significant role in protecting a host against respiratory in-
fections. Thus, the effects of air pollutants on the respiratory tract are
of special importance in relation to respiratory infections.
Health effects studies of air pollutants have been usually concerned with
the causal association between a single pollutant and a disease state. How-
ever, it is well known that frequently more than one factor is responsible
for the occurrence of natural diseases. Therefore, multiple causalities must
be considered in the assessment of biological effects of air pollutants. One
such interaction is depicted by the animal model system which reflects the
exacerbation of bacterial respiratory infections by inhalation of air pollu-
tants. Indeed, changes in the resistance to respiratory infections provide
a highly sensitive experimental model which is with increasing frequency used
in studies of health effects of air pollutants.
The relationship between exposure to air pollutants and the resistance to
respiratory infections has been investigated in our laboratories since 1955.
The gaseous pollutants included in these studies were ozone, nitrogen di-
oxide, sulfur dioxide, and peroxyacetyl nitrate. Particulate pollutants in-
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eluded sulfuric acid, various sulfates and nitrates, and oxides of platinum,
palladium, manganese, and nickel. The studies were designed to determine the
effects of single and multiple exposures to a single pollutant or to pollu-
tant mixtures.
MATERIALS AND METHODS
The experimental protocol used in the studies calls for exposure of a
group of animals, usually 6- to 8-week-old mice, to a pollutant and another
group to clean air. After the exposure the two groups of mice are combined
and within less than an hour challenged by the respiratory route with an
aerosol of the infectious bacteria, either Streptococcus pyogenes or Klebsiella
pneumoniae. The challenge usually lasts for 10 to 15 minutes and results in
the deposition of approximately 750 to 1500 viable bacteria per lung. After
the challenge the mice are kept in an isolated clean-air room for a 14-day
observation period. The changes in resistance to the infection are usually
measured by two parameters, namely, changes in mortality rates and in survival
time when compared to those seen in infected mice exposed to filtered air.
In addition to mortality and survival, several other parameters are measured.
These include clearance rate of inhaled viable bacteria from the lungs, damage
to the respiratory tract tissue as observed by conventional histopathological
and scanning electron microscopic examination, development of lung edema,
activity and function of alveolar macrophages, humoral and cell-mediated
immune responses, and various clinical pathology measurements. To determine
the statistical significance of the differences in responses between animals
exposed to the pollutants and those exposed to clean air the chi-square
analysis, Student's t-test, analysis of variance, and linear regression
analysis were used, as appropriate.
The details of experimental methods used for preparation of the environ-
mental atmospheres, monitoring of the pollutants, infectious challenge with
the bacterial aerosols, and the assay of the various health parameters were
described in previous publications (2-4).
RESULTS
Effects of Gaseous Pollutants
Figure 1 shows mortalities resulting from a single 3-hour exposure of
mice to either nitrogen dioxide or ozone and challenge with Streptococcus
aerosol (3). Statistically significant increases (p .< 0.05) in actual
are seen after the single exposure to either 3.76 mg/m NO or 0.2 mg/m
These data indicated the high sensitivity of this assay system, whereby a
significant physiological response can be attained at concentrations of air
146
-------
i
I
100
90
80
70
60
50
40
30
20
10
0
Significant Mortality Change (p<0.05)
( ) Number Of Mice
(790) (237) '(280K s(256)N
^. .y V y, ^ \_ X. V X_
o
0
1.5
2.82
2.0
3.76
NO,
3.5 5.0 ppm
6.58 9.49 rng/nr
0 0.05 a I 0.5 ppm
0 0.10 0.20 0.98 mg/m3
0,
Figure 1. Mortality rates in mice exposed for 3 hours to O or NO and
challenged with Streptococcus aerosol (3).
pollutants frequently found in urban environments• The mortality rates of
mice exposed to ozone are in a close agreement with those reported by Coffin
et al. (1). In their work, the death rate in control mice was 10.6%, in those
33 3
exposed to 0.14 mg/m 0 20.0%, to 0.2 mg/m O 35.0%, and to 0.98 mg/m O
80.0%. Thus the reproducibility of this animal model system between two dif-
ferent laboratories and during two different time periods is apparent.
In an urban environment the concentrations of air pollutants vary con-
siderably with their rate of emission and meteorological conditions* This,
frequently results in presence of low baseline concentrations of a pollutant
with superimposed short-duration peaks of higher concentration of the same
pollutant. For example, a large portion of nitrogen oxides present in an
urban environment is generated by the vehicular traffic. Morning rush hour
traffic results in high concentrations of nitric oxide and hydrocarbons which
in presence of sunlight are converted to nitrogen dioxide. Subsequent sun-
light irradiation of nitrogen dioxide results in increase in concentrations
of ozone. Human populations therefore can be exposed over extended periods
of time to low concentrations of NO or to low concentrations of this pollu-
tant with superimposed peaks of elevated concentrations of NO and 03 mixtures.
Figure 2 shows the effects of continuous 24 hours/day, 7 days/week exposure
to 0.94 mg/m (0.5 ppm) or 2.82 mg/m (1.5 ppm) NO on the susceptibility to
K. pneumoniae infection (6). The increased mortality rates in mice exposed
147
-------
LU
50
40
30
20
10
Hi Significant Excess Mortality
( ) Number of Animals
0.5 ppm
1.5 ppm
7 14 3O 60 90 ISO 270 360
1/12 1/3
Duration of Continuous N02 Exposure, Days
Figure 2. Excess mortalities in Swiss albino mice after chronic ex-
posures to 0.5 ppm and 1.5 ppm NO and challenged with K. pneumoniae (6).
to 0.94 mg/m NO for 3 months or longer, and to 2.82 mg/m NO for 8 hours
or longer were significant (p _< 0.05). Excess mortality was also present in
mice exposed to nitrogen dioxide for the shorter time periods, but the dif-
ferences were not statistically significant.
To determine the effects of inhalation of nitrogen dioxide and ozone
mixtures, groups of mice were exposed for 3 hours to various concentrations
of each pollutant, a mixture containing the same concentration of the two
pollutants, or to filtered air. The four groups were then simultaneously
challenged with Streptococcus aerosol. The differences between mortality
among mice exposed to the pollutants and the corresponding control mice,
challenged with the bacterial aerosol but exposed to filtered air, are shown
in Table 1 (3). The numbers in parentheses show the mortalities expected to
result from the exposure to the mixtures. The data indicate that the effects
of the 3-hour exposure to the N0_ and O mixture were additive. In most in-
stances the excess mortalities were equivalent or somewhat higher than the sum
induced by exposure to each individual pollutant.
To further simulate the realistic condition of urban population exposure,
groups of mice were exposed continuously 24 hours/day (7 days/week) to a
background concentration of 0.19 mg/m NO with superimposed 3-hour daily
peaks (5 days/week) of either 0.94 mg/m NO or a mixture consisting of 0.94
33 ^
ma/m NO and 0.20 mg/m O . Inasmuch as under true environmental conditions
£ . **
148
-------
Table 1. Actual and Expected Changes in Mortality in Mice Exposed for
3 Hours to Nitrogen Dioxide and Ozone Mixtures and Challenged with
Streptococcus Aerosol
Concentration
N°2
ppm
0
1.5
2.0
3.5
5.0
A
. 3/ nu
mg/mY
0
2
3
6
9
.82
.76
.58
.40
j PP» 0
j/m 0
A
0
- 1.
14.
28.
35.
Mortality change, %
0.05
0.10
0
0
A (E) A
7
3*
2*
7*
5.4
4.6 (3
22.0* (19
(33
(41
7.2
.7) 4.2
17)
.6) 38.5*
.1)
.1
.20
(E)
(5
(21
(35
(42
.5)
.5)
.4)
.9)
0
0
A
28
23
56
68
65
.6*
.9*
.2*
.7*
.3*
.5
.98
(E)
(26.9)
(42.9)
(56.8)
(64.3)
Pollutant mortality - control mortality. A - actual mortality change;
E - expected mortality change.
Significant change in mortality compared to infected mice exposed to
filtered air (*p <_ 0.05).
exposure to the pollutants can be expected also to occur after the inhalation
of the infectious bacteria, groups of mice were exposed to the pollutants
before and for the 14-day period following the infectious challenge. Results
shown in Figure 3 indicate that the combination of extended exposure to the
pollutants and continuation of the exposure after the respiratory challenge
with Streptococcus aerosol was most deleterious. Upon such an exposure se-
quence increased mortality and shortened survival time (see Figure 4) were
seen in almost all exposure regimens. Moreover, these changes in resistance
appeared sooner than when mice were exposed to clean air after the challenge.
Another important factor appeared to be the stress of repeated daily exposure
to the NO and 0. mixture. Irrespective whether or not the mice were reex-
posed to the pollutants or kept in clean air after the infectious challenge,
increased mortality was seen in this group after 2 months or longer exposures*
The reduced mortality rates during the first 3 months of continuous ex-
posure to 0.1 ppm NO with the superimposed daily 3-hour peaks of either
nitrogen dioxide and ozone mixture or 0.5 ppm NO could possibly represent
initial adaptation or a protective effect of the continuous exposure to
nitrogen dioxide. This response occurred only after withdrawal from the
polluted atmosphere and appeared to be negated by continuation of the exposure
to the pollutants either for an additional 3 months or for the 14-day period
after the infectious challenge*
149
-------
1 MONTH
2 MONTH
i 3 MONTH
6 MONTH
»0
20
10
o
V
3
I -20
-J
§
* W
UJ
o
MORTALITY CH
S S S
-10
•20
*><0.03
POLLUTANT
CHALLENGE
CLEAN AIR
h
-—
i
Eg^
If K
i
POLLUTANT
CHALLENGE
LLUTANT
2» hr./d«y: AIR
.9
3 hr./d.y
1
AIR
Figure 3. Changes in mortality from streptococcal pneumonia in mice
subjected to various NO. and 0, exposure regimens (5).
150
-------
5
« 3
1
3 * h
O
-l
-2
-3
1 MONTH
I 2 MONTH
3 MONTH
D
6 MONTH
POLLUTANT
CHALLENGE
POLLUTANT
2» hr./dcy: AIR
3 Nr./
AIR
Figure 4. Changes in mean survival time in mice exposed to various NO,
t
and O_ regimens and challenged with Streptococcus aerosol.
151
-------
Table 2. Mortality and Survival Time of Mice Exposed to 5.0 ppm (13.1 mg/m3)
Sulfur Dioxide and Challenged with Streptococcus Aerosol
Mortality, %
SO exposure
1x3 hr/day
5x3 hr/day
10 x 3 hr/day
15 x 3 hr/day
1 mo x 24 hr/day
2 mo x 24 hr/day
3 mo x 24 hr/day
Air
24.1
27.1
39.6
31.9
9.9
5.0
8.3
so2
26.1
27.1
38.3
30.4
7.8
12.5
10.4
Change
+2.0
0
-1.3
-1.5
-2.1
+7.5*
+2.1
Survival time
Air
12.1
11.6
10.4
11.5
13.4
13.7
13.4
so2
12.0
12.0
10.5
11.5
13.5
13.1
13.3
» day
Change*
-0.1
+0.4
+0.1
0
+0.1
-0.6
-0.1
Pollutant mortality - control mortality.
Significant change compared to infected mice exposed to filtered ait
(*p £ 0.05).
The third gaseous pollutant included in the studies was sulfur dioxide
(SO ). These experiments encompassed single and multiple 3-hour exposures
2 ^
as well as continuous 24 hours/day, 7 days/week exposures to 13.1 mg/m (5
ppm) SO . As seen in Table 2 exposure to SO had no effect on the resistance
to streptococcal pneumonia as measured by changes in mortality rates or sur-
vival time. The one statistically significant increase in mortality seen
after 2 months of continuous exposure appears to be of no practical or true
importance since all the other values obtained in these exposures did not indi-
cate any changes in resistance. Other health effect parameters measured durino
these studies, such as viability and phagocytic activity of macrophages,
development of lung edema, body weights, rectal temperature, and SEN exami-
nation of lungs, trachea, and nasal cavities, also did not show any changes
which could be ascribed to the exposure.
Effects of Particulate Pollutants
Although considerable information is now available on the effects of
photochemical oxidant pollutants on the resistance to infection, there is a
paucity of data pertaining to the effects of particulate pollutants, especially
sulfates or nitrates. The following tables and figures summarize results
more recently obtained in our laboratories. The results further confirm the
utility of this experimental animal model system for studies of air pollutants
as well as environmental contaminants of importance to occupational health.
152
-------
Table 3. Mortality and Survival Rate of Mice Exposed for 3 Hours
to Sulfates and Challenged with Streptococcus Aerosol (4]
ZnSO,
4
go Mortality
mg/m
0
<1.
1.
2.
3.
><•
1
2-2.0
1-3.0
1-4.0
1
D/T
373/1689
125/599
369/813
186/278
-
-
%
22. 1
20.9
45.4*
66.9*
-
-
MST
day
12.1
12.2
9.1*
6.2*
-
-
Zn(NH.
4
)2(so4)
Mortality
D/T
165/756
29/120
112/445
67/192
-
45/48
%
21.8
24.2
25.2
34.9*
-
93.8*
2
MST
day
12.3
11.9
12.0
10.9*
-
4.0*
-------
70i-
60
£150
.r c
0$
r i 40-
I!
«A *-
£ 2 30H
o "3
,*, &•
UJ —
20h-
10
0
__ ZINC SULFATE
I
0.5 1.0 1.5 2.0 2.5
S04, mg/m3
_ ZINC NITRATE
0.5 1.0 1.5 2.0 2.5
N03,mg/m3
Figure 5. Excess mortality in mice exposed for 3 hours to zinc sulfate
and zinc nitrate and challenged with Streptococcus aerosol.
^"-«
o
°.C 4
o o
VI *-
§§
£20
•- 2
II -2
-4
-6
ZINC SULFATE
I
0.5 1.0 1.5 2O 2.5
S04 ,mg/m3
- ZINC NITRATE
0.5 1.0 1.5 2.0 2.5
NO 3 tmg/m3
Figure 6. Changes in survival time of mice exposed for 3 hours to zinc
sulfate and zinc nitrate and challenged with Streptococcus aerosol.
154
-------
Table 4. Mortality and Survival Rate of Mice Exposed for 3 Hours
to Sulfates and Challenged with Streptococcus Aerosol
A1_SO.
2 4
Mortality
mg/m
0
0
1.
2
2
.8
5-1.9
.1
.5
D/T
26/192
2/47
-
18/96
27/48
13
4
18
56
% Change
.5
.3 -9.2
-
.8 +5.3
.3 +42.8*
A1NH . (
4
MST
day
12
13
12
9
.9
.8
-
.6
.2*
S°4>2
Mortality
D/T
19/287
-
11/144
11/96
6/48
6.
-
7.
11.
12.
%
6
6
5
5
Change
-
-
+ 1.0
+4.9
+5.9
MST
day
13.4
-
13.5
13.2
13.0
MST - mean survival time.
Significant change from infected mice exposed to filtered air (*p £ 0.05).
Figure 7. Excess mortality in mice exposed for 3 hours to sulfates and
challenged with Streptococcus aerosol.
in mean survival time. On the other hand, a similar 3-hour exposure to the same
concentration of aluminum ammonium sulfate resulted in somewhat increased
mortality which, however, was not statistically significant.
Figure 7 shows the relationship between the concentration of several
155
-------
Table 5. Maximum Tested Concentrations of Sulfates and Nitrates
Which Did Not Alter the Resistance to Streptococcal Infection
Compound mg/m
Aluminum ammonium sulfate - A1NH.(SO.)_ 2.4
4 42
Ferric ammonium sulfate - Fe(NH.)_SO 2.5
42 4
Ferric sulfate - Fe_(SO ) 2.9
2 42
Sodium sulfate - Na.SO. 4.0
2 4
Ammonium sulfate - (NH.).SO 5.3
42 4
Ammonium bisulfate - NH.HSO 6.7
4 4
Lead nitrate - Pb(N03>2 2.0
Calcium nitrate - Ca(NO3)2 2.8
Sodium nitrate - NaNO 3.1
Potassium nitrate - KNO 4.3
Ammonium nitrate - NH.NO- 4.5
sulfates and excess mortality. The least square lines are based on linear
regression analyses of excess mortalities resulting from a single 3-hour ex-
posure to these compounds and infectious challenge. It is apparent from these
data that cadmium sulfate was most effective and magnesium sulfate least
effective in reducing the resistance to Streptococcal pneumonia. The estimat-
ed concentrations of the compounds which induced 20% excess mortality (ED )
3 3 3 20
were 0.2 mg/m cadmium sulfate, 0.6 mg/m copper sulfate, 1.5 mg/m zinc sul-
fate,. 2.2 mg/m aluminum sulfate, 2.5 mg/m zinc ammonium sulfate, and 3.6
mg/m magnesium sulfate. Estimated on the same basis, exposure to 1.3 mg/m
of zinc nitrate similarly resulted in 20% excess mortality.
Table 5 shows preliminary data on the maximum concentrations of sulfates
and nitrates tested to date which, after a 3-hour exposure, did not result
in significant changes in mortality or survival time in mice challenged with
Streptococcus aerosol•
The data shown in Figure 7 and Table 5 add another dimension to this
animal model system. It is apparent that changes in resistance to respiratory
infection can be used to rank the effects of inhalation of pollutants having
a related chemical structure. In all experiments the exposure conditions,
including the particle size of the aerosol, were kept identical. Thus, the
differences in response can only be ascribed to the toxicity of the com-
pounds .
156
-------
§^ Significant Excess Mortality
( ) Number of Animals
Squirrel
Mice Hamsters Monkey
too
90
eo
70
60
50
40
30
20
10
0
NO2 Concentration Range,ppm
Figure 8* Effects of 2-hour acute exposure to NO and challenge with
K. pneumoniae on mortality of mice/ hamsters, and squirrel monkeys (6).
EXPERIMENTAL VARIABLES
Several experimental variables can affect the responses in this animal
model system. They include the animal host, infectious agent, and exposure
sequences.
Animals
The selection of the animal host in this model system is of major impor-
tance. Figure 8 shows the response of mice, hamsters, and squirrel monkeys
exposed to nitrogen dioxide and challenged with K. pneumoniae aerosol (6).
The figure shows the percent mortality in the three species of animals and the
numbers of animals used to obtain these means. Within each animal species,
the mortality data were subdivided into three groups. The first is mortality
of the control group of animals challenged with the infectious agent but
exposed to filtered air. The second group represents mean mortality of
animals challenged with the infectious agent and exposed to concentrations
of nitrogen dioxide which did not significantly enhance the mortality. The
157
-------
Table 6. Mortality in Different Mouse Strains after Exposure
to 5 ppm NO2 and Challenge with Klebsiella Aerosol
Air NO
Strain
BDF (black)
C57BL (black)
Swiss (albino)
BALE (albino)
D/T
31/120
24/70
164/390
48/100
%
oe oa
f. o . o
ab
34.3
42.1b
48. Ob
D/T
40/120
36/70
262/390
72/100
%
33.3
51.4
67.2
72.0
Change
%
7.5a
h*
17. 1b
c*
25.1
24. 0C*
Means in a column with the same superscript are not significantly dif-
ferent (p £ 0.05).
Significant change in mortality compared to corresponding infected mice
exposed to filtered air (*p £ 0.05),
third group is the mean mortality of animals at nitrogen dioxide concen-
trations at which significant enhancement in mortality was observed.
The mortality of control animals indicates the range of natural resis-
tance to bacterial pneumonia in different species of animals. The respective
death rates in control mice, hamsters, and monkeys were 41%, 11%, and 0%.
The respiratory dose for monkeys and hamsters was approximately 10 organisms,
a dose of the infectious agent which repeatedly killed the Swiss albino mice.
Thus, the higher concentrations of nitrogen dioxide necessary to induce a
significant increase in mortality in monkeys and hamsters can, at least in
part, be ascribed to their innate resistance to this infectious agent.
Differences in natural resistance to bacterial pneumonia were also seen
in studies of the effects of nitrogen dioxide in four strains of mice and are
shown in Table 6. The BDF mice showed significantly higher natural resis-
tance to the infection than the other strains. However, excess mortalities
ranging from 8 to 25% were observed in all four strains of mice after 3-hour
exposure to 5 ppm of NO. and the infectious challenge.
Infectious Agent
Table 7 shows the effects of a single 2- or 3-hour exposure to nitrogen
dioxide on the resistance of Swiss albino mice to bacterial pneumonia induced
by inhalation of K. pneumoniae or £5. pyogenes aerosols. A direct comparison
between the two groups of results cannot be made because of the time interval
separating the two studies. The work with Klebsiella was reported in T966
(2), the one with Streptococcus in 1977 (3). Nevertheless, it is apparent
that the mice were somewhat more susceptible to the Klebsiella than to the
Streptococcus infection.
158
-------
Table 7. Effect of a Single Exposure to Nitrogen Dioxide on Mortality
of Mice Challenged with Klebsiella or Streptococcus Aerosol
NO Concn.
*
-------
Exposure Sequence
The sequence and interval between the exposure to the pollutants and
challenge with the infectious agent are yet another factor of importance.
In all of the studies discussed previously, the experimental plan required
exposure to the pollutants followed within less than one hour by the in-
fectious challenge. Figure 9 shows that, at least for the short-term exposures
to nitrogen dioxide, the extension of the interval between exposure and chal-
lenge allows a recovery from the effects of the pollutants.
CONCLUSIONS
In discussing the importance of this animal model system in studies of
health effects of air pollutants two aspects must be considered. First is
the value of the model as a highly sensitive indicator of in vivo effects.
The changes in resistance to respiratory infection can be seen at concen-
trations of oxidant and other pollutants markedly lower than those detected
by any other in vivo method. The model depicts the responses of the respira-
tory system and reflects the overall damage to the pulmonary defense mechanisms,
Therefore, it indicates that basic defense mechanisms of the lungs have been
impaired. A major part of this impairment appears to result from the damage
to the alveolar macrophage system and the ensuing inability to remove the in-
haled microorganisms from the lungs.
The second aspect is that this model may simulate a situation which
could occur in man. Decreased resistance to respiratory infections as a
consequence of exposure to air pollutants was observed in mice, hamsters, and
monkeys. In spite of the functional and anatomical differences of the res-
piratory tract and the differences in innate and acquired immunity between
man and these animal species, under the proper set of circumstances man could
be expected to respond to these stresses in a very similar manner. The ne-
cessary components for the occurrence in man are the presence of a suffi-
ciently high concentration of pollutants and the presence of an infectious
microorganism capable of invading and colonizing in the human host, exploit-
ing the state of reduced resistance.
160
-------
REFERENCES
Coffin DL, Bloomer EJ, Gardner DE, Holzman R: Effect of air pollution
on alteration of susceptibility to pulmonary infection. Proc 3rd Ann
Conf Atmospheric Contamin in Confined Space. Dayton, OH, 1967, pp 71-
80
Ehrlich R: Effect of nitrogen dioxide on resistance to respiratory in-
fection. Bacteriol Rev 30:604-614, 1966
Ehrlich R, Findlay JC, Fenters JD, Gardner DE: Health effects of short-
term inhalation of nitrogen dioxide and ozone mixtures. Environ Res
14:223-231, 1977
Ehrlich R, Findlay JC, Gardner DE: Susceptibility to bacterial pneumonia
of animals exposed to sulfates. Toxicol Letters 1:325-330, 1978
Ehrlich R, Findlay JC, Gardner DE: Effects of repeated exposures to
peak concentrations of NO. and 0 on resistance to streptococcal pneumonia.
J Tox Environ Health (in press)
Ehrlich R, Henry MC, Fenters JD: Influence of nitrogen dioxide on resis-
tance to respiratory infections. AEC Symposium Series 18, 1970, pp
243-257
161
-------
DISCUSSION
DR. DREW: What was the basis for the selection of 5 ppm for SO ? I noted
that in several of your experiments you were looking at 5 ppm ozone and SO ,
knowing that the toxicities of these compounds are different.
DR. EHRLICH: It was just an arbitrary selection which was in part guided by
the SO limits in the atmosphere as suggested by the EPA. It was not a
scientific or technical selection, but simply a practical selection to de-
termine the effects of SO at this concentration.
DR. DREW: In that last slide it appeared that there is no dose response,
that there is a very flat dose response essentially, once you get above 3.5
ppm of nitrogen dioxide. Regardless of what the concentration was, the per-
cent in mortality did not increase any further.
DR. EHRLICH: The slide showed excess mortalities rather than actual death
rates.
DR. DREW: I understand, but they were all the same after 3.5 ppm, at least
in the first column of that last slide.
DR. EHRLICH: This is rather old data obtained with K. pneumoniae. We found
that with K. pneumoniae we could not obtain a meaningful dose response,
inasmuch as this agent is highly infective to mice, and mice appear to be
highly susceptible. One of the reasons we changed to Streptococcus sp« was
because of this. It1s a much more reproducible infectious system providing
a better dose-response mortality rate.
DR. DAWSON: One of the problems that1s been raised in connection with the
preparation extrapolating to a human case is the number of organisms that
are delivered. Can you comment on that?
DR. EHRLICH: I never suggested to use this model for direct extrapolation
to man. On the other hand, I feel strongly that it's a highly sensitive
model, which presents a total biological response reflecting the various
defense mechanisms involved. I think that it is the only model which, in a
162
-------
way, puts all those things together. As far as the human infections are con-
cerned/ I think they are different/ in terms of man's susceptibility to
infection* Thus I don1t think we could expect to see the same type of response
in humans. Your criticism is valid only if one just wants to take the
laboratory information and extrapolate it directly into man* I am presenting
it as a highly sensitive biological model system most appropriate to studies
of health effects of pollutants. The data on SO and some of the sulfates
and nitrates are important and reinforce the validity of this model system
because they demonstrate that the model does not respond to every pollutant.
It certainly responds only to compounds which affect the resistance to in-
fection.
163
-------
Experimental Infection of the Respiratory Tract
with Mycoplasma pneumonias
E. P. C. Hu, J. M. Kirtz, D. E. Gardner, and *D. A. Powell
U. S. Environmental Protection Agency
Research Triangle Park, North Carolina
"University of North Carolina
Chapel Hill, North Carolina
Mycoplaama pneumoniae is one of the most common pathogens of the human
respiratory tract. Infection with this agent is the leading cause of acute
pneumonitis in high contact environments such as college campuses and military
bases, as well as being a common cause of respiratory illness in children (6).
Illnesses associated with M. pneumoniae infections range from mild upper respira-
tory infection, through tracheobronchitis, to acute pneumonia (2). These agents
have been associated with exacerbations of chronic bronchitis in adults with
chronic obstructive pulmonary disease and with severe illnesses in compromised
hosts such as patients with B-cell immunodeficiency (8) or sickle cell anemia
(19). Because of the prevalence and seriousness of jH. pneumoniae associated
respiratory illness, it is important to determine the effects of air pollution
on the interaction between this organism and host respiratory tissues.
Fortunately, the Golden Syrian hamster provides an established animal model for
M. pneumoniae infection (5). Ihe purpose of this paper is to review briefly the
features which have made this animal model so ideal for studying M. pneumoniae
pathogenesis, to describe a recently developed method for studying this model
using aerosolized, radioactively labeled M* pneumoniae, and finally, to de-
scribe some observations on the deposition and clearance of aerosolized myco-
plasmas within hamster lungs.
165
-------
REVIEW OF ANIMAL MODEL MYCOPLASMA PNEUMONIAS INFECTION
Studies on the pathogenesis of M. pneumoniae infection in the Golden
Syrian hamster have been conducted both in vitro and in vivo. One main thrust
of the ^n vitro work has involved the study of isolated tracheal rings infected
with M. pneumoniae (3, 4). Through these studies it has been established that
M. pneumoniae is a filamentous organism/ lacking a cell wall, but possessing a
unique tip structure responsible for its attachment to its primary host target
tissue, ciliated respiratory epithelium (4). This attachment causes a variety
of adverse effects on the ciliated cell, including alterations in cellular
metabolism (10, 11), changes in cellular ultrastructural morphology (4), and
eventually, disorganization of ciliary activity and loss of cilia from the
epithelial surface (15).
Additionally, examination of the in vitro interaction between M. pneumoniae
and isolated alveolar macrophages (16, 17) has shown that M. pneumoniae. like
several other mycoplasma species (14), has the capacity to resist phagocytosis
unless opsonized with specific antibody or complement. This feature exists de-
spite the absence of an identifiable capsule or cell wall, the key components
essential for bacterial antiphagocytic capacity.
Studies of the effects of M. pneumoniae infection in the Golden Syrian
hamster began with the work of Dajani et al. (5). Following intranasal inocu-
lation of 0.2 ml of virulent organisms, the hamster develops a bronchopneumonia
which is very similar to hunan disease. At the peak of infection, the histologic
picture consists of scattered areas of peribronchial and to a lesser extent
perivascular round cell infiltrate. The intraluminal exudate which develops is
composed of mononuclear and polymorphonuclear phagocytes (1). Pulmonary disease
is evident by the third to seventh post-infection day, peaks on the tenth to
fourteenth day, and begins to resolve by the third week of infection. All
animals ultimately recover from the primary infection with a return to normal
of the lung histology and complete clearance of the organisms. The development
of IgG antibodies, as measured by complement fixation and growth inhibition,
follows a time course similar to that of hunan disease. When animals have re-
covered from a primary infection, they are left with a long-lasting relative
immunity against reinfection. While the precise mediators of this immunity re-
main undefined, it is known that stimulation of local pulmonary immune mechanism a
is more important than stimulation of systemic immunity (7).
This concept was substantiated following the recent article by Jemski
et al. (13), outlining the role of aerosol particle size and site of deposition
of M. pneumoniae on the development of respiratory disease and immunity in
hamsters. In their report, representing the only published account of an animal
model aerosol infection by M. pneumoniae, the authors demonstrated that the re-
sponse of hamsters to M. pneumoniae infection was determined by the site of
166
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deposition of organisms and by the quantity of organisms introduced directly into
the lower respiratory tract. Hie variables which were studied included tj»
pneumoniae delivered as a large particle aerosol, with a mass median diameter
of 8 urn, a small particle aerosol with a mass median diameter of 2.3 urn, or
intranasal inoculation of 200 ul, 20 yl, or 2 yl of organisms. Only the large
volume intranasal inoculum and the small particle aerosol resulted in deposition
of mycoplasmas into the lungs. The other three routes of infection colonized
only the upper airways. The most significant disease, as measured by the per-
centage of animals with pneumonia and overall mean pneumonia score, was found
in animals with the large volume intranasal inoculum. Animals receiving small
volumes but containing the same number of mycoplasmas, administered only to the
upper airway, developed little or no pneumonia. This observation suggested to
these authors that the development of disease in the hamster may depend largely
on the initial number of organisms delivered directly to the lower respiratory
tract. Thus, it would seem that initial deposition and rate of organism clear-
ance may be very important determinants of disease development.
Similarly, the initial location of organism deposition within the respira-
tory tract was critical in the generation of a protective immune response. In
this segment of the study, all animals were allowed to recover 4-8 weeks follow-
ing primary infection and then were challenged with a large volume inoculum of
intranasal organisms. While any route of infection conferred at least some pro-
tection against disease development, maximal protective immunity was seen only
in those animals given initial exposure which provided inoculation of the lower
respiratory tract—that is, either by large volume intranasal inoculation or by
small particle aerosol. Thus, again, initial deposition and pulmonary clearance
would seem to be crucial events in determining the generation of protective
immunity.
Aerosol Chamber Development
With these data in hand, we decided that the evaluation of air pollutant
effects on disease and immune response to M. pnevmoniae infection in the hamster
must be studied such that the initial deposition and clearance could reproducibly
be evaluated. To accomplish this, an aerosol exposure system was developed in
which viable mycoplasmas, tagged with a radioactive marker, could safely and
efficiently be delivered in an aerosol with a particle mass diameter that would
assure deposition deep within the lower respiratory tract. One of the main
criteria considered in designing the aerosol inhalation chamber was to be able
to have a high concentration of radioactively labeled M. pneumoniae introduced
into the lungs to make deposition and clearance studies possible. Thus, we de-
signed a chamber of small volume with a low total air flow rate to maximize the
organisms' air concentration during a 25-minute exposure period. Figure 1 shows
167
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VALVE 8
PRESSURE REGULATOR 9
SILICA GEL SCRUBBER 10
CHARCOAL SCRUBBER 11
CANISTER HEPA FILTER 12
ROTAMETER 13
PRESSURE GAUGE 14
SVRINGE IS
NEBULIZER 16
MIXING COLUMN 17
EXPOSURE CHAMBER 18
IMPACTOR 19
SAMPLING ORIFICE 20
SAMPLING PUMP
CONTAINMENT CHAMBER
HEPA FILTER
ORIFICE
FUME SCRUBBER
EXHAUST BLOWER
TRANSFORMER
O>
CO
CD- CC CO
COMPRESSED
AIR
TO
EXHAUST
H2O DRAINS
Figure 1. Schematic diagram of Mycoplasma pneumoniae aerosol inhalation chamber.
-------
a schematic diagram of the inhalation chamber. Compressed air is filtered and
passed into rotometers which control air flow rate into the nebulizer and the
drying column. Air enters this column through two tangential inlets causing a
turbulence which causes evaporation of water from the nebulized aerosol reducing
the particle size* From the drying column, the combined air streams enter the
center of the chamber and then flow into the two side exposure sections where
inhalation occurs.
Since M. pneumoniae are human pathogens and were radioactive in many ex-
periments, stringent safety precautions were taken to filter all exhaust air
and provide adequate containment of the aerosolized organisms. This was accom-
plished by housing the nebulizer, mixing column, and exposure chamber inside a
stainless steel Rochester chamber, and venting all exhaust air through several
HEPA particle filters. Sampling of the aerosol particle size was performed
with a seven-stage low flow rate cascade Lovelace impactor.
To prepare radioactively labeled M. pneumoniae for aerosolization, organisms
were grown on the inner surface of glass bottles, labeled with tritiated oleic
acid, washed extensively to remove unincorporated radiolabel, and scraped into
M199 containing 1% horse serum. During a single run, 4 ml of suspended organisms
containing approximately 10 colony-forming units (CPU) were aerosolized over
a 25-minute period at 21°C and 27% relative humidity. Following this, 4
ml of saline were aerosolized for an additional 25 minutes to complete the
total exposure. To attain the desired particle size, the nebulizer flow rate
and dilution air flow rate were 5.5 and 11.5, respectively.
Table 1 shows the high degree of particle size reproducibility accomplished
with this chamber. Here, results of experiments occurring over a one-year period
are presented to demonstrate the consistency of the count median aerodynamic
diameter. The latter value, reflecting the particle size reaching the hamster
lung/ is within the size range which would assure deposition within the distal
airways of the lower respiratory tract.
The correlation between the radioactive concentration in the nebulizer
solution and the radioactive counts measured within the central aerosol chamber
and found in the hamsters' lungs at the end of aerosol exposure is shown in Table
2. As shown by the regression analyses at the bottom of the table, there was
greater than 90% correlation between the concentration of mycoplasmas within
the chamber air and that found in hamster lung*
Of potentially more importance, however, was the similarity of deposition
for all animals during each aerosol exposure. If any ports were found to pro-
vide variable exposure or exposure consistently above or below the mean, the
value of this exposure chamber for comparative studies would be questionable.
Figure 2 demonstrates graphically the excellent correlation of exposure in all
eight animals studied in each of four successive experiments. These data are
tabulated in Table 3 where it can be seen that the coefficient of variation of
169
-------
Table 1. Size Distribution Data for M. pneumoniae Aerosol
in 8-Port Chamber
Exposure date
08-19-77
10-11-77
10-20-77
10-25-77
12-01-78
01-17-78
01-19-78
01-21-78
08-04-78
08-07-78
08-14-78
08-16-78
Average + S.D.
Count median
aerodynamic
diameter (CHAD)
1.8
1.6
1.7
1.7
1.7
1.4
1.4
1.3
1.5
1.5
1.6
1.6
1.57 ± 0.15
Geometric standard
deviation (erg)
1.5
1.4
1.4
1.4
1.5
1.4
1.5
1.4
1.4
1.4
1.4
1.4
1.42 + 0.05
Mass median
aerodynamic
diameter (MMAD)
2.9
2.2
2.5
2.5
2.7
2.0
2.1
1.9
2.2
2.1
2.2
2.3
2.3 + 0.3
the average cpm/mg of dry lung weight for the eight animals in any one ex-
periment varied from 0.14 to 0.19.
One important question in an aerosol system such as this is the extent
to which aerosolized organisms remain viable. To determine how many organisms
were killed by the aerosolization process, the percentage of mycoplasmas
found to be viable upon deposition in the hamster lung immediately at the end
of aerosol exposure was multiplied by the specific activity (CPU/cpm) of the
initial aerosol suspension to determine the theoretical number of viable or-
ganisms delivered to the respiratory tract. The CFU of organisms recovered
divided by this theoretical number revealed the approximate actual survival
rate which averaged 55%.
DEPOSITION AND CLEARANCE OF MYCOPLASMA PNEUMONIAE IN THE HAMSTER LUNG
Following the achievement of technical reproducibility in the mycoplasma
aerosol chamber, clearance studies were undertaken in nonimmune hamsters.
In these studies, pulmonary clearance, as measured by the removal of radio-
actively tagged mycoplasmas from the lung, was compared with pulmonary myco-
plasmacidal capacity, as measured by the rate of decline of mycoplasma viability
within the lung. One aim in these studies was to compare the clearance and
170
-------
Table 2. Comparison of Mycoplasma pneumoniae Concentrations in
Nebulizer Solution,
Air Sample, and Hamster Lungs
Nebulizer solution Air sample
concentration,
Exposure date cpm/10
8-19-77
8-22-77
8-31-77
9-08-77
10-11-77
10-13-77
10-20-77
10-25-77
12-01-77
01-17-78
01-19-78
01-21-78
08-04-78
08-07-78
08-14-78
08-16-78
Regression
X
vs. Y
Nebulizer
2.
0.
2.
2.
1.
1.
1.
2.
0.
1.
1.
0.
3.
5 x
85 x
4 x
8 x
2 x
2 x
2 x
2 x
94 x
2 x
0 x
ul
1
1
1
o5
105
o5
o5
1.05
1
1
1
1
1
78 x
6 x
4.5 x
3.0 x
3.
2 x
1
1
1
1
o5
o5
o5
105
o5
o5
105
o5
o5
o5
o5
Analysis
solution
A
-995
Average
hamster
concentration, lung content
cpna/ml
15.
4.
13.
32.
8.
7.
•i
13.
••
12.
6.
41.
air
6
4
6
1
9
9
4
6
9
5
39.6
, 26.7
at To, cpm/lung
6,
2,
6,
13,
4,
4,
2,
6,
6,
5,
5,
4,
15,
241
358
097
135
453
210
403
138
807
424
661
815
937
20,283
15,581
22,010
Y - A + BX
B
0.0483
r *
correlation coefficient
2
0.76
vs. lung content
Nebulizer solution -3.08
vs. air concentration
Air concentration 719
vs. lung content
1.00 x 10
431
-4
0.84
0.92
viability curves with similar data published on animal models infected with
aerosolized bacteria (9, 18). Prior studies have examined in vivo clearance
of radioactively labeled bacteria administered to animal lungs by aerosol ex-
posures which provide particles of approximately 2 um mass median diameter.
Under these circumstances, deposition occurs primarily at the alveolar level and
171
-------
xjix
O
3.0
2.0
1.0
0.8
0.6
0.4
0.2
0.1
O 8-4-78
A 8-7-78
tf 8-14-78
O 8-16-78
* X - CPM/mg DRY LUNG
X - AVERAGE OF Xs FOR 8 ANIMALS
I
I
I
3456
CHAMBER PORT NUMBER
8
Figure 2. Chamber position vs. normalized deposition of radioactively
labeled Mycoplasma pneumoniae.
Table 3. Specific Activity of Radioactively Labeled
Mvcoplaana pneumoniae Deposited in Hamster Lungs
Average cpm
Exposure date for 8 animals
Average cpm/mg
dry lung for 8
animals
Standard
deviation of
individual
cpm/mg dry
lung
Coefficient
of
variation
8-04-78
8-07-78
8-14-78
8-16-78
15,937
20,283
15,581
22,010
122.5
166.9
119.0
162.4
19.8
32.0
22.6
22.2
0.16
0.19
0.19
0.14
172
-------
provides the capacity to analyze simultaneously physical removal and viability
of the intra-alveolar organisms.
With most bacteria, the rate of transport or physical clearance of radio-
isotope out of the lung occurs more slowly than the decline in the number of
viable bacteria. This has led to the theory that physical removal of bacteria
plays a relatively minor role in the early defense of the lung and that a more
important pulmonary defense mechanism involves alveolar macrophage engulfment
and intracellular killing of the inhaled bacteria (12).
Figure 3 shows the viability of M. pneumoniae deposited in the total lung
tissue. There was little decline in the viability of M. pneumoniae during the
first eight hours after aerosolization. Only after this time period was there
a decline in viability such that by 24 hours the viable mycoplasmas in the lung
were reduced by 90%. Thus, during the initial eight hours, when macrophage en-
gulfment and intracellular killing play the chief role in intrapulmonary removal
of inhaled bacteria, little change in M. pneumoniae viability was noted. However,
the number of viable organisms began to increase significantly by 48 hours
TIME AFTER INFECTION
Figure 3. Clearance of viable Mycoplasma pneumoniae from hamster lungs
following aerosol infection.
173
-------
100
5
LU
CC
H-
>
g
a
Z
UU
U
QC
LU
a.
10 12
TIME, hours
24
48
Figure 4. Clearance curve of radioactivity deposited as
Labeled Mycoplasma pneumonias•
H-oleic acid-
following the initial infection which indicates that the rate of replication
was faster than the rate of clearance by host defense mechanisms* At 10 days
post-infection, the number of viable mycoplasmas reached a plateau of 10 organ-
isms per lung, and then started to decline by six weeks after the infection.
Histological sections taken from animals 20 days post-infection showed typical
pathology including extensive peribronchial infiltration*
Figure 4 shows the clearance rate of radioisotope, which was found to be
significantly more rapid than the decline in mycoplasma viability, most likely
due to the rapid mucociliary clearance of nonattaching, inactivated aerosolized
organisms. There was no point on the curve at which the percent of remaining
radioactivity was greater than the percent of viable organisms. From these data
then, it would seem that alveolar macrophages in nonimmune hamsters play a limit«»,»
role in the early pulmonary clearance of aerosolized M. pneumoniae, and the
interaction of M. pneumoniae and alveolar macrophages noted from in vitro studi
is most likely representative of the relationship occurring Ln vivo, on the
other hand, the role of mucociliary clearance would seem to be a more important
nonspecific host defense mechanism in minimizing the initial number of viable
organisms capable of establishing residence within the host lung tissues.
174
-------
With the establishment of this technique for the reproducible aerosolization
of viable, radiolabeled M. pneumoniae we are encouraged that many features of
this important respiratory infection can be examined in the hamster animal model
and the adverse effects of air pollution exposure determined. In addition to
the ready measurement of deposition and clearance with this system, we have demon-
strated that aerosol infected hamsters eventually develop histologic changes and
serologic responses comparable to animals infected by 0.2 ml intranasal inocula-
tion. Thus, air pollutant effects on the development of this form of chronic
pneumonia, as well as on the generation of protective immune response, will also
be among our future research objectives.
REFERENCES
1. Clyde WA Jr: Models of Mycoplasma pneumoniae infection. J Infect Dis
127:569-572, 1973
2. Clyde WA Jr: Mycoplasma pneumonia. In: Medical Care of the Adolescent
(Gallagher JR, ed), 3rd ed. New York, Appleton-Century-Crofts, 1976
3. Collier AM, Clyde WA Jr, Denny FW: Biologic effects of Mycoplasma
pneumoniae and other mycoplasmas from man on hamster tracheal organ culture.
Proc Soc Exp Biol Med 132:1153, 1969
4. Collier AM, Clyde WA Jr, Denny FW: Mycoplasma pneumoniae in hamster
tracheal organ culture: Immunofluorescence and electron microscopic studies.
Proc Soc Exp Biol Med 136:569, 1971
5. Dajani AS, Clyde WA Jr, Denny FW: Experimental infection with Mycoplasma
pneumoniae (Eaton's agent). J Exp Med 121:1071, 1965
6. Denny FW, Clyde WA Jr, Glezen WP: Mycoplasma pneumoniae disease: Clinical
spectrum, pathophysiology, epidemiology, and control. J Infect Dis 123:74,
1971
7. Fernald GW, Clyde WA Jr: Pulmonary immune mechanisms in Mycoplasma
pneumoniae disease. In: The Immunologic Reactions of the Lung {Kirkpatrick
CX, Reynolds HRY, eds). New York, Marcel Dekker, 1976
8. Foy HM, Ochs H, Davis SD, et al: Mycoplasma pneumoniae infection in patients
with immunodeficiency syndromes: Report of four cases. J Infect Dis 127:388,
1973
9. Green GM: integrated defense mechanisms in models of chronic pulmonary
disease. Arch Int Med Sym 6:500, 1970
10. Hu PC, Collier AM, Baseman JB: Alterations in the metabolism of hamster
tracheas in organ culture after infection by virulent Mycoplasma pneumoniae.
Infect Immun 11:704, 1975
175
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11. Hu PC, Collier AM, Baseman JB: Further studies on the interaction of
virulent Mycoplasma pneumoniae with hamster tracheal organ culture. Infect
Immun 14:217, 1976
12. Jakab GJ, Green GM: Regional defense mechanisms of the guinea pig lung.
Amer Rev Resp Dis 107:776, 1973
13. Jemski JV, Hetsko CM, Helms CM, et al: Immunoprophylaxis of experimental
Mycoplasma pneumoniae disease: Effect of aerosol particle size and site
of deposition of M. pneumoniae on the pattern of respiratory infection,
disease, and immunity in hamsters. Infect Immun 16:93, 1977
14. Jones TC, Minick CR, Yang L: Attachment and ingestion of Mycoplasma
pulmonis by mouse peritoneal macrophages—scanning electron microscopic
observations. Am J Pathol 87:347, 1977
15. Muse "KE, Powell DA, Collier AM: Mycoplasma pneumoniae in hamster tracheal
organ culture studied by scanning electron microscopy. Infect Immun 13:229
1976
16. Powell DA, Clyde WA Jr: Opsonin-reversible resistance of Mycoplasma
pneumoniae to in vitro phagocytosis by alveolar macrophages. Infect Immun
11:540, 1975
17. Powell DA, Muse KB: Scanning electron microscopy of guinea pig alveolar
macrophages: In vitro phagocytosis of Mycoplasma pneumoniae. Lab Invest
37:535, 1977
18. Rylander R: Studies of lung defense to infection in inhalation toxicology.
Arch Int Med Sym 6:496, 1970
19. Shulman ST, Bartlett J, Clyde WA Jr, Ayoub EM: The unusual severity of
mycoplasmal pneumonia in children with sickle-cell disease. New Engl j
287:164, 1972
176
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DISCUSSION
DR. JAKAB: I'd like to propose another reason why the curves differed. First
of all/ you used a different tracer than we did, and second of all, your
animals are restrained, they incubate in the aerosol chamber. This morning
Joe Brain presented some very nice studies showing that if you have a shallow
breathing animal—a rabbit is a shallow breathing animal—whatever was de-
posited was primarily in the bronchi, whereas with deep breathing it would
go in the lung parenchyma. I was wondering whether restraining affected
the breathing pattern that Joe showed, and therefore your animals inhaled
mycoplasma and that position was preferentially in the bronchi, and therefore
(inaudible) did not reach the alveoli.
DR. POWELL: It's certainly a possibility. We have autoradiographs, which
we're waiting to develop, to determine the site of deposition in the alveoli.
DR. HU: We also did some experiments not shown here where we actually take
out the individual lobes and determine radioactivity. We see that there is a
constant, even distribution of radioactivity in all parts of the lung.
DR. DAWSON: I'm still worried about this question of extrapolation to man.
You started off by saying that hamsters were extremely good experimental
models, and I wonder why that is. What is so good about hamsters? They're
extremely resistant compared to squirrel monkeys. Are these levels of
exposure of organisms that you were using like those man might get in his
environment?
DR. POWELL: Let me start by answering the first question. I think that a
hamster is fortuitous in the sense that it does take a human pathogen.
Relatively few mycoplasmas are required to be deposited in the lung to develop
a pneumonia along a similar time course thought to be due to the incubation
in humans, that is, two weeks. We have a limited knowledge of histology
of mycoplasma disease in humans because it's rarely a fatal disease. When
compared to the pathology in hamsters, it's basically the same kind of his-
tologic picture, that is, of peribronchial round cell pneumonia. Serologi-
cally, the time course is almost identical to that in the human. It mimics
human disease with the exception that the hamster does not become overtly ill.
In that sense, it provides a very good model to manipulate.
177
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Is it artificial to be aerosolizing? Is it more appropriate to be put-
ting the stuff right in the nose? Do all infections occur with a primary
upper respiratory infection that leads then only to the lower respiratory
tract? I don't think that we really know the answer to that, to be quite
honest. In fact, the paper that I cited regarding infection and immunity
would say that at least in the hamster that is not the case. If you deposit
large numbers of organisms in a very small dose, they stay limited to the upper
airway, they do not progress to involve the lower respiratory tract. There are
some fascinating fortuitous studies in humans in dental clinics where people
grind the teeth of individuals with mycoplasma infection. This generated an
outbreak of M. pneumoniae disease in people working in the clinic, suggesting
that aerosol infection is probably the most efficient way to infect the hunan
host as well. In that sense this may not be all that far from the way much
of human disease originates.
DR. DAWSON: How about numbers of organisms?
DR. POWELL: I don't know what kind of numbers of organisms it takes to cause
a primary infection in the hunan. We do know that infections occur repeatedly
in humans, and of those repeated infections few probably cause disease. Whether
many of those occur at a subliminal dose or whether they require a certain
number of infections to generate Immune responses leading to disease just
is not known. Clearly, there are a lot of infections that are subclinical
or cause very mild upper respiratory illness.
DR. GARDNER: I might mention in the bacterial model system that it takes only
between 400 and 4,000 microorganisms in the lung at zero time to show the
enhancement of mortality.
PARTICIPANT: In mice?
DR. GARDNER: In mice.
DR. COFFIN: In any species you have to have enough organisms to cause the
disease. If you can then do some other manipulations to make them more
susceptible, a greater number of animals should have that disease regardless
of the number of organisms required. For instance, in Streptococcus of
human variety, it is impossible to infect a mouse by inhalation. You can show
that a mouse becomes more susceptible to the human Streptococcus, but it takes
more of the secondary problem, the ozone or whatever you're using, because
the mouse is not as susceptible to human Streptococci* What you have here
178
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is a model which mimics disease in another species. I think'that the actual
number of organisms doesn't make all that much difference.
It was interesting to see your data, which had to do with the relative
physical clearance in the bacterial system and in the mycoplasma system, which
was striking. It seemed to me that there could be several mechanisms that
might explain that. One is that for some reason or another, the mycoplasms
were not going down into the deep lung. Most of them were being implanted on
the ciliated epithelivm, therefore they would disappear faster. Would it be
explained by the difference in which the macrophages handled these organisms?
We know that the phagocyte handles bacteria very quickly and efficiently.
What is the status of the phagocytosis of mycoplasms by macrophages?
DR. POWELL: Both your points are well taken. The first point was raised by
Dr. Jakab, and we need to prove that the particle size of our aerosol gets
to the alveolar level. We know that radioactivity is evenly distributed through
the whole lung. That doesn't disprove that particles may be only going to the
bronchiolar level. We've got to prove that they, in fact, reach the alveoli,
which we're attempting to do. In terms of the macrophage, the only data that
exist are the data that I showed you, and that's been corroborated by other
people. Dr. Bredt in Germany has similarly shown that M. pneumoniae as well
as other mycoplasma species are not rapidly phagocytized, despite not having a
capsule or a cell wall. The mechanism by which they tend to avoid this
phagocytosis has not been delineated, but clearly once they're opsonized
with a specific antibody or complement they are engulfed.
179
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Experimental Models for Study of Common
Respiratory Viruses
W. A. Clyde, Jr.
University of North Carolina
Chapel Hill, North Carolina
Many epidemiologic investigations have shown that there is excess mortal-
ity (10) and respiratory disease morbidity (16) in areas of high atmospheric
pollution. These adverse health effects can be categorized in part on the
basis of the toxic substance(s) involved; however, the mechanisms through
which the pollutants exert their effects are poorly understood. While elimi-
nation of cause in a cause-and-effect relationship can be curative, this is
not always possible or practical. A more complete understanding of the effect
limb can suggest other approaches to modulate the end result of the cause-and-
effect equation for benefit of the human host.
It is recognized that infections with common viruses are responsible for
the vast majority of acute respiratory diseases. The viral agents involved
are similar in nature and distribution in areas of both low and high atmospheric
pollution. It follows/ therefore/ that an interactive effect of toxic exposure
and infectious agent may be involved in the excess respiratory disease morbidity
that has been described in polluted areas. Research on this problem has been
limited by the ethical/moral constraints of human experimentation, and further
by lack of clear indications for approaches to this issue. Use of experimental
models of the common respiratory virus infections could provide the necessary
foundation/ by suggesting hypotheses to test in humans.
The purpose of this presentation is to review information concerning the
development of practical animal models for important human respiratory viral
infections. Following a brief discussion of the relative importance of dif-
ferent respiratory viruses, four examples of experimental model systems will
181
-------
be described. In the context of this discussion consideration will be given
to study parameters which could be meaningful for examination of the interac-
tive effects of viruses and toxic substances.
IMPORTANT HUMAN RESPIRATORY VIRUSES
While the list of viruses that can infect the human respiratory tract is
extensive, a relatively small number constitute those that are encountered
most frequently (Table 1) (9). The relative importance of the different viruses
varies according to host age and other epidemiologic factors. In infancy, res-
piratory syncytial virus (RSV) is of paramount importance and is the principal
etiologic agent of epidemic bronchiolitis. Natural immunity to RSV appears to
be minimal, and annual reinfections are frequent during the first years of life
(8). Parainfluenza type 3 virus (P3) is another common cause of bronchiolitisi
infections with this agent are seen in both epidemic and endemic patterns (7) .
Among pre-school children, parainfluenza type 1 (and type 2 to a lesser extent)
virus infections are related to the occurrence of epidemic croup. The influenza
viruses assume relatively greater importance as causes of respiratory disease
morbidity in older children and adults. Experimental models for investigation
of influenza virus infections have been available for many years, and will not
be considered further in this presentation. Although outside the scope of this
discussion, it should be noted that Mycoplasma pneumoniae infections are a very
ccnunon cause of tracheobronchitis and pneumonia in older children and young
adults. Experimental models of mycoplasma infection are described elsewhere in
this symposium (D. A. Powell, P. C. Hu, A. M. Collier). Adenovirus respiratory
diseases occur at all ages, although the types involved differ: in children,
types 1, 2, 5, and 6 are encountered most frequently; in adults, types 3, 4
and 7 are seen particularly as causes of acute respiratory disease (ARD) of
military recruits. Rhinoviruses assume importance as agents of significant res-
piratory disease morbidity primarily in adults.
From the foregoing it is apparent that research related to the interactive
effects of pollutants and respiratory viruses should be targeted on those agent:
having the greatest human health impact. Toward this end, information will
be summarized on experimental models of respiratory syncytial virus, para-
influenza type 3 virus, adenovirus, and rhinovirus infections.
MODELS OF RESPIRATORY SYNCYTIAL VIRUS INFECTIONS
Respiratory syncytial virus is an enveloped RNA-containing virus sharing
some biologic properties of the paramyxovirus group* The agent is difficult to
handle in the laboratory because of its lability and the limited range of sus-
ceptible cell culture types available for isolation and propagation of the viru
The natural host range includes at least the higher primate and bovine species
182
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Table 1. Non-bacterial Agents of Importance
in Human Respiratory Diseases
Agent
Host
Disease syndrome
Respiratory syncytial virus
Parainfluenza viruses
Type 3
Infants
Children, adults
Infants
Children, adults
Bronchiolitis, pneumonia
Rhinitis
Bronchiolitis, pneumonia
Rhinitis
Types 1, 2
Infants, children
Adults
Croup
Rhinitis
Mvcoplasma pneumoniae
Older children,
adults
Tracheobronchitis,
pneumonia
Influenza viruses
Adenoviruses
Types 1, 2, 5, 6
Children, adults
Adults
Infants
Influenza
Pneumonia
Rhinitis, bronchiolitis,
pneumonia
Types 3, 4, 7, 14, 21
Rhinoviruses
Adults
Adults
ARD, pneumonia
Rhinitis
in addition to humans. Initial work in search of animal models for RSV had
only limited success (4). It was found that infection could be established
in ferrets, although disease was limited to the nasal passages; hamsters and
guinea pigs showed little evidence of disease manifestations after infection,
although immune responses were measured. More recently, further studies of
ferrets of different ages and of the cotton rat have yielded promising results.
The Perret-RSV Model
Coates and Chanock first demonstrated susceptibility of the ferret to RSV
in a search for animal models involving several other species (4). After intra-
nasal inoculation, ferrets yielded virus from their tracheas for four days and
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nasal turbinates for one week. Maximum viral replication occurred within
three to four days, and the animals demonstrated brisk serologic responses
in three to four weeks by the neutralization and complement fixation tech-
niques . Histopathologic changes were limited to the nasal passages; after
one week there was destruction of the ciliated epithelium, formation of multi-
nucleated cells/ and appearance of intracytoplasmic inclusions. Healing took
place in 30 days, but submucosal cellular hyperplasia and irregularities in
cartilage were seen as residua. Little use was made of this model subsequently
due to the lack of significant pulmonary involvement.
In 1976, Prince and Porter (14) reexamined the possible use of ferrets
for experimental RSV infections. They employed animals of various ages and
reproduced the findings described above in adult animals. Moreover, it was
found that virus replicated in the lungs of infant ferrets and that this event
was age-dependent, disappearing by age four weeks. Viral antigen was demon-
strated by immunofluorescence in the surface epithelium of the nose, and in
alveolar cells within the lungs. Lack of bronchiolar involvement makes the in-
fant ferret model an imperfect human counterpart; however, the age—dependent
organ localization mimics the lower respiratory tract involvement with RSV
seen in infants, and the upper respiratory tract disease of older children and
adults. The model thus has usefulness for long-term studies and for examining
the determinants of virus host-cell tropism.
The Cotton Rat-RSV Model
In 1971, Dreizen and co-workers (6) explored use of the common cotton rat
(Sigmodon hispidus) as an experimental host for RSV infection. In contrast
to the ferret, RSV replicated to high titer throughout the respiratory tract
of the cotton rat. The time course of viral replication was similar to that
seen in the ferret nasal turbinates, with maximal production around the fourth
day following inoculation. Pathologic changes of rhinitis, bronchitis, and
bronchiolitis were present which prominently involved the surface epithelium.
Recently Prince and co-workers (13) confirmed Dreizen1s findings and added
new information about the cotton rat model. It was found that age of the ani-
mal had no influence on susceptibility of the lung to RSV, unlike the ferret.
Ciliated epithelial cells showed morphologic evidence of injury, with changes
including cytoplasmic ballooning, loss of cilia, nuclear contraction, and
cellular desquamation. Epithelial injury was most prominent in the nasal turbi-
nates, but was present also in bronchioles. Viral antigen was present through-
out the nasal epithelium as shown by immunofluorescence; additionally, antigen
was identified in bronchial and bronchiolar epithelium, but not in the trachea.
Although information on human lung pathology in RSV disease is not exten-
sive (5), the cotton rat observations appear to supply a reasonable counter-
part in terms of the areas involved in the respiratory tract. Autopsy material
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reveals a component of interstitial pneumonia and extensive peribronchiolar
round cell infiltration, which has not been characteristic of the cotton rat
lesions. However, the microbiologic similarities and host cell specificity
provide a basis for many kinds of meaningful experiments; the increased volume
of the bronchiolar epithelium may even compromise flow through small airways,
making pathophysiological studies possible. Together, the cotton rat and the
infant ferret provide the basic tools required to initiate an understanding of
the pathogenesis of RSV disease. Unfortunately, cotton rats are not available
commercially at the present time.
Other RSV Models
The initial recovery of RSV was from a chimpanzee with rhinitis; for a
time the virus was called the "chimpanzee coryza agent." Apparently this ani-
mal is naturally infected with RSV and thus could serve as an experimental
host. However, usefulness of the model is limited by cost and the difficulties
attending studies with large primates. Limited experience also suggests that
chimpanzees do not have pulmonary disease after inoculation with RSV. Sero-
logic surveys have shown that Cebus monkeys regularly possess RSV antibodies,
suggesting another animal which experiences natural disease with the virus (1).
Recently, a bovine RSV has been described, and experimental infection of
calves is reported (11). Inoculated animals develop fever, serous rhinitis,
peribronchiolitis, and pneumonia. While the calf could be valuable for cer-
tain kinds of studies, there are practical limitations for extensive labora-
tory investigations with animals of this size. Bovine antiserum against RSV
is available commercially (Burroughs Wellcome, Ltd.) and has been used success-
fully for rapid immunofluorescence diagnosis of human infections; this suggests
close similarity or identity of the human and bovine viruses.
MODELS OF PARAINFLUENZA TYPE 3 VIRUS INFECTIONS
As the name implies, P3 virus resembles influenza virus in some features;
notable is possession of a hemagglutinin and neuraminidase, but these do not
show the antigenic diversity of influenza virus. The virus grows readily in
a number of cell culture types commonly used in the diagnostic laboratory. It
can be recognized by characteristic cytopathic effects or by adsorption of
erythrocytes, and can be identified by hemadsorption inhibition with type-
specific antiserum. The natural host range includes man, guinea pigs, and
monkeys; a similar but antigenically distinct virus is the etiologic agent of
shipping fever in cattle and is widespread among many cloven-hoofed species.
Practical experimental models to be discussed are the Syrian hamster and the
guinea pig.
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The Hamster P3 Model
Experimental P3 virus infections using hamsters were described in detail
by Buthala and Soret (3), who examined virologic, serologic, and pathologic
parameters. In initiating these studies, they found variable susceptibility
to infection of animals from different sources. This variability was unrelated
to age, sex, or weight of the animals and was thought to be due to partial im-
munity. Although P3 virus is not known to cause natural disease in the hamster
it is possible that human to hamster transmission occurs, or that they have
endemic infection with a related agent such as Sendai virus (murine parain-
fluenza type 1)« In any event, source of supply is a critical matter requiring
careful control and monitoring.
Optimal infection of the hamster with P3 was achieved with intranasal
inoculation of cill culture-passaged material. Virus replication reached a peak
in the lung at th'r'ie days, clearing spontaneously by one week. Pathologic
changes were maximal at five to seven days, resolving by two weeks* Necrotizino
nasal lesions were observed first, followed by extension of changes into the
lower respiratory tract. The airway epithelium became hyperplastic with for-
mation of multinucleated cells. Peribronchial and perivascular leukocytic
infiltration was pronounced, but alveolar involvement was seen infrequently.
Intraluminal exudates of leukocytes and desquamated epithelial cells were noted.
Infected animals developed a brisk serologic response detectable with the com-
plement fixation method which rose between the first and second weeks post-
inoculation. The pattern of changes which accompanies P3 infection of the
hamster is reproducible with regularity in susceptible animals. There are
close similarities between the pathologic findings and information concerning
P3 bronchiolitis and pneumonia in humans (5). Until animal models of RSV in-
fections are developed further, the P3 hamster model can serve as a good system
for studies on the pathogenesis of acute viral bronchiolitis.
The Guinea Pig P3 Model
Guinea pigs are known to be susceptible to P3 infection, and have been
used extensively as donors of antiserum for studies on interrelationships of
the paramyxoviruses. Details of the experimental disease produced in this
animal have not been described, but unpublished observations (15) indicate a
virologic and pathologic course very similar to that in the hamster. The guinea
pig offers several advantages over the hamster for use as an experimental model.
Many technical manipulations are done more readily in the guinea pig because
of its size. Commercial reagents are available for immunologic studies. Also
the animals are highly sensitive to many phannacologic agents, which could
make them good models for pulmonary physiologic changes in consequence of
infection. As with the hamster, guinea pigs are commonly immune to P3 which
186
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probably reflects a combination of their sensitivity to infection and the
ubiquitous distribution of the virus in nature. It is possible to produce
colonies free of P3 experience, and methods for this purpose have been
reported (17).
MODELS OF ADENOVIRUS AND RHINOVIRUS INFECTIONS
Although adenoviruses cause less respiratory disease morbidity than some
other agents that have been discussed, they were among the first respiratory
viruses discovered and have been studied extensively. These DNA-containing
viruses constitute a large family of specific serotypes whose natural hosts
include man, monkeys, cattle, rodents, dogs, and chickens* Generally, there
is species specificity between host and given serotypes such that experimental
models are not readily available. Considering the types most frequently en-
countered in humans (see Table 1), hamsters, dogs, and rabbits may be infected
but show no overt signs of illness. Latent infections may become established
in hamsters; several serotypes are carcinogenic, resulting in sarcomas at the
site of inoculation. Pulmonary lesions can be produced in pathogen-free,
colostrum-deprived piglets with serotypes 1, 2, 5, and 6 (2). A fatal disease
can be produced in newborn hamsters with serotype 5 (12).
The rhinoviruses are etiologic agents of many common colds, especially
among adults, and thus are important for study in the context of this symposium.
These RNA-containing viruses demonstrate a multiplicity of serotypes, which
complicates their identification and the performance of serologic testing.
Special conditions are required for rhinovirus isolation, including selected
cell cultures, and low incubation temperature and medium pH relative to other
groups of respiratory viruses. The host range of these viruses appears
limited (9). Cattle experience natural infection with biologically similar
agents; chimpanzees and gibbons can be infected with some of the human sero-
types. Experimental infection of a variety of common laboratory animals has
been unsuccessful.
Progress in the development of techniques to maintain organ or specialized
cell cultures (see A. M. Collier, this symposium) provides the potential for
use of human cells as hosts for the adenoviruses and rhinoviruses of interest.
These model systems provide easily controlled circumstances for detailed study
of the host-parasite interaction, but obviously lack the ability to respond
like the intact host. Nevertheless, simplified models could prove very useful
in dissecting the cellular basis of the interactive effects of toxic substances
and infectious agents.
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RESEARCH STRATEGIES USING EXPERIMENTAL MODELS OF RESPIRATORY VIRUS INFECTIONS
The foregoing discussion has emphasized the development of animal models
of the more common human respiratory virus infections. Consideration will now
be given to meaningful ways in which these model systems might be used to study
the interactive effects between the viruses and pollutants of interest. This
symposium has addressed a wide variety of host defense mechanisms and pharma-
cologic reactions, all of which could be evaluated in the experimental infec-
tions described.
Information accumulating on the pathogenicity of different infectious
agents suggests mny mechanisms by which host defenses are bypassed. Depending
upon the target cell for a given agent within the respiratory tract, the micro-
bial offense can relate primarily to evasion of muco-ciliary clearance, avoid-
ance of phagocytosis, exertion of immunosuppressive effects, and the like. it
is probable that effects of toxic substances have a similar specificity;
accordingly, the most appropriate study parameters must be chosen considering
both infectious agent and pollutant. Concerning respiratory viruses, the
microbiologic, immunologic, pathologic, and physiologic dimensions of the
experimental models could be explored to determine the superimposed effects of
toxic exposures.
The virologic aspects of the experimental models have been defined in
terms of the time course of infection, amount of viral replication, and prin-
cipal host cell types involved. Adverse effects of toxins might be reflected
in longer viral persistence, higher titers, and more extensive involvement of
host cells. The role of toxins in these effects could include alterations in
host cell receptor sites for the viruses, and in the immune and phagocytic
mechanisms required for control and elimination of infection.
Studies on the immunologic aspects of the experimental infections are
facilitated by the information developed in recent years concerning the
respiratory tract. The presence of virus-specific secretory IgA in the upper
respiratory tract correlates well with the presence of protective immunity.
Antibodies of other classes can be produced locally by the bronchus-associated
lymphoid tissue, and probably function in neutralization of free virus, preven-
tion of host cell attachment, or opsonization for phagocytosis of infected cell
debris. Since intracellular virus is protected from the effects of antibody,
another important mechanism concerns cellular immunity provided by thymus-
derived lymphocytes. Immune cells which are specific for both virus and host
cell type are capable of lysing virus-infected cells, thereby contributing to
control of the infection. The immune mechanisms described are operative in
or on the respiratory mucosal surface, and thus may be particularly vulnerable
to the effects of atmospheric pollutants.
The pathology of experimental infections could be altered in a variety
of ways by the superimposed effects of toxic exposure. Attention could be
188
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focused on the degree, extent, and persistence of histopathologic changes in
the lung. The models described in this presentation are self-limited diseases,
so that the occurrence of mortality would provide meaningful information.
Sublethal effects in regard to the healing and repair processes also should
receive attention, since the interaction of infection and toxin might produce
long-term sequelae.
Lastly, physiologic studies are important to accomplish in the experimen-
tal model systems. Correlates are needed for the pathophysiologic changes in
humans which have been important indicators of the health effects of pollutants.
Methods are available to perform respiratory physiologic studies in small
laboratory animals, but thus far application of these procedures to models of
infectious diseases has been limited. Some of the anatomic considerations
that have been described suggest that pathophysiologic changes could be a very
sensitive measure of the modulation of disease by toxic exposure.
In summary, several practical laboratory animal models for common human
respiratory virus infections have been described. These models lend themselves
to studies on the interactive effects of pollution and infection, thus address-
ing an important human health problem. Although findings from such studies
cannot be extrapolated directly to the human situation, they can serve as in-
dicators of health effects which should be sought and of appropriate methods
of study that could be employed.
REFERENCES
1. Belshe RB, Richardson LS, London WT, Sly DL, Lorfeld JH, Camargo E,
Prevar DA, Chanock RM: Respiratory syncytial virus infection of four
species of primates. J Med Virol 1:157-162, 1977
2. Betts AO, Jennings AR, Lament PH, Paze Z: Inoculation of pigs with
adenovirus of man. Nature (Lond) 193:45-46, 1962
3. Buthala DA, Soret MG: Parainfluenza type 3 virus infection in hamsters:
Virologic, serologic and pathologic studies. J Infect Dis 114:226-234,
1964
4. Coates HV, Chanock RM: Experimental infection with respiratory syncytial
virus in several species of animals* Am J Hyg 76:302-312, 1962
5. Collier AM, Clyde WA Jr: Model systems for studying the pathogenesis
of infections causing bronchiolitis in man. Pediat Res 11:243-246, 1977
6. Dreizin RS, Vyshnevetskaia LO, Bagdamian EE, Yankevich OD, Tarasova LB,
Klenova AV: [Study of experimental respiratory syncytial virus infection
in cotton rats: Virologic and immunofluorescent studies.] Vopr Virusol
16:670-676, 1971
189
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7. Glezen WP, loda PA, Clyde WA Jr, Senior RV, Sheaffer CI, Conley WG,
Denny FW: Epidemiologic patterns of acute respiratory disease of children
in a pediatric group practice. J Pediat 78:397-406, 1971
8. Henderson FW, Collier AM, Clyde WA Jr, Denny FW: Respiratory syncytial
vims infections, reinfections and immunity: A prospective, longitudinal
study in young children. New Engl J Med (In press)
9. Jackson GG, Muldoon RL: Viruses Causing Common Respiratory Infections
in Man. Chicago, University of Chicago Press, 1975, p 248
10. Lave LB, Seskin EP: Air Pollution and Human Health. Baltimore, Johns
Hopkins University Press, 1977, p 368
1 1. Mohanty SB, Ingling AL, Lillie MG: Experimentally induced respiratory
syncytial viral infection in calves. Am J Vet Res 36:417-419, 1975
12. Pereira HG, Allison AC, Niven JSF: Fatal infection of new born hamster
by an adenovirus of human origin. Nature (Lond) 196:244-245, 1962
13. Prince GA, Jenson AB, Horswood RL, Camargo E, Chanock RM: The pathogene-
sis of respiratory syncytial virus infection in cotton rats. Am J Pathol
93:771-791, 1978
14. Prince GA, Porter DD: The pathogenesis of respiratory syncytial virus
infection in infant ferrets. Am J Pathol 82:339-350, 1976
15. Sanders RS, Clyde WA Jr: Unpublished data
16. Shy CM, Goldsmith JR, Hackney JD, Lebowitz MD, Menzel DB: Health Effects
of Air Pollution. New York, American Lung Association, 1978, p 48
17. Welch BG, Snow EJ Jr, Hegner JR, Adams SR Jr, Quist KD: Development of
a guinea pig colony free of complement-fixing antibodies to parainfluenza
virus. Lab Animal Sci 27:976-979, 1977
190
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Session V:
Regulation of Mucus Secretion
and Cellular Differentiation
-------
Mucous Membrane of Respiratory Epithelium
L M. Reid and R. Jones
Children's Hospital Medical Center
Boston, Massachusetts
Although there are animal models in which the airway damage La associated
with necrosis and sloughing of epithelium, this report concentrates on animal
models in which the nature and dose of the damaging agent are such that the
epithelium remains intact. Such models are especially relevant to understanding
the effect of a relatively low dose of irritants delivered in either acute or
chronic exposure. It is a remodeling of the cell population of the normal
intact epithelium that occurs, one that is particularly related to mucus hyper-
secretion, the most obvious single feature of the response and almost certainly
the most important functionally (24, 30).
Even when the injury is serious enough to cause ulceration, we have shown
healing to occur while the administration of the irritant continues and, in
the new epithelium, remodeling is seen similar to that discussed below (31).
Our most recent studies show that changes that we identify with chronic irrita-
tion occur quickly, much more quickly than previously suspected (11, 18).
In such studies it is essential to use animals with "clean" lungs (1, 2).
The normal human airways are sterile and even with mucus hypersecretion this
state often continues. We find that histological criteria are more sensitive
to determine satisfactory cleanness than bacteriological or viral studies.
Lymphocyte infiltration may be of an unacceptable degree even when the lungs
are sterile.
The mixed nature of the mucus or total bronchial liquid, and the variety
of epithelial cells responsible for the special mucus or epithelial glycopro-
teins (9, 10, 33) call for a multidisciplinary approach. Animal models or
193
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in vivo studies with which we are particularly concerned here can be complemented
by in vitro studies of the tissue cells (3, 37, 38). Histochemical studies (11
16, 17, 21, 22, 23, 35) are complemented by biochemical analysis of total secre-
tion, both its bronchial and serum components (7, 20, 25, 26, 27). These in
turn need to be correlated with rheological behavior (19, 25, 27).
CELLS OF THE RESPIRATORY. EPITHELIUM
Within the airway epithelial lining eight epithelial cell types have been
identified (9, 10) (Fig. 1 ). All cells touch the basement membrane) only two
do not reach the lumen—the basal and Kultschitsky cells. The serous, Clara, and
mucous are the secretory cell types. The undifferentiated or intermediate cell
and the ciliated and brush cells are not secretory. "Mucous" is the term we now
prefer to goblet cell, since we can define it by reference to the nature of the
secretory granules, whereas "goblet" refers essentially to the amount and shape
of the secretory mass which resembles a chalice or goblet. The serous and Clara
cells are characterized by electron dense and discrete granules: the Clara cell
has a considerable amount of smooth endoplasmic reticulum and its apex often
bulges above the tight junctions with neighboring cells. Two mesenchymal cells—
the globule leukocyte and the lymphocyte—are found.
Reconstruction of the human submucosal gland reveals a duct about 1 mm lona
lined by the duct cell—a tall cell, packed with mitochondria and reminiscent of
the striated duct of the salivary gland but lacking the striations (Fig. 2) (29)
The tubules are lined by mucous or serous cells—the "serous" regions being alvav
distal to a mucous region, i.e., farther from airway lumen, and arising either
as a lateral pouch or from the distal ends of a mucous tubule (28).
Nerve fibers are found within the airway epithelium (8). In the rat, they
are non-myelinated and only in the extrapulmonary airways. They are numerous
particularly in the young animals, and are closely associated with basal secre-
tory and ciliated cells. The nature of the vesicles they contain suggests that
some are sensory, some are motor, and of the latter, some are adrenergic and
some are cholinergic. Nerve fibers are found within the basement membrane of
the submucosal glands. Myoepithelial cells and "clear" cells, probably immune—
blasts, are also found in the secretory tubules of the gland.
In the rat, mitotic activity represents turnover of the airway epithelium
that is, in some regions, as fast as 10 days and in others 250 days. This ranae
takes into account all regions, males and females, and three ages (1) (Fig. 3)
NORMAL HOMEOSTASIS
Under basal conditions homeostasis of the airway epithelium is maintained
with regard to several of its features, the normal pattern varying from region
to region. The nature of the control mechanisms is not understood. Homeoetaaia
194
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Epithelial
Figure 1. Eight epithelial cell types present in rat airway epithelium.
is maintained with respect to the cell types present. For example, in the trachea
the basal cell is numerous, making up about 20% of the cell population, where-
as it is virtually absent in peripheral airways. The Clara cell, the secretory
cell of small airways, is not seen in the large central ones. The proportions
of the cells that are present vary, the ciliated cell being about 1 in 5
centrally and 2 in 3 peripherally.
At a given airway level/ the pattern of glycoprotein within the secretory
cells varies (11, 13, 15, 21). The epithelial glycoprotein is either neutral or
acid (21-23): it is acid either because of sialylation or sulfation of the glyco-
protein, and the sialic acid may or may not be susceptible to digestion by the
enzyme sialidase (Fig. 4). Sometimes within a single granule only one type of
glycoprotein is identified or there may be a mixture. A cell can be characterized
by its predominant granule type and by the "amount" of secretion, i.e., by the
number of granules it includes. Regional differences in the normal pattern of
glycoprotein distribution are also seen.
Cell division is another feature having a basal level of activity (1).
The oldest group of animals we have studied did not show a regional pattern, in
that the percentage of cells incorporating H-thymidine was similar at all air-
way levels. Since concentration of cells is less in peripheral airways, this
means a slower replacement per unit area in small than in large airways, in the
young animals, in males more than in females, mitotic activity is more marked in
trachea than in peripheral airways. This sex difference was apparent in the
young animals although male and female animals were gaining weight at the same
rate.
195
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2OOyum
V /
>/ f
7 /
/ A
/ Ax. ..••
1 I >>s
' /
r~~
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.r
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i
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i
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i
i
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i \
i \
/ \
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— r
V
\
^
ii_^
^™»«
^
\
^/
\\ T ^.
B
b
Figure 2, a and b. Reconstruction of human bronchial gland, drawn to scale
shows collecting duct with tubules arising from it. The branching patterns of
two tubules arising from the duct are shown (A and B). Mucous cells (continuous
lines), serous cells (broken lines). Secretion from the serous cell thus passes
over the mucous cell before reaching the collecting duct. Reprinted with
permission (29).
196
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15-
UJ
3
z
o
o
9.
TOSE
NUM
a male
• female
33 days
±11
1
47 days
rl
Hi J ri :°5dn.ys
i n m
AIRWAY LEVELS
Figure 3. Mitotic index (number of mitotic nuclei per 1000 nuclei) in male
and female rats of three age groups and at five airway levels. Levels I-III:
proximal/ mid, and distal trachea; Level IV: main intrapulmonary axial pathway;
Level V: distal airway. Reprinted with permission (1).
Horneostasis is maintained, on a regional basis, with respect to types of
cells present, their proportion, their rate of mitosis, and the pattern of in-
tracellular glycoprotein identified histochemically.
ANALYSIS OF MUCUS SECRETION, CELLULAR DIFFERENTIATION, AND THEIR CONTROL
The mucus glycoprotein cones from several cell types both within the sub-
mucosal glands and the surface epithelium; the mucus is then mixed with serum
components. The structure of the airway wall changes with age and disease.
Because of its complexity, to understand airway secretion and its control we
have used a multidisciplinary approach in its study* Tissue studies of the
normal human airway and its modification in hypersecretory states offer an im-
portant reference. In organ culture systems of human airway, the behavior of
197
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Neutral ISS3 Sialytated Fl Sialylated ( I Sulphated
Sialldase [M Staltdas*
Sensitive Resistant
D
EXTRAPULMONARY AIRWAY - Level III
CON
U t
18 11
53
18
[PR
INTRAPULMONARY AIRWAY - Level IV
33 8 40
IB^B^Bl
CON
9 9
37
IPR
Zl
100
Figure 4. Percentage of secretory cells containing four types of glyco-
proteins in control (CON) and isoproterenol-treated (IPR) rats. Level III:
distal trachea; Level IV: proximal region of intrapulmonary axial pathway.
the various cell types and the way their activity is controlled can be explored
(5, 6). The total secretion can be analyzed as sputum and as material collected
in organ culture (3). Environmental conditions can be altered in animal models.
By organ culture of tissue obtained from such animals in vivo and Ln vitro
studies can be correlated (4, 34).
Our more recent organ culture studies of human epithelium show that secre-
tory cells of the surface epithelium, at least of the large airways, unlike the
submucosal glands, are not stimulated to secrete by acetylcholine or its analogues,
H-threonine transport into mucous and serous cells is largely ouabain-sensitive,
that of H-glucose only partially so. Post-pulse addition of several agents has
established that precursor uptake and glycoprotein synthesis and discharge by
mucous and serous cells are not coupled events (6). In a series of experiments
the effects of colchicine and cytochalasin B have been followed (5). Neither
agent influences the basal rate of secretion of either mucous or serous cells.
When incubated with the tissue and radioactive precursor, both reduce acetyl-
choline-stimulated secretion. It seems that the cytochalasin B-sensitive fila-
ment system must be intact for the discharge effect of a cholinergic agent.
RESETTING THE HOMEOSTATIC MECHANISM
The homeostatic mechanism is reset to a different level by a variety of
stimuli-irritants, infections, and drugs.
198
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Irritants
The biological response of the epithelium is illustrated first by its re-
action to tobacco smoke (4, 14, 15) with or without the addition of an anti-
inflammatory agent (Fig. 5). Phenylmethyloxadiazole (PMO) is the agent illus-
trated although similar results have been produced by phenylbutazone. Rats
were exposed to a smoke from cigarettes, delivered from a Wright auto-smoker
over a period of four hours (Table 1). In the first experiment described, this
4-hour daily exposure was repeated for four days a week for six weeks• The
tobacco produced an increase in the concentration of secretory cells and of
ciliated cells at all airway levels. Phenylmethyloxadiazole prevented this in
the extrapulmonary airway but had no such "protective" effect on intrapulmonary
airways• Analysis of intracellular types of glycoprotein showed a shift in
their pattern. In the trachea the shift is from a population of cells contain-
ing predominantly neutral glycoprotein to one that is acid-producing. This
shift occurs whether or not PMO is administered, that is, whether or not there
is an increase in secretory cell number. From thesje studies we know that this
shift can be detected within 24 hours, making it the most sensitive marker of
change in airway epithelium.
Tobacco also increases mitotic activity. A burst of mitosis is seen 24
hours after starting exposure to tobacco smoke; PMO gives partial protection
against this increase. The increase in secretory cells, particularly mucous
cells, arises, in part, from conversion of undifferentiated cells to secretory
cells, from new cells that appear as the result of mitosis, and within the secre-
tory cell population by interconversion of serous or Clara cells to mucous cells.
The interconversion is apparent from cell counts and from identification of
intermediate cell forms. Electron dense granules become mixed with electron
lucent granules until, finally, all are electron lucent and "confluent." By
light microscopy it is possible to identify an increase of Alcian Blue staining
granules. When a cell switches to secrete acid glycoprotein, the AB positive
granules appear first at the apex of the cell. The "switch-on" of enzyme re-
sponsible for the attachment of sialic acid seems to start near the cell apex
after the secretion is already packaged in its granules. The shift is first to
sialylation and then to sulfation. When the latter occurs, the sulfated granules
also appear first at the apex, with sialylated granules being present nearer the
nucleus.
In studies of shorter exposure time, it emerged that this lability of the
cell population, particularly of its secretory cells, is apparent after only
one exposure to tobacco smoke (18) (Fig. 6). In this experiment the response
to tobacco smoke was monitored at intervals up to 14 days. Animals were sacri-
ficed 20 hours after the last exposure. Extrapulmonary and intrapulmonary air-
ways showed different patterns of response. In the case of the trachea, one
period of exposure to tobacco produced a "discharge" effect so that the total
199
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Tobacco
Tobacco & PMO
Control
4O
8O
I2O
160
Figure 5. Mean number of secretory cells in 6 mm of rat tracheal epithelium
after inhalation of tobacco smoke (tobacco), alone or with phenylmethyloxadiazole
(tobacco + PMO) , or after air alone (control).
Table 1. Mean Number of Secretory Cells in 3 mm of Epithelium in Control
Rats and After a Single 4-Hour Period of Inhalation of Tobacco Smoke
Level
Extrapulmonary
airways
Intrapulmonary
airways
I
II
III
IV
V
VI
VII
Control
34+8.2
41 + 8. 1
64 + 6.5
27 + 6.4
6 + 2.8
28 + 6.4
6 + 2.0
Tobacco
5 + 1.9**
7+0.9**
53 + 4.6
64 + 7.4*
17 + 5.3
29 + 9.7
7 + 0.2
*p < 0.05, **p < 0.01.
secretory cell number was reduced. One exposure produced tolerance to the dis-
charge effect so that after two days of exposure the secretory cells had in-
creased significantly above control values. In the intrapulmonary airways no
discharge effect was seen: the one 4-hour exposure period significantly increased
the secretory cell number above control values. Some further increase developed
during the second week, when the concentration of secretory cells was virtually
the same as that seen after six weeks of exposure* (This was a separate experi-
ment .)
In the trachea, tolerance to exposure is quickly lost. One day of rest
from exposure restores tolerance so that the next exposure produces a discharge
effect. Adaptation to the environment is rapid and in a short time the maximal
effect is produced. Although tolerance is quickly achieved, it also seems to
200
-------
EXTRAPULMONARY AIRWAYS
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Figure 6. Mean number of secretory cells in 3 mm of airway epithelium of
control (CON), isoproterenol-treated (IPR), or salbutamol-treated (SBM) rats at
seven levels. Levels I-III: proximal/ mid, and distal trachea with main bronchi;
Levels IV and V: proximal and distal region of main intrapulmonary axial path-
way; Levels VI and VII: lateral branches. Reprinted from Fed Proc 38:191-196, 1976
be quickly lost. The homeostasis mechanism is reset for each of its features,
secretory cell type and population, mitotic activity, and glycoprotein pattern;
a regional pattern is still apparent.
The anti-inflammatory agent PMO has modulated the response, offering pro-
tection from some aspects of the irritant but not from others; PMO given alone,
by parenteral injection, caused increase in submucosal gland size. In vitro
study of submucosal gland from these animals showed reduced precursor uptake,
as well as decreased discharge. The animals that had received tobacco plus PMO
showed normal secretory rate, whereas tobacco alone produced an increase in
secretory rate.
Drugs, Beta-adrenergic Agents—Isoproterenol and Salbutamol
We have shown that isoproterenol causes hypertrophy of the bronchial sub-
mucosal gland and increases surface epithelial secretory cell number even under
germ-free conditions (12, 32, 33, 39). The secretory cells producing acid glyco-
protein are particularly increased in the surface epithelium. In a recent ex-
201
-------
periment, we have extended these studies to study the effect of isoproterenol -
over shorter times and at various airway levels and to compare it with that of
salbutamol (a nonselective beta and a selective beta-2 agonist, respectively)
(Fig. 6).
The two drugs produce an increase in secretory cell number, but with strik-
ing regional differences. At all levels, both drugs cause a shift to acid glyco-
protein production, whether or not secretory cell number is increased. With
respect to secretory cell number, the drugs show different regional specificities,
Isoproterenol causes increase in secretory cell number at all levels except the
mid-trachea (so this pattern is not based on the distribution of nerves). Sal-
butamol produces an increase only at the most proximal tracheal level and the
most distal part of the axial pathway of left lung. At each level the sal-
butamol effect is less marked than that of isoproterenol. The effect can be
detected after one injection of isoproterenol. These studies also show that
the lability of the epithelial cell population is striking and rapid, and the
"maximal" response is quickly achieved.
RECOVERY
After administration of the drug was ceased, "recovery" was followed and
here also major regional differences are seen (Fig. 7). In the trachea the
secretory cell concentration returns virtually to normal within a day or so.
In other regions differences persist. In the main bronchus, even 12 weeks
later, secretory cell number has not returned to normal. At this level there
is a stage when secretory granules are seen as ghost structures, and an ir-
regular mixture of granules of acid and neutral glycoprotein is seen, different
from the orderly way in which a "switch-on" to acid glycoprotein occurs, it
seems that the airway does not become tolerant of the stimulus, the tissue does
not "adapt" permanently since on withdrawal of the isoproterenol the airway re-
verts to normal. During recovery a greater variance is seen between individual
animals in the same group than during adaptation to the drug. This is particu-
larly true of surface epithelium* In the submucosal gland hypertrophy is also
produced by isoproterenol, and has been shown to revert to normal within six
weeks (12).
It seems that this pattern of response reflects regional differences in
sensitivity, based perhaps on regional differences in the nature and concen-
tration of receptors. Since these drugs mimic naturally occurring agents it
could be that the adrenergic system, beta and probably alpha, is part of the
normal control system. Either way, the use of the appropriate agonists and
antagonists offers a way to modulate secretion, certainly experimentally and
perhaps also clinically.
202
-------
UPPER TRACHEA
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Figure 7. Mean number of secretory cells in 3 mm of epithelium in the
upper trachea and left main bronchus of control (CON) and isoproterenol-treated
(IPR) rats after single daily injections on 6 consecutive days of either normal
saline (controls) or isoproterenol (10 mg/100 g body weight). Number of cells
present 24 hours after the last injection and after periods of 1 to 12 weeks
recovery.
Intra- and Extracellular Conditions
The histochemical studies of the glycoprotein within the cell are of
special significance in showing the shift to increased acidification of the
oligosaccharide side chains, with a relative increase in sulfated forms (Fig. 4)
and, within the granules that are only sialylated, to those in which more of
the sialic acid is resistant to sialidase. Understanding the composition and
structure of bronchial mucus glycoproteins (as of other epithelial glycoproteins,
e.g., gastric and cervical) is an actively expanding field, both as regards the
polypeptide core and the oligosaccharide side chains (7, 27, 36).
The histochemical shift seen in states of hypersecretion is associated
with the biochemical finding of increased concentration of both N-acetyl neu-
raminic acid (syn. sialic acid) and sulfate in the oligosaccharide fraction.
203
-------
Sometimes both occur on the same chain, the largest chains are usually the
most heavily sulfated. It seems that whereas sialic acid is a terminal sugar,
sulfation permits further glycosylation.
Animal studies reveal the striking lability of the intact epithelium,
both as regards its cell mix and its glycoprotein product. These adjustments
occur in the airway cells whether or not increased mitotic activity is also
part of the response to a given stimulus. The speed of adaptation and its nature
raise questions about the mechanisms of homeostasis responsible for stability
in such a complex population of cells that yet shows such striking regional
variation. These questions then lead to those concerned with the basis of the
resetting of this regulation by irritants and drugs. Understanding these
changes can be expected to indicate what is needed to control the features of
adaptation that are undesirable clinically.
REFERENCES
1. Bolduc P, Reid L: Mitotic index of the bronchial and alveolar lining of
the normal rat lung. An Rev Resp Dis 114:1121-1128, 1977
2. Bolduc P, Reid L: The effect of isoprenaline and pilocarpine on mitotic
index and goblet cell number in rat respiratory epithelium. Brit J Exp
Path 59:311-318, 1978
3. Coles S: Regulation of the secretory cycles of mucous and serous cells
in the human bronchial gland. In: Mucus in Health and Disease. Advances
of Experimental Medicine and Biology (Elstein M, Parke DV, eds).
New York and London, Plenum Press, 1977, pp 155-168
4. Coles SJ, Levine L, Reid L: Hypersecretion of mucus glycoproteins in rat
airways induced by tobacco smoke. Am J Path 94:459-472, 1978
5. Coles SJ, Reid L: Effect of colchicine and cytochalasin B on glycoprotein
secretion by human and canine airway. Fed Proc 38:522, 1979
6. Coles S, Reid L: Glycoprotein secretion in vitro by human airway:
Normal and chronic bronchitis. Exp Molec Path 29:326-341, 1978
7. Creeth JM, Bhaskar KR, Horton JR, Das I, Lopez-Vidriero MT, Reid L: The
separation and characterization of bronchial glycoproteins by density-
gradient methods. Biochem J 167:557-569, 1977
8. Jeffery P, Reid L: Intra-epithelial nerves in normal rat airways: A
quantitative electron microscopic study. J Anat 114:35-45, 1973
9. Jeffery P, Reid L: New observations of rat airway epithelium: A quantita-
tive and electron microscopic study. J Anat 120:295-320, 1975
10. Jeffery P, Reid L: The respiratory mucous membrane. In: Respiratory
Defense Mechanisms (Part 1), Vol. 5 in Series Lung Biology in Health and
Disease (Brain JD, Proctor DP, Reid L, eds). New York, Basel, Marcel
Dekker, 1977, pp 1«3-245
204
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11. Jones R: The glycoproteins of secretory cells in airway epithelium, in:
Respiratory Tract Mucus. Ciba Symposium 54 (new series) (Porter R,
Rivers J, O'Connor M, eds). Amsterdam, New York, Elsevier/Excerpta Medica,
North Holland, 1978, pp 175-188
12. Jones R, Baskerville A, Reid L: Histochemical identification of glyco-
protein in pig bronchial epithelium: (a) normal and (b) hypertrophied
from enzootic pneumonia. J Path 116:1-11, 1975
13. Jones R, Bolduc P, Reid L: Goblet cell glycoprotein and tracheal gland
hypertrophy in rat airways: The effect of tobacco smoke with or without
the anti-inflammatory agent phenylmethyloxadlazole. Brit J Exp Path
54:229-239, 1973
14. Jones R, Bolduc P, Reid L: Protection of rat bronchial epithelium against
tobacco smoke. Brit Med J 2:142-144, 1972
15. Jones R, Reid L: Secretory cells and their glycoproteins in health and
disease. Brit Med Bull 34:9-16, 1978
16* Jones R, Reid L: The effect of pH on Alcian Blue staining of epithelial
acid glycoproteins. 1. Sialomucins and sulphomucins (singly or in simple
combinations). Histochem J 5:9-18, 1973
17. Jones R, Reid L: The effect of pH on Alcian Blue staining of epithelial
acid glycoproteins. 2. Human bronchial submucosal gland. Histochem J
5:19-27
18. Jones R, Reid L: Secretory cell hyperplasia and modification of intra-
cellular glycoprotein in rat airways induced by short periods of exposure
to tobacco smoke, and the effect of the anti-inflammatory agent phenyl-
methyloxadiazole. Lab Invest 39:41-49, 1978
19. Keal EE: Physiological and pharmacological control of airways secretions.
In: Respiratory Defense Mechanisms, Vol. 5 in Series Lung Biology in
Health and Disease (Brain JD, Proctor DF, Reid L, eds). New York, Basel,
Marcel Dekker, 1977, pp 357-401
20. Keal EE, Reid L: Pathological alterations in mucus in asthma within and
without the cell. In: New Directions in Asthma (Stein M, ed). Park Ridge,
Illinois, American College of Chest Physicians, 1975, pp 223-239
21. McCarthy C, Reid L: Acid mucopolysaccharides in the bronchial tree in the
mouse and rat (sialomucin and sulphate). Quart J Exp Physiol 49:81-84, 1964
22. Lamb D, Reid L: Histochemical and autoradiographic investigation of
the serous cells of the human bronchial glands. J Path 100:127-138, 1970
23. Lamb D, Reid L: Histochemical types of acidic glycoprotein produced by
mucous cells of the tracheobronchial glands in man. J Path 98:213-229,
1969
24. Lamb D, Reid L: Mitotic rates, goblet cell increase and histochemical
changes in mucus in rat bronchial epithelium during exposure to sulphur
dioxide. J Path Bact 96:97-111, 1968
205
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25. Lopez-Vidriero MT, Das I, Reid L: Airway secretion: Source, biochemical
and Theological properties. In: Respiratory Defense Mechanisms (Part 1),
Vol. 5 in Series Lung Biology in Health and Disease (Lenfant C, ed).
New York, Basel, Marcel Dekker, 1977, pp 289-356
26. Lopez-Vidriero MT, Reid L: Bronchial mucus in health and disease. Brit
Med Bull 34:63-75, 1978
27. Lopez-Vidriero MT, Reid L: Chemical markers of mucus and serum glyco-
proteins and their relation to viscosity in mucoid and purulent sputun
from various hypersecretory diseases. Am Rev Resp Dis 117:465-467, 1978
28. Meyrick B, Reid L: ultrastructure of cell in the human bronchial sub-
mucosal glands. J Anat 107:281-299, 1970
29. Meyrick B, Sturgess JM, Reid L: A reconstruction of the duct system and
secretory tubules of the human bronchial submucosal gland. Thorax 24:729-
736, 1969
30. Reid L: An experimental study of hypersecretion of mucus in the bronchial
tree. Brit J Exp Path 44:437-445, 1963
31. Reid L: Animal models in clinical disease. In: Respiratory Tract Mucus.
Ciba Symposium. Amsterdam, Elsevier/Excerpta Medica, North Holland, 1977
pp 297-310
32. Reid L: The cell biology of mucus secretion in the lung. In: The Lung:
IAP Monograph No. 19. Baltimore, Williams and Wilkins, 1978, p 7
33. Reid L: Secretory cells. In: Symposium on Non-respiratory Aspects of
Lung Physiology (Lenfant C, ed). Fed Proc 36:2703-2706, 1977
34. Reid L, Meyrick B, Coles S: Glycoprotein synthetic pathways in, and drug
effects on, the human bronchial mucosa in vitro. In: Organ Culture in
Biomedical Research. British Society for Cell Biology Symposium 1 (Balls M
Monnickendam M, eds). London, Cambridge University Press, 1976, pp 463-480
35. Roberts GP:. Histochemical detection of sialic acid residues using periodate
oxidation. Histochem J 9:97-102, 1977
36. Roberts GP: The role of disulfide bonds in maintaining the gel structure
of bronchial mucus. Arch Biochem Biophys 173:528-537, 1976
37. Sturgess J, Reid L: An organ culture study of the effect of drugs on the
secretory activity of the human bronchial submucosal gland, din Sci
43:533-543, 1972
38. Sturgess J, Reid L: Secretory activity of the human bronchial mucous
glands in vitro. Exp Molec Path 16:362-381, 1972
39. Sturgess J, Reid L: The effect of isoprenaline and pilocarpine on (a)
bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart
thymus, liver and spleen. Brit J Exp Path 54:388-403, 1973
206
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DISCUSSION
PARTICIPANT: Do you find that isoproterenol changes the nature of the air-
ways in that it increased secretory cells of different types? For instance/
where the Clara cells are, do you find new types of secretory cells or an
increase of those types?
OR* REID: We find new types. The first thing I should say is that with
tobacco, the increase in secretory cells was at the expense of the intermediate
cells: both ciliated and secretory cells increased. With isoproterenol,
the first counts indicate that it's only the secretory cells. When the
cells change/ Clara cells virtually disappear, dara cells develop into
cells that look just like mucous cells* You see the stage where even the
smooth endoplasmic reticulum becomes less abundant. That1s one feature of the
cell* Electron dense granules develop, then a mixture of these and electron
lucent granules is seen, and ultimately all become electron lucent.
DR. ADLER: With exposure to tobacco smoke/ do you find a massive shift in the
secretory serous cells to the mucous cells before you get an increase of
electron dense granules in the cells?
DR. REID: Yes/ because we can pick the increase at 24 hours, and the first
wave of mitosis occurs at about 24 hours. It is clearly not as obvious
because the dramatic increase of cell number occurs about two days in the
tobacco group.
DR. NETTESHEIM: In the literature there is a lot of confusion about terms.
Do I understand you correctly that you consider the goblet cells/ secretory
cells, and mucus-producing cells in the upper airways are cells of different
functional states of the same cell type?
DR. REID: I would feel that at the moment. First of all, how we came to use
the three names. The serous cell is electron dense with mainly rough ER. The
Clara cell differs widely in different species, but for this purpose, we can
pick the main feature as being electron dense granules. That is the basis for
the three terms* At normal baseline/ with homeostasis at work/ those three
cell types are fixed, because there are only certain cell types at a given
207
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level of the airway, and much that these cells are "end" cells, as it were;
they are fully differentiated, and do not change. Under conditions of
irritation, by drugs, by infection, then you will get a shift of one cell type
to another. To say "stages" would imply that a cell will automatically go
through all its stages, and I don't think the cells always go through all their
stages. If we talk about secretory cell forms, it might remind us that under
conditions of irritation or drug treatment, you can convert one of those cell
types to another. Does that seem reasonable?
DR. GOLDSTEIN: What is known about the physiological consequences of the
various types of glycoprotein that might give some teleological reasons for
these very interesting and rapid shifts?
DR. REID: Not a lot* The fetus produces only sol fated glycoprotein, not
sialylated at all. It isn't until about four months after birth that the
sialylation of the bronchial glycoprotein occurs. I'm talking about the hvaaan.
One of the epithelial glycoproteins that we've been able to study, which is
only sulfated, is of the fish, and it had very low viscosity. Those are
facts. Now to be teleological and to make a hypothesis. It's quite clear
that you don't want the normal bronchial tree to be blocked by thick secretions
soon after birth. One could suggest that the sulfation is to keep viscosity
low. In disease, for example, chronic bronchitis, the gland is hypertrophied.
We know that both sialylation and sulfation increase, and viscosity increases
as the concentration of the mucus arises.
So there is evidence that viscosity increases as sialylation and sulfation
increase. The longer oligosaccharide side chains tend to have more sulfated
but sialic acid always comes in a terminal position. The sialic acid content
of the glycoprotein increases in the bronchitic, compared with normal secretion
from a normal airway—in particular when compared with prostaglandin P
23
stimulated secretion. It's difficult to be more precise than that.
208
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New Methods Used To Investigate the Control
of Mucus Secretion and Ion Transport
in Airways
B. Davis and J. A. Nadel
Cardiovascular Research Institute, UCSF
San Francisco, California
Mucins secreted from submucosal glands and surface epithelial cells con-
bine with water to form the respiratory tract secretions* The secretions
with trapped inhaled particles are moved up the airway to the mouth by the
sweeping action of the cilia. This clearance process may be controlled by
neurotransmitters or mediators acting (a) on secretion of mucin from sub-
mucosal glands and from surface epithelial cells; (b) on the composition
or consistency of mucus; (c) on the interaction of the cilia with the mucous
layer; and (d) on the rate of ciliary beating. These controls may be altered
by environmental toxins, infections/ inflammatory responses, or abnormal
biochemical pathways and cause disease.
Knowledge gained from previous studies of airway epithelium has recently
been reviewed (25). In the past/ studies of respiratory tract secretions in
man depended on the collection of sputum (2, 17, 28) which is contaminated by
saliva and nasal mucus, or the collection of samples of mucus secretion via a
tracheostomy (28). Knowledge of the action of drugs and mediators on mucus se-
cretion was inferred from measurements of respiratory tract secretions in
animals (3, 4, 16) or from changes in volume in a fluid-filled segment of
trachea (14). These older methods do not localize the source of the secretions
to submucosal glands or surface epithelial cells; therefore, they cannot be
used to examine the control of secretion*
In this review we describe new methods for studying the control of mucus
secretions that we developed using preparations of trachea in vivo and in
209
-------
Figure 1. Photograph obtained through a dissecting microscope of hillocks
on tantalum-coated canine tracheal epithelium produced by electrical stimula-
tion of a superior laryngeal nerve. The tantalum prevented the normal
dispersion of the secretions from the mucous glands. The hillocks/ caused
by accumulated secretions, averaged 0.2 mm in diameter.
vitro. Preliminary descriptions of some of these methods have been published
previously (7, 9, 18, 22-24, 30). The methods provide direct evidence of
secretion from the submucosal glands and allow us to study the functions of
airway epithelium under we11-controlled conditions.
_IN VIVO TECHNIQUES
To study the control of secretion of individual mucous glands, we de-
veloped techniques that allow us to study the submucosal gland as a physio-
logical subunit in vivo. We anesthetize dogs and ventilate the lungs arti-
ficially using a Harvard pump. Both vagus nerves are dissected free and
placed on stimulating electrodes. We make an incision in the anterior mid-
line of the upper two-thirds of the trachea and pull the cut edges apart
210
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widely to expose the epithelial surface. A fine powder of inert metal (tan-
talum) is then sprayed onto the epithelial surface. Since the normal disper-
sion of secretions from the submucosal glands is prevented, elevations in
the tantalum layer at the sites of the submucosal gland duct openings appear
during secretory activity (Fig. 1). We call these elevations which are
caused by submucosal gland secretion "hillocks." We have demonstrated that
our new technique is suitable for neurophysiological studies. In each ob-
servation period, after the previously secreted mucus has been removed by
wiping, the mucosal surface is coated with powdered tantalum and photographed
through a dissecting microscope (magnification, 6X). We photograph the
tantalum layer over the same area of epithelium at one-half minute intervals
during baseline conditions and after stimulating either the superior laryngeal
or recurrent laryngeal nerve electrically. The number of "hillocks" on each
photograph is counted and divided by the area of epithelium to obtain the
number of "hillocks" per square centimeter. These studies show that submu-
cosal gland secretion is increased by electrical stimulation of the superior
laryngeal and recurrent laryngeal nerves (7). We are able to prevent the
secretomotor effect of stimulating the motor branches of the vagus nerve by
atropine sulfate given intravenously or locally, indicating that the secreto-
motor effect involves postganglionic cholinergic pathways.
The measurement of the number of hillocks per square centimeter of
trachea! epithelium does not provide evidence about secretion from individual
submucosal glands; therefore we modified our photographic measuring system
(10). Now we visualize the hillocks with a television camera attached to a
dissecting microscope, record their images with a videotape recorder/ and
view the field during the experiment on a television monitor. A record of
time is simultaneously displayed on the monitor and recorded on videotape.
Thus, we make a continuous record of the secretion from individual submucosal
glands; the rate of secretion can be estimated from the increase in the size
of a hillock with time. We divide the observed field into six sections and
measure the diameter of one round hillock in each section.
We measure the diameters of the hillocks using a split-video image
measuring device (Instrumentation for Physiology and Medicine/ San Diego).
The images of the hillocks are played back from a videotape recorder onto
the screen of a modified television monitor. The image is held on the screen
by stopping the videotape recorder at any desired time. To measure the
diameter of a hillock, we select the line in the video image which runs along
the diameter of the hillock, offset electrically the upper part of the image
of the hillock, and displace it from the lower part of the image of the
hill°c^ by the length of one diameter (Fig. 2). The distance moved, which
equals the diameter of the hillock, is shown on a digital scale, which we
convert to millimeters with a calibration factor. This measuring device
211
-------
Figure 2. Enlargement of the screen of a video-image measuring monitor
showing a picture of hillocks projected from a tape recorder. The image of
one hillock has been split along its diameter by offsetting electrically the
upper part of the picture from the lower part. The distance moved by the off-
set image was measured electrically.
allows us to measure the diameters of hillocks at frequent short time intervals
(Fig. 3). To estimate the rate of secretion of individual glands we assume
that each hillock is hemispherical, and calculate the volumes of the hemi-
spheres from the measured diameters. Although this provides only an estimate
of the volume of secretion, it emphasizes the large changes in volume that
occur with small changes in the diameter of a hillock (volume of a hemisphere »
~~TT w D )•
To examine the secretory response of the submucosal glands to nervous
or pharmacological stimuli, we measure the increase of number of hillocks
per cm during baseline conditions and after a stimulus. This method allows
us to measure the pattern of the secretory response of individual glands to
different stimuli (Fig. 3). Our studies with the method show that an a-adre-
nergic agonist, phenylephrine, causes secretion from, canine tracheal mucous
glands. The effect is smaller than the stimulatory effect of electrical
stimulation of cholinergic efferent fibers in the superior laryngeal nerve
(11). The difference in the response to the two stimuli may be due to the
fact that phenylephrine has a vasoconstrictive effect on the local blood
supply to the trachea (Fig. 4).
212
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i.or
Diameter
of
Hillocks
(mm)
0.5
I 2
Minutes
Figure 3* Graph of measurements of diameters of hillocks made over 2.5
seconds during a control period of 60 seconds and during a 60-second period
after injection of 40 ug of phenylephrine (P.E.) into a cranial thyroid
artery of the dog.
Another technique for studying submucosal gland secretion, which has
been developed in our laboratory (31), allows the secretion from a single
submucosal gland to be collected into a micropipette. Cat tracheal glands
are studied because the secretions from these glands are watery and can be
collected easily into a capillary tube. In anesthetized cats ventilated via
the lower trachea/ the tracheal mucosa is exposed and covered with HEPES
oil equilibrated with water. To visualize the gland duct openings we stain
them JLn vivo with neutral red dye (0.1% solution) applied to the luminal
surface of the epithelium and observe them through a dissecting microscope.
With experience we are able to recognize the gland duct openings by their
ellip^cal shape and therefore we no longer use neutral red dye. To collect
the secretion we use oil-filled constant bore pipettes bent at their ends
to an angle of 30°-45°. The tips of the pipettes are fire polished to pre-
vent damage to the epithelial surface. The tip of the pipette is placed over
a gland duct opening, so that the opening is surrounded and can only discharge
213
-------
B
Figure 4. Angiograms of canine tracheal vessels produced by injecting
dye through a cannula in the right cranial thyroid artery 1 minute after
injection of 2 ml of saline (A) and 1 minute after injection of phenylephrine,
40 ug in 2 ml of saline (B). Phenylephrine caused constriction of the tra-
cheal vessels (arrows).
its secretion into the capillary lumen. We start the flow by applying slight
negative pressure with a syringe to the end of the micropipette. Secretion
is collected for 1 minute and then oil is aspirated into the capillary lumen
to isolate the sample; two or three samples are collected into one micropipette
and the volume of each sample is calculated by measuring the length of the
fluid column using a Vernier micrometer and a stereo-microscope. The volumes
of sequential 1-minute samples collected from the same gland duct vary very
little over periods up to 4 hoursc We used this method to show that the
rate of secretion from the glands was increased by electrical stimulation of
the vagus nerves and by intravenous injection of phenylephrine (30, 31).
214
-------
K
Figure 5. Ussing chamber, glass perfusion chamber/ and electrical
2
circuits. A piece (1.2 cm ) of trachea (A) bathed on each surface by Krebs-
Henseleit solution wanned to 37°C in a heating jacket (B) which is connected
to a water pump and heater (C). The solution is circulated and oxygenated
by bubbling it with 95% O , 5% CO (D). Electrical potential difference is
measured with KCl-agar bridges (E) connected via calomel half cells (F) to
an electrometer (G). The trachea is short-circuited by passing current to
the electrolyte solution via saline-agar bridges (H) from an external source
(I) regulated by an automatic voltage clamp (J) to keep the spontaneous
electrical potential difference at zero. The short-circuit current is mea-
sured by a milliammeter (K).
215
-------
VITRO TECHNIQUES
The effect of physiological and pharmacological agents on secretion of
mucus cannot at present be adequately studied using only tissues ^.n vivo.
Therefore, we developed an _in vitro preparation of cat trachea for the study
of mucus secretion. We mount pieces of the anterior part of the trachea
2
(exposed area, 1.3 cm ) as flat sheets between the two halves of a modified
Ussing chamber (Fig. 5). The pieces of tissue, composed of pseudostratified
epithelium, submucosal glands, connective tissue, and cartilage, are approxi-
mately 1 mm thick. We place a thin parafilm ring between the epithelial
surface of the tissue and one-half of the Ussing chamber to help form a seal
between the tissue and the chamber, and to help minimize edge damage.
One-half of the Ussing chamber has six sharpened pins equally spaced
around the medial edge which we push through a section of the trachea, pinning
it to a block of paraffin wax. The pins hold the tracheal section flat across
the chamber. After cutting the section free from the remaining trachea, we
insert the pins into corresponding holes in the other half of the chamber
to close it. We connect each half of the Ussing chamber to a glass perfvision.
chamber (MRA, Clearwater, Florida) and perfuse both sides of the chamber with
10 ml of oxygenated (95% 0 , 5% C02) Krebs-Henseleit solution, warmed to
37°C to maintain viability (Fig. 5).
We measure electrical potential difference across the epitheliun via
two agar-KCl (1 M) bridges positioned in the electrolyte solution on either
side of the trachea 2 mm from the surface and connected through calomel half
cells to a high impedance millivoltmeter. We measure the short-circuit current
2
(expressed as uA/cm of tissue) via two agar-NaCl (0.15 M) bridges which
carry direct current to the electrolyte solution from an automatic voltage
clamp connected to the agar-salt bridges by silver/silver chloride junctions.
To calculate the resistance of the tissues, we divide the potential difference
by the short-circuit current. The relationship between the clamped voltage
and the current needed to clamp it is linear over the range +40 mV to -60 mV.
Potential difference and short-circuit current, measured every 15 minutes
during the experiment, are used to monitor the viability of the tissue.
To study the effects of a drug on sulfated mucin secretion, and Cl~ and
Na fluxes, we measure sulfated mucin secretion, bidirectional fluxes of Cl~,
and bidirectional fluxes of Na in separate pieces of tissue before and after
the addition of the test drug.
35
[ S]sulfate is taken up by the mucus-secreting cells of the cat trachea
and is released into the airway lumen bound to glycoproteins (15). For this
reason, we use the secretion of bound s into the luminal side of the chamber
as a measure of sulfated glycoprotein secretion. We add 1.0 md sodiun [35sj-
sulfate to the submucosal side of the tissue; every 30 minutes we collect the
216
-------
solution bathing the luminal side of the tissue and replace it with fresh Krebs-
Henseleit solution* Samples are collected for a control period of 3-4 hours.
The test drug is added to the submucosal side of the tissue for one 30-minute
sampling period and subsequently washed out. Samples are collected for 1-2
hours after the drug is washed out. The samples obtained are placed in dia-
o
lysis tubing bags (average pore radius permeability 24 A, VWR Scientific)
and dialyzed against distilled water to remove unbound [ S]sulfate. Up to
30 of these samples are placed in 4 liters of distilled water; this water is
exchanged six times during a 48-hour period. To each of the first four
volumes of dialysis water we add nonradioactive sodivn sulfate (0.5 g) to
displace noncovalently bound [ S]sulfate, and we add sodiun azide (0.5 g)
to prevent fungal and bacterial growth. The last two dialyses are against
20 mM phosphate buffer, which helps to disperse the mucins. On the completion
of dialysis, a 0.6 ml aliquot of each sample is taken, mixed with 4.0 ml of
scintillation fluid (PCS; Amersham) and the bound S is counted in a 8-
scintillation counter (Liquid Scintillation System HK III, Searle Analytic).
The output is expressed as counts per minute per square centimeter of tissue
per hour (cpm/on .hr).
f _ ^.
To measure the fluxes of Cl and Na across the tissue, we add Krebs-
36 22
Henseleit solution which contains either 5-10 uCi Cl or 2-5 uCi Na to
one side of the chamber, and measure the rate of appearance of radioactivity
on the other side. We obtain samples by collecting the solution from the
appropriate side of the chamber and replacing it with fresh Krebs-Henseleit
solution every 30 minutes. We collect samples for a control period of 2 hours,
add the test drug to the submucosal side of the tissue for one 30-minute
sampling period, and subsequently remove the drug by washing* Samples are
collected for 1 hour after the drug is removed. A 0.6 ml aliquot of each of
these samples is taken, mixed with 4.0 ml scintillation fluid, and the radio-
activity counted in a ft-scintillation counter. From the specific activities
36 22
of the Cl and Na source solutions we are able to calculate the ion fluxes
in micro-equivalents per square centimeter of tissue per hour (uEq/cm .hr).
When we have measured the flux of an ion in one direction, we wash out both
sides of the chamber and measure the flux of the same ion in the opposite
direction, before and after the test drug, in the same piece of tissue.
All fluxes are measured under open-circuit conditions, except for a few
seconds (less than 5) every 15 minutes when short-circuit current is measured.
Electrical potential difference and short-circuit current are measured every
5 minutes during the 30-minute period that the test drug is in the chamber.
This new method for studying respiratory tract secretion has the following
advantages over other in vitro methods: We can measure mucus output and ion
transport in similar pieces of trachea from the cat, under well-controlled
conditions for periods up to 10 hours. We can assess the viability of the
217
-------
.> & w ^PJTe cW»*. '•> -frVvjk .JS&fS 9 „'
SL^ktff^«^^M^§,
:T
Figure 6. Electron micrograph of a ciliated cell prepared from canine
tracheal epithelium which had been mounted in an Ussing chamber for 8 hours,
The ultrastructure of the cell and junctions appear normal.
218
-------
tissue by electrical measurements during the experiments and by electron
microscopy after the experiments. Using this method we found that an a-
adrenergic agonist (phenylephrine) and a &-adrenergic agonist ( terbutaline)
both stimulated the secretion of sul fated mucus and net ion movement into the
lumen (9, 27). The effects of a-adrenergic and {J-adrenergic stimuli were
different. Phenylephrine caused large increases in the output of sulfated
mucin and in the net fluxes of Cl and Na toward the lumen; terbutaline
caused a moderate increase in the output of sulfated mucin and a small in-
crease in net Cl flux toward the lumen; it did not affect either unidirectional
flux of Na . From these findings we predict that phenylephrine will cause
more ion-mediated water secretion than terbutaline. Thus, phenylephrine may
act to produce more water in the respiratory tract secretions.
Originally we used trachea mounted in an Ussing chamber to study ion
movement across canine tracheal epitheliun (26) (Fig. 6). In epithelia, ion
movement induces transepithelial movement of water by creating local osmotic
gradients in the lateral intercellular spaces (13, 29). Therefore, we rea-
soned that active ion transport was likely to regulate the rate of water
secretion into the airway. Changes in ion-mediated secretion of water may
alter the consistency of respiratory tract fluid and change the depth of the
periciliary fluid layer. The periciliary fluid layer is important for the
regulation of mucociliary clearance. With too much periciliary fluid, the
propulsive effect of the cilia will not reach the mucus gel layer; with too
little periciliary fluid, the ciliary motion will be blocked by the weight
and resistance of the gel layer. Thus, the regulation of water secretion into
the periciliary fluid could be an important determinant of mucociliary clear-
ance rates.
To study ion movement we used Ussing1 s short-circuit current method and
the posterior membranous part of the trachea of dogs. We found net movement
— 2 +
of Cl toward the lumen (2.7 + 0.6 wEq/cm .hr) and net movement of Na toward
the submucosa (0.8 +_ 0.2 uEq/on .hr) . These net ion fluxes measured under
short-circuit conditions were associated with a spontaneous transepithelial
2
electrical potential (30.7 + 2.7 mV) and a short-circuit current (108 ^ 8 uA/cm )
(26). Thus, Cl and Na must be actively transported across canine tracheal
epithelium. By forming local osmotic gradients ions may regulate movement
of water into the tracheobronchial sections (5, 24).
Mediators and drugs which mimic the actions of the autonomic nervous
system affect ion movement across the trachea. Histamine increased net move-
ment of Cl and Na toward the tracheal lumen; the response was prevented by
an H -antagonist but not by an H -antagonist (20). Acetylcholine increased
1 - -t- ^
net movement of Cl and Na toward the lumen; the response was prevented by
small concentrations of atropine (19). Terbutaline, a specific 8 -adrenergic
— +
agonist, increased net movement toward the lumen of Cl , but not of Na ; the
219
-------
Figure 7. Freeze-fracture through the tight junction of a ciliated cell in cat trachea which was
removed from an Ussing chamber after 6 hours. The junction is formed by parallel strands with a few inter-
connections*
-------
effect was prevented by propranolol (6, 12). We speculate that ion-mediated
water secretion into the airway can be altered by mediators and neurotrans-
mitters.
Active transport in tracheal epithelium was affected by drugs which in-
hibit ion transport in other epithelia. Furosemide reduced net Cl movement
toward the lumen when added to the submucosal side/ but not when added to the
luminal side of the trachea (8). Ouabain was bound to more sites on the sub-
mucosal membranes than the luminal membranes of the tracheal surface epithelial
cells (32); the drug inhibits Na transport when added to the submucosal side
of the trachea but has little effect when added to the luminal surface of the
epithelium. The transepithelial movement of Cl depends on co-existing active
Na transport, since net Cl movement is greatly reduced by replacement of
Na in the bathing solution (1, 21, 33) and by adding ouabain, an inhibitor
of sodium pumps, to the submucosal bathing solution (1, 33). Further studies
will be necessary to determine the cells responsible for the active ion trans-
port and the exact systems involved.
We use the in vivo preparation of cat trachea in an Ussing chamber to
study epithelial permeability and electrical properties and the pattern of
tight junction strands in the same piece of epithelium (18). In each tissue
we measure electrical potential difference, short-circuit current, and elec-
trical resistance for periods up to 6 hours. Simultaneously, we measure
14
the flux of C-sucrose (an extracellular tracer) from submucosa to lumen dur-
ing 30-minute periods. At a predetermined time during the experiment, we re-
move the tissue, fix it, and prepare it for freeze fracture. Small pieces of
the trachea are fractured and replicated in a freeze-etch device (Balzers,
Liechtenstein) with a double replica stage. We use electron micrographs of
freeze-fractured tracheal tight junctions (Fig. 7) to quantitate the changes
in tight junctions associated with changes in electrical resistance and
permeability of the epithelium. We used this method to study the effect of
removing Ca from the bathing solutions on the properties of the epithelium.
The Ca -free solution caused a decrease in electrical resistance, an increase
14
in permeability to C-sucrose, and produced areas of disarray and reorientation
of the tight junction strands (18).
SUMMARY
The control systems for the production and removal of respiratory tract
secretions may be altered in disease. For instance, patients with cystic
fibrosis do not adequately clear the sticky secretions which form in their
airways, patients with severe asthma plug their small airways with altered
mucus, and patients with chronic bronchitis produce excessive amounts of se-
cretion that must be removed from the airway. Direct methods for studying
221
-------
the regulation of the system in man are not available because the mucous glands
are inaccessible and the secretions obtained at the mouth are contaminated.
Previous methods for studying the respiratory tract secretions in animals
did not identify the sources of the secretions* We have developed new tech-
niques using animal tracheas in vitro and in vivo which allow us to study the
submucosal glands. We have shown that the glands have a secretory response
to cholinergic agonists and stimulation of the vagus nerve, and to a-adrenergic
and fj-adrenergic agonists.
Vfe have shown that tracheal epithelium actively transports Cl~ and Na+
and that the net movement of these ions toward the lumen is increased by
autonomic agonists. Net movement of these ions may be an important control
of ion-mediated water flux into the lumen. Furthermore, changes in the per-
meability of the tissue may modify ion-mediated water movement. We have shown
that tight junctions can be reversibly altered by changes in their external
milieu. We speculate that epithelial permeability may also be modified by
the autonomic nervous system. In the future, we expect that a better under-
standing of the controls of airway epithelial functions will lead to advances
in the treatment and prevention of airway disease.
ACKNOWLEDGMENTS
We thank our colleague Todd Lempert for preparing the electronmicrographs,
The studies on tight junctions were performed in collaboration with Dr. D. s.
Friend. Dr. Davis is the recipient of a NHLBI Young Investigator Research
Award HL-21150. Some of the reported studies were supported by grants from
the U.S. Public Health Service—Program Project HL-06285, Pulmonary SCOR
Grant HL-19156, and HD-10445—and by Cystic Fibrosis Foundation Grants.
REFERENCES
1. Al-Bazzaz FJ, Al-Awqati Q: Interaction between sodiun and chloride
transport in canine tracheal mucosa. J Appl Physiol 46:111-119/ 1979
2* Alstead S: Potassium iodide and ipecacuanha as expectorants. Lancet
2:932-933, 1939
3. Boyd EM, Jackson S, Ronan M: The effect of sympathomimetic amines upon
the output of respiratory tract fluid in rabbits. Am J Physiol 138:
565-568, 1943
4. Boyd EM: in: Respiratory Tract Fluid. Springfield, Illinois, Charles
C. Thomas, 1972, pp 100-122
5. Davis B, Marin MG, Olver RE, Nadel JA: Active ion transport across
canine tracheal epithelium: A possible control system for mucociliary
clearance. Chest 67:57(3), 1975
222
-------
6. Davis B, Marin MG, Nadel JA: 8-Adrenergic receptor in canine trachea
epithelium. Am Rev Respir Dis 111:947, 1975
7. Davis B, Marin MG, Fischer S, Graf P, Widdicombe JG, Nadel JA: New
method for study of canine mucous gland secretion in vivo; Cholinergic
regulation. Am Rev Respir Dis 113:257, 1976
8. Davis B, Ueki I, Bruderman I, Marin M, Nadel JA: Submucosal action of
furosemide on chloride ion movement across canine tracheal epithelium.
Am Rev Respir Dis 115:320, 1977
9. Davis B, Phipps RJ, Nadel JA: A new method for studying tracheal se-
cretions ^n vitro; Effect of adrenergic agonists in cats. Chest 75:
224-225, 1979
10. Davis B, Chinn R, Graf P, Popovac D, Nadel J: Effects of phenylephrine
and superior laryngeal nerve stimulation on submucosal gland secretion
in canine trachea jln vivo. Clin Res 27;55A, 1979
11. Davis B, Chinn R, Graf P, Popovac D, Nadel J: Mucous gland secretion
in canine trachea: Effects of superior laryngeal nerve stimulation
versus phenylephrine injection. Fed Proc 38:1329, 1979
12. Davis B, Marin MG, Yee JW, Nadel JA: Effect of terbutaline on move-
ment of Cl and Na across the trachea of the dog _in vitro. Am Rev
Respir Dis (In press, 1979)
13. Diamond JM, Bossert WH: Standing gradient osmotic flow: A mechanism
for coupling of water and solute transport in epithelia. J Gen Physiol
50:2061-2083, 1967
14. Florey H, Carleton HM, Wells AQ; Mucus secretion in the trachea.
Br J Exp Pathol 13:269-284, 1932
15. Gallagher JT, Kent PW, Passatore M, Phipps RJ, Richardson PS: The
composition of tracheal mucus and the nervous control of its secretion
in the cat. Proc R Soc London (Biol) 192:49-76, 1975
16. Gunn JA: The action of expectorants. Br Med J 2:972-975, 1927
17. Hamilton WFD, Palmer KNV, Gent M: Expectorant action of bromhexine in
chronic obstructive bronchitis. Br Med J 3:260-261, 1970
18. Lempert T, Chinn R, Friend D, Nadel J, Davis B: Effects of Ca on
epithelial permeability and tight junctions in cat trachea in vitro.
Fed Proc 38:922, 1979
19. Marin MG, Davis B, Nadel JA: Effect of acetylcholine on Cl" and Na
fluxes across dog tracheal epithelium _in vitro. Am J Physiol 231:1546-
1549, 1976
20* Marin MG, Davis B, Nadel JA: Effect of histamine on electrical and
ion transport properties of tracheal epitheliun. J Appl Physiol 42:
735-738, 1977
223
-------
21. Marin MG, Zaremba MM: Influence of Na on stimulation of net Cl
flux by acetylcholine and terbutaline in dog tracheal epithelium. Fed
Proc 37:514, 1978
22. Nadel JA, Davis B: Autonomic regulation of mucus secretion and ion
transport in airways. In: Asthma: Physiology, Inununophannacology,
and Treatment (Lichtenstein LM, Austen KF, eds). Second International
Symposium. New York, Academic Press, 1977, pp 197-210
23. Nadel JA: Autonomic control of airway smooth muscle and airway secretions,
Am Rev Respir Dis 115 (6, part 2 of 2 parts):117-126, 1977
24. Nadel JA, Davis B: Regulation of Na and Cl transport and mucous gland
secretion in airway epitheliun. In: Respiratory Tract Mucus (Ciba
Symposium). Amsterdam, Elsevier, 1978, pp 133-147
25. Nadel JA, Davis B, Phipps RJ: Control of mucus secretion and ion trans-
port in airways. Ann Rev Physiol 41:369-381, 1979
26. Olver Re, Davis B, Marin MG, Nadel JA: Active transport of Na"1" and
Cl across the canine tracheal epitheliun in vitro. Am Rev Respir Dis
112:811-815, 1975
27. Phipps R, Davis B, Nadel JA: Effect of terbutaline on mucin secretion
in cat airway using a new _in vitro method. Fed Proc 37:221, 1978
28. Potter JL, Matthews LW, Lemm J, Spector S: Human pulmonary secretions
in health and disease. Ann NY Acad Sci 106:692-697, 1963
29. Schultz SG: The role of paracellular pathways in isotonic fluid trans-
port. Yale J Biol Med 50:99-113, 1977
30. Ueki I, German V, Nadel J: Direct measurement of tracheal mucous gland
secretion with micropipettes in cats: Effects of cholinergic and
a-adrenergic stimulation. Clin Res 27:59A, 1979
31. Ueki IF, German VF, Nadel JA: Micropipette measurement of airway sub-
mucosal gland secretion: Autonomic effects. Am Rev Respir Dis (In
press, 1979)
32. Widdicombe JH, Yee JY, Nadel JA: Site of Na-pumps in dog tracheal
epithelium. Fed Proc 37:221, 1978
33. Widdicombe JH, Ueki IF, Eruderman I, Nadel JA: The effects of Na-sub-
stitution and ouabain on ion transport by the dog's tracheal epitheliun.
Am Rev Respir Dis (In press, 1979)
224
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DISCUSSION
DR. WASHERMAN: You've presented your mucus secretion in terms of S label,
although your autoradiographs would make you think that it -was in mucus.
How do you, in fact, know that your counts were in mucus?
DR. DAVIS: Gallagher e_t al. showed that S is incorporated into mucus
glycoproteins secreted into cat trachea in vivo. [Gallagher JT, Kent PW,
passatore M, Phipps RJ, Richardson PS: The composition of tracheal mucus
and the nervous control of its secretion in the cat. Proc R Soc London
(Biol) 192:49-76, 1975].
225
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Mucus Glycoprotein Secretion by Tracheal
Explants: Effects of Pollutants
J. A. Last and T. Kaizu
University of California
Davis, California
We have been using the tracheal explant system, developed several years
ago in the laboratories of Dr. Lynn Reid (13) and others (2, 3, 5-7, 15),
as a quantitative assay for monitoring effects of air pollutants and other
pneumotoxins on the respiratory epithelium. We have used an "in vivtro"
approach, as indicated schematically in Ficjure 1, featuring exposure of
animals (usually rats) in vivo to known amounts of pneumo toxin, followed
by removal of their tracheae for assay of effects in vitro* With small
animals such as rats, we routinely incubate the tracheae in tissue culture
medium containing radioactively labeled precursors. The culture medium is
then removed (Fig. 1) and an aliquot is treated with 5% trichloroacetic acid
to precipitate the proteins present. With a suitable choice of labeled
precursor and incubation time for the rat tracheal explants, essentially
all of the acid-precipi table radioactivity in the medium is mucus glycopro-
teln, as we will demonstrate. What are the characteristics of such an assay?
Figure 2 shows the time course of incorporation of [ H] glucosamine by tracheal
in such an assay system. The appearance of acid-precipi table gly-
coproteins in the culture medium can first be observed after about three
hours. The incorporation rate is linear between about 8 hours of incubation
and at least 50 hours, the maximum duration of incubation, as shown in Figure
2. In some experiments the time course has remained linear for as long as
96 hours (data not shown). For routine assay purposes we generally incubate
tracheal explants for about 24 hours. This is a convenient duration of in-
227
-------
Figure 1. Schematic diagram of a typical "in vivtro" experiment. Rats
are exposed to a pneumotoxin (in this case, ozone) in vivo« Tracheae are
then excised and their secretion rates are determined in vitro in tissue
culture medium containing suitable labeled precursor(s). After incubation,
an aliquot of medium is precipitated with trichloroacetic acid and filtered.
The filters are dried and counted by liquid scintillation (cf., ref. 12).
I28
20 30
HOURS
Figure 2. Time course of incorporation of [ H]glucosamine by rat tracheal
explants into acid-precipitable glycoproteins released into the culture medium
(cf., ref. 12).
228
-------
0 140
c
o
O
•s
o" !20
•—
0
0
-------
Table 1. Effect of Exposure of Rats to Ozone on the Rate
of Glycoprotein Secretion by Their Tracheal Explants
Ozone
Secretion rate,
concentration Control
(ppm)a (cpm)
0.6
0.4
0.2
19,110 + 2,230
14,913 + 2,309
16,870 + 2,770
16,
14,
17,
mean
- SD
Exposed
(cpm)
972 +
327 +
034 +
2,269
2,118
2,503
Total
(n)
24
24
22
Exposed/
control
89
96
101
P value
$
NS
02
aExposures were for 8 hours per day for 3 days to the indicated concen-
tration of ozone.
Not significant.
exposure of rats to ozone, but also to the concentration of ozone to which
the rats are exposed. The dose-response characteristics of this system to
ozone after 3 days are shown in Table 1. A statistically significant de-
crease in secretion rate is observed with 0.8 parts per million of ozone
(see Figure 3) and with 0.6 parts per million of ozone (Table 1). An in-
significant decrease is observed at 0.4 parts per million and no effect is
seen at 0.2 parts per million (Table 1). The data of Table 1 also illustrate
one of the difficulties in working with the tracheal explant system. Com-
parisons between matched controls and experimental rats are very precise with-
in a given experiment. In a single experiment the size of tissue slices
and the matching of rats can be controlled rigorously. A single batch of
medium can be used so there are minimal errors introduced by pipetting and
addition of labeled precursor to the medium. On the other hand, comparisons
between separate experiments are much more difficult, as shown in Table 1.
Small differences in technique from day to day result in variations in the
absolute value for control rats such that data within a chronic exposure
regimen must usually be normalized to control values; hence, the expression
of the data in Figure 3 as percent of control. In addition, growth of the
rats within a given chronic exposure experiment is appreciable and can also
affect these results.
The tracheal explant system can be used with animals other than rats.
We have demonstrated elsewhere (10) that tracheal explants from a wide variety
of experimental animals, as well as human tracheal and/or bronchial explantsv
can be used in this system. In Table 2 we present a comparison of tracheal
explant secretion rates for multiple tracheal slices prepared from individual
bonnet monkeys exposed to either 0, 0.5, or 0.8 parts per million of ozone
for 7 days. While the increased secretion rate after 7 days suggests that
230
-------
Table 2. Tracheal Explant Secretion Rates for Tracheal
Slices from Monkeys Exposed to Ozone
Controls 0.5 ppm 0 0.8 ppm 0,
Secreted precipitable
labeled molecules
after 7 days of
O exposure 12,362 + 2,108 16,679 + 3,767 21,949 + 6,383
there may be a species difference between monkeys and rats, we obviously
need to look at many more monkeys to confirm these results. Nonetheless,
clearly the technique is applicable to animals other than rats, especially
non-human primates.
We can also use this system to study effects of pollutants other than
ozone, For example, we have studied effects of exposure of rats to sulfuric
acid aerosols, alone and in combination with ozone (11). Some representative
results we have observed in this assay with sulfuric acid aerosols and/or
ozone are presented in Table 3. While 0.5 ppm of ozone by itself causes a
decrease in the observed secretion rate after three days, the combination
of sulfuric acid aerosol with ozone causes a significant increase in this
measured value. Similarly/ these agents potentiate one another after 14
days of continuous exposure (Table 3). Sulfuric acid aerosol, by itself,
is without affect on matched rats, as measured by their tracheal explant
secretion rate, at these levels tested (11). Thus, exposure of rats to the
combination of ozone and sulfuric acid aerosol (0.5 ppm of ozone; 0.01*5
mg/m of sulfuric acid aerosol, ref. 11) causes changes in the tracheal ex-
plant secretion rate that are not only quantitative, but are qualitative over
the 2- to 3-day exposure interval. We are presently vigorously studying the
biochemical and/or cellular basis of such pollutant-induced changes in se-
cretion rates.
Once we had documented quantitative changes in mucus secretion rates
by tracheal explants from rats exposed to various pollutants, we turned our
attention to the question of whether there was also a qualitative change in
the mixture of glycoproteins being secreted by the explants. To separate
the mucus glycoproteins into various fractions on ion-exchange columns, it
was first necessary to solubilize these compounds. In the absence of prior
treatment to solubilize the glycoproteins, yields from ion-exchange columns
were very low and erratic. Other workers have used reduction and alkylation
of sulfyhydryl groups in the protein backbone involved in glycoprotein cross-
linking for this purpose (1, 14); however, since we were most interested in
231
-------
Table 3. Rate of Secretion of Mucus Glycoproteins by Tracheae
from Rats Exposed to H2804-03 Mixtures
Days exposed
in vivo
3
14
3
3
14
H_SO.
2 43
(mg/m )
1.1
1.1
1.1
None
None
03 (ppm)
0.5
0.5
None
0.5
0.8
Rate of secretion
(% of control value)
127
132
100
84
112
P value
<0.005
<0.001
NSb
NS
NS
Tracheal explants were incubated 22-24 hours; in all cases, n
slices.
NS, not significant.
14
determining changes in the carbohydrate side chains rather than the protein
backbone, we chose limited digestion with papain as our initial solubilization
step, as shown schematically in Figure 4. After papain digestion we can
chromatograph the resultant mixture of glycoproteins on DEAE-cellulose and
recover the applied radioactivity essentially quantitatively. As shown in
Figure 5, an idealized version of such a column, the [ H] glucosamine- labeled
glycoproteins separate into one neutral and two acidic glycoprotein fractions
under the conditions we initially used for this purpose ( a linear gradient
from 0 to 1 molar lithium chloride in 50 mM sodium acetate-acetic acid buffer
at pH 4.4). We observe three peaks (A, B, and C) that are eluted at the same
positions from the DEAE-cellulose column whether we use [ H] glucosamine or
14 35
[ C] threonine as the precursor for the glycoproteins. When [ S] sulf ate
is used as the labeled precursor, most, if not all, of the label appears in
the most acidic fraction from the DEAE-cellulose column, the fraction that
we call peak C. This material is chrcmatographically indistinguishable from
3 14
peak C labeled with [ H] glucosamine or with [ C] threonine. Both of these
two acidic peaks are eluted from the column at positions distinctly different
from those of hyaluronic acid or chondroitin sulfate treated in an identical
manner, added as internal standards and assayed chemically by determination
of their constituent uronic acid components. If a mixture of the two acidic
fractions is incubated at 37° in an appropriate buffer overnight for 18-24
hours, then rechromatographed, their elution positions are unchanged from
the original chromatographic mobilities observed. If the same mixture of
fractions B and C is incubated in buffer containing neuraminidase from
Vibrio cholerae , an enzyme that specifically cleaves sialic acid residues
from the non-reducing end of oligosaccharide chains/ we then observe only
one peak after incubation; this single peak is chrcjiatographically indistin-
232
-------
Tracheal Explant
Tisl
1 Incubate for 24
1 hours at 37*
Incubated tissue
(Extract with HO
fat 100* x 2 min.
sue
ract
SpAit Non-dia
tissue
Culture medium
I Dialysis
bluffer
Ly cable- 1
against
Diffusate
Treat with papain
Papain treated
Precipitate
Super
ute, Cl3CCOOH-treated
Dialysis against
buffer
Non-dialyzable-2
Chromatography on
DEAE-cellulose with
gradient elution
Diffusate
Fraction A
T
Fraction B
T
Fraction C
Figure 4. Diagrammatic representation of the techniques used to purify
mucus glycoproteins from the culture medium of tracheal explants.
§
"S
1.0
.01
Fraction number
Figure 5. Idealized representation of the elution of labeled glyco-
proteins, treated as in Figure 4, from a column of DEAE-cellulose.
233
-------
OLIGOSACCHARIDE SIDE CHAINS
(Gal NAc, Glc NAc, Gal + Pucose, sialic acids)
Figure 6. Idealized structure of tracheobronchial mucus glycoproteins.
guishable from the less acidic peak, peak B of Figure 5. Thus, we can con-
clude that the more acidic peak (peak C) from the DEAE-cellulose column
contains glycoproteins containing oligosaccharide side chains with terminal
sialic acid residues in their structure. The sulfur-35 labeling experiment
suggests that, in addition to its sialic acid content, the more acidic fraction
is also more heavily sulfated than is fraction B.
In Figure 6 we indicate schematically the structure of the tracheobron-
chial mucus glycoproteins that we are studying in such an experiment. They
are long extended proteins in a backbone configuration with short oligosaccha-
ride side chains coming off, perhaps as frequently as every fifth or sixth
amino acid residue of the protein. The protein backbones are high in serine
and threonine, to the extent of about 40-50% of their total amino acid content
and the oligosaccharide side chains occur attached to serine or threonine
residues in ester linkages through the hydroxyl groups of the serine or threo-
nine residues. The oligosaccharide side chains contain N-acetylglucosamine,
N-acetylgalactosamine, galactose, fucose, and, when present, sialic acids.
These are presumably the only sugars present. The length of the side chains
is a subject of some controversy, but they are usually assumed to be approxi-
mately six or eight residues long (8). We have evidence, based on cleavage
of the serine or threonine linkages to the oligosaccharides under alkaline
conditions, that the oligosaccharide side chains that we are studying are
relatively homogeneous in size, as indicated by their mobility on columns
of Sephadex G-50 (data not shown).
234
-------
4
o
* B
E
o.
u
6
C-H
10 20
FRACTION NUMBER
30
3
9 10 15 20
FRACTION NUMBER
Figure 7. Elution of [ HJglucosamine- labeled glycoproteins , prepared
from exhaustively dialyzed tracheal culture medium, from a column of BioGel
AlSOm (cf . ref . 12) .
Figure 8* Elution of S-labeled glycoproteins, prepared as in Figure
4, from a BioGel A 150m column. '•
One technique that has been used extensively for the characterization of
mucus glycoproteins secreted by cultured tracheal explants has been fraction-
ation upon sizing columns, usually either agarose or polyacrylamide (4-6, 9).
Under appropriate conditions of chromatography, two peaks of mucus glyco-
proteins are observed to be eluted from such sizing columns. The first peak,
which elutes at approximately the void volume of the column, is extremely
large and presumably represents aggregated glycoproteins. The second peak,
which is included in the column, may also be shown to be a glycoprotein
fraction (6). The ratio of glycoproteins in the two peaks has been used
by others (5), as well as by ourselves (12), as a probe for observing changes
in the structure of glycoproteins being secreted by tracheal explants. We
have published (12) evidence that chronic exposure of rats to ozone causes
changes in the ratio of the two peaks from a BioGel A150m column such that
the lower-molecular-weight included peak is augmented at the expense of the
first peak, as shown in Figure 7. In simpler terms, there was a shift in the
relative number of counts from peak I to peak II upon exposure of the rats
235
-------
to ozone in those experiments. We therefore turned our attention to the
question of whether the peaks from the BioGel A150m column corresponded in
any way to the fractions we could isolate by chromatography on DEAE-cellulose
using the techniques illustrated schematically in Figure 4. However, this
experimental design required that we omit the papain digestion step. Thus,
the isolated fractions from the BioGel columns were applied directly to
DEAE-cellulose and eluted under the gradient conditions illustrated in Figures
4 and 5. Since we anticipated low yields by this procedure, we labeled the
glycoproteins with sulfate rather than with [ H]glucosamine to enhance the
counting efficiency. Under these conditions, we observed a third fraction
eluted from the BioGel column, as a shoulder on the high-molecular-weight
35
side of peak II, as shown in Figure 8. Thus BioGel chromatography of s-
labeled material gave us three fractions, more or less well resolved from
each other, which we labeled ^, S, and C as shown in the figure. To make a
long story short, each of the three fractions A, j), and c: corresponded to
one of the peaks from the DEAE-cellulose column, with only minor cross-con-
tamination of the fractions with one another. Thus fraction A, the high-
molecular-weight peak I, corresponded to the neutral glycoprotein fraction
from the DEAE-cellulose fraction. Similarly fraction Ct the included peak II
corresponded to the first acidic glycoprotein fraction B from DEAE-cellulose.
Finally fraction B, the high-molecular-weight shoulder on peak II, correspond-
ed to fraction C, the most acidic glycoprotein fraction from DEAE-cellulose.
Thus, we can express the results of our earlier experiments, in which we had
shown a shift in the ratio of the glycoproteins from peak I to peak II after
exposure of rats to ozone, in more biochemical terms. The chronic exposure
of rats to ozone apparently induces a shift in the composition of the mucus
glycoproteins being secreted by their tracheal explants such that the product
is relatively more acidic than is the mixture of mucus glycoproteins from
control rat explants. This result is not unexpected (11, 13), considering
the results of others who have exposed rats to various irritant stimulants
under chronic conditions.
We have recently improved our DEAE-cellulose chromatographic techniques
by changing to a less steep lithium chloride gradient. Under these conditions
one neutral fraction is still eluted, but four acidic fractions are now
eluted rather than two. Each of these four acidic fractions is different
from the other as evaluated by cellulose acetate electrophoresis or by re-
chroma tography on DEAE-cellulose. We are currently using immobilized lectin
columns as a technique for probing the structure of the sugars on the oligo-
saccharide side chains within the components of the various fractions. We
have been able to document shifts in the ratios of these chromatographic peaks
relative to one another upon exposure to ozone in this new system. For example
236
-------
as shown earlier (cf. Figure 3, Tables 1 and 3), short-term acute exposure
to ozone at 0.5 parts per million for three days results in a significant de-
crease in the mucus secretion rate by the tracheal slices. When the papain-
treated glycoproteins from control and exposed rat tracheal explants are chro-
matographed on DEAE-cellulose, we find a shift to a mixture that is relatively
deficient in acidic mucus glycoproteins from the tracheae of ozone-exposed
rats. Experiments are currently in progress to try to elucidate which acidic
fraction is being decreased under these circumstances; preliminary data im-
plicate the sialic acid-containing fraction as being present in relatively
smaller amounts.
In conclusion, we have developed the tracheal explant system as a tech-
nique for quantitating the effects of exposure to air pollutants and other
pneumotoxins on the respiratory airways of small laboratory animals, and on
non-human primates. These techniques are relatively easy to perform and give
reproducible quantitative results. Thus, any airborne noxious agent can be
evaluated with this system. We are currently concentrating our attention
on trying to elucidate the molecular basis for changes in secretion rates
both in terms of decreased secretion rates in short-term acute exposures to
ozone and increased secretion rates in longer-term chronic exposure regimens
to various pollutants. These techniques promise to have a great deal of value
both for mission-oriented research (screening of airborne pollutants, gasses,
and chemicals for effects of the respiratory airways) and for basic research
studies (elucidation of the structure of tracheobronchial mucus in health
and disease).
REFERENCES
1. Boat TF, Cheng PW, Iyer RN, Carlson DM, Polony I: Human respiratory
tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial
secretions, and sputum of patients with cystic fibrosis. Arch Biochem
Biophys 177:95-104, 1976
2. Boat TF, Iyer RN, Macintyre MD, Carlson DM, Matthews LW: Evaluation
of a culture method for study of human tracheobronchial secretion.
Amer Rev Resp Dis 103:915, 1971
3. Bonanni F, Levinson SS, Wolf G, De Luca L: Glycoproteins from the hamster
respiratory tract and their response to vitamin A. Biochim Biophys
Acta 297:441-451, 1973
4. Chakrin LW, Baker AP, Christian P, Wardell JR: Effect of cholinergic
stimulation on the release of macromolecules by canine trachea in vitro.
Amer Rev Resp Dis 198:69-76, 1973
237
-------
5. Chakrin LW, Baker AP, Spicer SS, Wardel JR, DeSanctis N, Dries C:
Synthesis and secretion of macromolecules by canine trachea. Amer
Rev Resp Dis 1-05:368-381, 1972
6. Ellis DB, Stahl GH: Biosynthesis of respiratory-tract raucins: Incorpor-
ation of radioactive precursors into glycoproteins by canine tracheal
explants in vitro. Biochem J 136:837-844, 1973
7. Formijne P, Van der Schoot JB, De Nie I: On the formation of nucin in
tracheal mucosa of the rat. Biochim Biophys Acta 83:239-241, 1964
8. Gallagher JT, Corfield AP: Mucin-type glycoproteins—new perspectives
on their structure and synthesis. Trends in Biochem Sci 8:38-41, 1978
9. Gallagher JT, Kent PW: Structure and metabolism of glycoproteins and
glycosaminoglycans secreted by organ cultures of rabbit trachea. Biochem
J 148:187-196, 1975
10. Jennings M, Cross CE, Last JA: Glycoprotein synthesis by tracheal ex-
plants from various mammalian species. Comp Biochem Physiol 57A:317-
320, 1977
11. Last JA, Cross CE: A new model for health effects of air pollutants:
evidence for synergistic effects of mixtures of ozone and sulfuric acid
aerosols on rat lungs. J Lab Clin Med 91:328-339, 1978
12. Last JA, Jennings MD, Schwartz LW, Cross CE: Glycoprotein secretion by
tracheal explants cultured from rats exposed to ozone. Amer Rev Resp
Dis 116:695-703, 1977
13. Reid L: Evaluation of model systems for study of airway epithelium,
cilia and mucus. Arch Int Med 126:428-434, 1970
14. Roberts GP: The role of disulfide bonds in maintaining the gel structure
of bronchial mucus. Arch Biochem Biophys 173:528-537, 1976
15. Yeager H, Massaro G, Massaro D: Glycoprotein synthesis by the trachea.
Amer Rev Resp Dis 103:188-197, 1971
238
-------
DISCUSSION
PARTICIPANT: Why did you seem to use electrophoresis more?
DR. LAST: If we take the materials before we digest with papain/ they don't
permeate into SDS or conventional polyacrylamide gels in our hands* After
papain digestion/ we found that the convenience and ease of electrophoresis more
than made up for the problems of quantitation where you can1t recover all the
materials* For routine work, we used cellulose acetate for screening purposes.
However/ for most of our studies we are trying to quantitate glycoproteins made;
we're much more comfortable with the ion-exchange columns, where we can get
quantitative recoveries* The literature on how to do the gel electrophoresis
for mucus glycoproteins is in agreement with most of our experience, and that is
that the yields are low--low and not reproducible.
DR. HU: The first question is about the trachea rings. Do you see any differ-
ence if you pick up the first ring or the last rings from the trachea?
DR. LAST: We're not using rings, we're using slices. For our normal size
rat, we get either two or three slices. The comparison is: Is there a dif-
ference between the laryngeal and the carinal end of the trachea? In our hands,
no.
DR. HU: There's no difference?
DR. LAST: Not in terms of the parameters that we're measuring. I don't
know how much of what we look at comes from the epithelial layer or how much
comes from glands. That, certainly, is a germane question, especially if the
labeled glycoproteins were coming from glands* But that kind of observation
leads me to believe that we are not dealing with a lot of glycoprotein coming
from the glands. I can go further than that. If we used bronchi from larger
animals, if we compared tracheae and bronchi, for example, from a monkey, we
did not see any difference, if we kept the slices identical in size.
DR. HU: What is the size of your column?
DR. LAST: Very small, between 1.5 and 3 cm of depth and about 1 cm in diameter
with the DEAE-cellulose. These are all very much micro techniques.
239
-------
DR. HU: What was the flow rate?
DR. LAST: About 1-1.5 milliliters per minute. The rate of flow affects the
resolution slightly.
DR. NETTESHEIM: I didn11 quite understand how you exclude in these _in vitro
studies the synthetic contribution of the connective tissue and the cartilage,
which are quite active and produce a lot of material that goes into the cul-
ture. Have you tried, for example, to strip the epithelium and see what the
contribution is of the stripped piece?
DR. LAST: We excluded proteoglycan synthesis because of the animal we chose
and the time of incubation in culture. Gallagher and Phipps1 work, for
example, in rabbits suggests that you would be seeing a good deal of proteo-
glycans if we incubated for longer times. Chemically, we can rule out more
than 5% proteoglycans in our system. We've looked for it and it's not there*
As I've said, the yields here are essentially quantitative, so we're not
losing any. When we did comparative studies with other animals, we did
see more acidic components coming off the DEAE-cellulose column, which I
presume may be from proteoglycans, but in the worst case it's no more than
20% of the total if incubation is for about 24 hours. The yield of presvmptivc
glycosaminoglycans increases if we incubated for 72 or 96 hours. We have
chosen our conditions, either luckily or fortuitously, so that proteoglycan
synthesis by the cultured tracheae is not a problem*
240
-------
Mucus and Surfactant Synthesis and Secretion
by Cultured Hamster Respiratory Cells
J. B. Baseman, N. S. Hayes, W. E. Goldman,
and A. M. Collier
University of North Carolina
Chapel Hill, North Carolina
Establishment of eukaryotic cells in monolayer culture as experimental
models has contributed significantly to our current understanding of growth
control and regulation of specialized functions in mammalian cells. The
possibility that single cell types can be examined free from the hetero-
geneity of host tissue and yet retain their biologic function is a major
advantage of tissue culture techniques. Unfortunately, the mammalian lung is
a complex structure with different cells possessing numerous physiologic
roles (3) and therefore provides a difficult primary source of tissue from
which to selectively dissociate particular cell types. Of special interest
are two classes of respiratory epithelial cells whose key functions are the
synthesis and secretion of mucus glycoprotein or pulmonary surfactant, each
essential to normal lung maintenance. Mucus is a viscous material which pro-
tects the tracheobronchial epithelium from inhaled particles and pathogenic
microorganisms by trapping them and permitting their removal via ciliary
activity. Surfactant or disaturated (primarily dipalmitoyl) phosphatidyl-
choline is released onto alveolar surfaces apparently by type II alveolar
cells to reduce the surface tension at the air-liquid interface, preventing
alveolar collapse. Mucus-secreting cells have until recently (6) not been
isolated and grown in pure culture, restricting basic studies on the biology
of the cell as well as properties of mucus production under normal and ab-
normal conditions. Attempts at cultivating type II alveolar cells, the major
in vivo contributor of surfactant, have met with limited success (4, 7, 10).
241
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Type II cells in culture lose their specialized function and characteristic
multilamellar bodies during early passage, and these cultures are often con-
taminated with fibroblasts. Transformed cells which apparently arise during
passage of the type II cell cultures have been investigated (12), but these
cells are clearly different from the original parent cell type. In this
paper we discuss selected properties of the mucus-secreting cell which we
have isolated and passaged in culture for the first time (6) and report on
the application of similar methodology to derive from hamster lung tissue
proliferating cells which maintain a specialized capacity to synthesize and
secrete surfactant.
MATERIALS AND METHODS
Respiratory Cell Cultures
Hamster tracheal cells were isolated by the dissociated cell rescue
technique previously described (6) and maintained as monolayer cultures in
nutrient mixture F-12 (GIBCO, Grand Island, N.Y.) with 10% fetal calf serum
(PCS; GIBCO) plus penicillin G (100 units/ml) and streptomycin sulfate (100
pg/ml). This isolation procedure requires repeated protease treatment of
tracheal rings and collection of dissociated cells by sequential gradient
centrifugation.
Primary cultures of hamster lung epithelial cells were prepared by a
similar isolation procedure using tissue from a newborn litter of Syrian
Golden hamsters (Engle Laboratory Animals, Farmerburg, Ind.) less than one
day of age. Lungs were pooled, rinsed several times in nutrient mixture F-12
containing 10% FCS and antibiotics, and minced into smaller fragments prior
to repeated protease treatment and gradient centrifugation. Dissociated
cells were suspended in F-12 medium containing 10% FCS and supplemented with
vitamin A (2 yg/ml; Sigma Chemical Co., St. Louis, Mo.) and epidermal growth
factor (10 ng/ml; Collaborative Research Inc., Waltham, Mass.) during initial
growth. These lung-originated cells as well as the initial tracheal cells
were seeded in appropriate tissue culture vessels and grown to confluence
with half-medium changes at two-day intervals. The latter procedure, which
provided cells with half-conditioned and half-fresh medium during the earli-
est stages of growth, was beneficial for monolayer formation. Cloning of
lung cells was accomplished by releasing cells from the vessel surface during
primary culture with 0.02% ethylenediaminetetraacetate (EDTA) in phosphate
buffered saline (PBS, pH 7.8) and plating them at low density in 100 mm petri
dishes. Individual penicylinders were placed over isolated cell growth and
sealed with silicone grease. Cells were washed in PBS and treated with EDTA-
242
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PBS prior to transfer to culture vessels containing fresh growth medium.
All subsequent growth and passage of cells occurred in nutrient mixture F-12
containing 15% PCS plus antibiotics. These lung epithelial cell cultures
were maintained for five to seven passages after which cells increased in
size/ became more granular, and failed to divide. On one occasion/ a trans-
formed cell clone spontaneously emerged during this senescence of the epithe-
lial cultures. Established lines of lung cells from rat (L2), cat (AKD),
and human (A549) reported to be of type II alveolar cell origin were obtained
from the American Type Culture Collection, Rockville, Md. These cells were
routinely grown in F-12 containing 10% PCS with antibiotics. Generally, all
cultures were kept at 37°C in an atmosphere of 5% CO. in air.
Other Cell Cultures
Primary cultures of chick embryo cells were prepared in Minimum Es-
sential Medium (MEM; GIBCO) plus 10% FCS as previously described (1) and used
during the third passage. Baby hamster kidney (BHK) and mouse (L25) estab-
lished cell lines were also maintained in this medium. These cells were ex-
amined for their ability to synthesize phosphatidylcholine and derivatives.
Radioisotopes
3 14
N-acetyl-D-galactosamine [acetyl- H, 1.5 Ci/mmol] and L-serine [U- C,
156 mCi/mmol] were purchased from ICN Radioisotope Division, Irvine, Calif.
Choline chloride [methyl- H, 69.5 Ci/mmol] was supplied by New England
Nuclear, Boston, Mass.
Detection of Glycoprotein Secretion
Hamster tracheal cells suspended in MEM with 10% FCS were seeded in
MultiWell (Falcon, Oxnard, Calif.) plates (0.5 ml volume/well, 150,000
cells/ml), and monolayer cultures were later radiolabeled with [ H]N-acetyl-
14
D-galactosamine (2.4 wCi/well) and [ C]L-serine (0.8 uCi/well) for 24 hours.
The culture fluid was removed from each well, clarified by centrifugation,
and precipitated with 10% trichloroacetic acid prior to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis as previously detailed (6). Gels
were sliced into 1 mm fractions and radioactivity determined by liquid scin-
tillation spectrometry.
Phospholipid Bioassay
Synthesis of lecithin (phospholipid) and its derivatives was measured
by incorporation of radioactive choline into phosphatidylcholine. For this
243
-------
biosynthetic determination, all cell types, including lung cell cultures
normally passaged in F-12 medium, were grown to confluence in MEM containing
10% PCS. Cell monolayers were washed once with serum-free MEM and incubated
3 2
under the latter conditions for 20 hours with 1.0 uCi [ H]choline per 75 cm
flask. Monolayers were then washed twice with PBS, gently trypsinized, and
total cell numbers determined by counting with a hemacytometer. Cells were
centrifuged at 400 x ^ for 10 minutes and each pellet transferred to a
Potter:Elvehjem tube and homogenized for 10 minutes at room temperature in a
2:1 chloroform-methanol mixture (v/v) according to the Folch method (5).
The extract was mixed with one-fifth its volume of 0.05% CaCl and allowed to
separate into two phases at 4°C overnight. The upper phase and interphase
were discarded. The lower phase was carefully washed three times with upper
phase solvent. The lipid sample was evaporated to dryness under a stream of
nitrogen and redissolved in 2:1 chloroform-methanol mixture (v/v). All samples
were stored at -20°C.
To compare total phospholipid synthesis with secretion among the various
lung cell cultures, each cell type was seeded in 50 cm petri dishes and
grown as described above. Twenty-four hours after addition of radioactive
choline, cells were separated from the culture fluid and lipids extracted
from the cell fraction by the Folch procedure. The fluid containing secreted
phospholipid was mixed with one volume of methanol and two volumes of chloro-
form, shaken well, and placed at -20°C overnight. Lipids in the lower phase
were isolated as previously indicated.
Phospholipid Separation by Thin-Layer Chromatography
Lecithin was identified by one-dimensional thin-layer Chromatography
(TLC). Radioactive samples were applied to a 20 x 20 cm precoated silica
gel 60 TLC plate (0.25 mm thickness). Dipalmitoyl phosphatidy1choline (Sigma
Chemical Co./ St. Louis, Mo.) and a mixture of lecithin and sphingomyelin
(Sigma) were routinely included as standards. Plates were developed for 90
minutes with a 65:25:4 chloroform-methanol-water mixture (v/v) and dried
for 30 minutes at room temperature. Spots were stained by 2,7-dichloro-
fluorescein (Sigma) dissolved in acetone and detected by ultraviolet light
illumination. The spot correlating with the R value of lecithin was scraped
into ACS scintillation fluid (Amersham, Arlington Heights, 111.) for radio-
active determination.
Isolation of Disaturated Phosphatidylcholine
Since lecithin and its derivatives have similar R values and are dif-
ficult to separate by TLC, the method of Mason and co-workers (8) was employed
to isolate disaturated phosphatidylcholine (DPC). In general/ the lipid
244
-------
sample was reacted with 3.1 mg osmium tetroxide (Sigma) in 0.5 ml of carbon
tetrachloride for 15 minutes at room temperature. This material was evapo-
rated to dryness under a stream of nitrogen and redissolved in 0.5 ml of 20:1
chloroform-methanol (v/v). Individual samples were then placed on an alumi-
num oxide (100-200 mesh, Bio-Rad Laboratories, Richmond, Calif.) column and
neutral lipids were eluted from the column with 20:1 chloroform-methanol
(v/v). DPC was then released with 70:30:2 chloroform-methanol-7M ammonium
hydroxide (v/v). The total amount of DPC in the original sample was measured
by counting an aliquot in ACS scintillation fluid. Values were expressed as
cpm per 10 cells.
Fluorescence Microscopy
Confluent cell monolayers were established on 12 mm cover slips in
MultiWell vessels. Cell preparations were washed once with PBS and stained
for 2 to 5 minutes in the dark with phosphine 3R at a concentration of 1 ug
per ml of PBS. Cover slips were drained and placed on slides, with the cell
side facing downward. Cells were examined for the presence of fluorescent
inclusion bodies in the cytoplasm, typically observed in type II alveolar
epithelial cells (9).
Transmission Electron Microscopy
Monolayer cultures grown to confluence in MEM supplemented with 10% FCS
were fixed and processed by the method of Stratton (13) to observe multila-
mellar bodies. Cells were fixed with 3% glutaraldehyde in 0.1M cacodylate
buffer, pH 7.3, for 3 hours at room temperature. Cells were then scraped
directly into the fixative without rinsing and centrifuged at 400 x cj for
10 minutes. The cell pellet was resuspended in 1% tannic acid and 1% glu-
taraldehyde in 0.1M cacodylate buffer and treated for 2 hours at room tem-
perature. Cells were then centrifuged, treated with 1% osmium tetroxide in
0.1M cacodylate buffer for 2 hours, and exposed to a series of acetone
dehydration steps prior to embedding in Epon and sectioning.
RESULTS
Morphology of Hamster Tracheal Cells
After selective dissociation of tracheal tissue as previously indicated,
specific cells attach to the vessel surface and appear to undergo extensive
structural modulation during the first week of growth. Figure 1 is represen-
tative of the homogeneous cell population that arises during In vitro incu-
bation. Note the considerable network of cytoplasmic granules (Pig. 1a).
When the outer surfaces of individual cells are observed by interference
245
-------
o A. .
v..
' ;
b
Figure 1. Cytoplasmic granules and secretory packets associated with
monolayer cultures of hamster tracheal cells. Packets are visualized in cells
by focusing in different planes using interference contrast microscopy, X85o.
In this set of photographs (a) represents the image in focal plane of the
cytoplasm and (b) represents a view just above the cell surface.
contrast microscopy, prominent packets which apparently derive from active
cell secretion are detected (Fig. 1b). These packets selectively react with
histochemical stains for acidic mucins (6).
Glycoprotein Synthesis by Tracheal Cell Monolayers
Consistent with the histochemistry and tissue origin of these monolayer
cultures, cells were readily capable of incorporating radioactive amino
acid and sugar precursors into macromolecules which were then secreted into
the medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
concentrated culture medium revealed a single radioactive peak with a mole-
cular weight of about 18,500 daltons (Fig. 2b). Tracheal ring organ cultures
-------
Table 1. Stimulation of Glycoprotein Secretion by Vitamin A
Radioactivity (cpm)
per 50 pg cell protein
a
Test system
- Vitamin A
+ Vitamin A
3H
291.0 + 16.4
835.3 + 120.6
14c
2313.7 +
4108.9 +
192.5
286.2
Cells were grown in MEM plus 10% PCS with or without vitamin A for
72 hours prior to addition of radioisotope for 24 hours.
Based upon incorporation of N-acetyl-D-galactosamine [acetyl- H] and
14
L-serine [U- C] into a secreted glycoprotein. Values were calculated
geometrically from cpm in the single radioactive peak on SDS-polyacrylamide
gel, and are reported as the mean + 1 standard deviation from the mean.
were radiolabeled under similar conditions, and an identical gel profile was
obtained (Fig. 2a). The influence of vitamin A on the morphological and
biochemical properties of this cell type was also examined. Absence of the
vitamin from culture medium dramatically decreased both the number of cyto-
plasmic granules (Fig. 3) and glycoprotein secretion (Table 1) in monolayer
cultures, correlating with the putative in vivo role of vitamin A (2).
Phospholipid Synthesis Among Various Cell Types
Cell monolayers of newborn hamster lung epithelium were monitored for
their ability to synthesize lecithin and disaturated phosphatidylcholine.
Most of the biochemical measurements were performed on cultures during the
third to seventh in vitro passage. As seen in Table 2, these cells readily
incorporated [ H]choline into phospholipid. Other animal cell cultures
were also included in this study since it was important to establish the bio-
logic specialization of the newborn lung epithelial cells. A significant
range in the capacity of the various cultures to synthesize lecithin and
disaturated phosphatidylcholine was observed. However, it was clear that
the hamster lung cultures were most proficient in this bioassay.
Phospholipid Synthesis and Secretion by Normal and Transformed Lung Cells
During early cultivation (three to five passages) of hamster lung
epithelial cells, increased ability to synthesize total phospholipid and
disaturated phosphatidylcholine was demonstrated (Table 3). This observation
appeared consistent with increases in both cell size and numbers of cyto-
247
-------
-------
b
(O
' • , /' r .'••'
I" '. f
Figure 3. Effect of vitamin A on numbers of intracellular granules in cultured hamster tracheal
cells. Interference contrast microscopy, X675. (a) Cells grown for 48 hours after removal of vitamin
A from culture medium, (b) Cells maintained continuously in medium containing vitamin A (2 ^g/ml).
-------
Table 2. Incorporation of Choline Chloride [methyl- H] into
Lecithin and Disaturated Phosphatidylcholine (DPC)
by Different Animal Cells
a
Cell type
Newborn hamster lung epithelium
Rat (L2)
Mouse (L25)
Cat (AKD)
Baby hamster kidney
Human A549
Chick embryo fibroblast
a
Confluent cell cultures were
MEM.
b . .
-4
cpm x 1 0 per
Lecithin
114.0
68.2
33.2
30.0
24.9
15.9
12.3
radiolabeled for 20
6
10 cells
DPC°
19.6
11.6
4.0
3.3
4.7
2.7
2.1
hours in serum-free
graphy. The appropriate spot correlating with the R value of lecithin was
removed and counted by scintillation spectrometry.
c
DPC was isolated from the lipid mixture by aluminum oxide chromato-
graphy after treatment of sample with osmium tetroxide.
plasmic granules. All cells examined in Table 2 with the exception of chick
embryo fibroblasts stained with phosphine 3R. The most intense staining
occurred in newborn hamster lung cells. As mentioned previously, naturally
transformed clones arose during late passage, and these cells demonstrated
a markedly reduced synthesis of phospholipid (Table 3). Note that the levels
of lecithin and surfactant synthesis in the transformed cell cultures are
similar to those of the established "type II alveolar" cultures (cat-AKD,
human-A549) presented in Table 2. Comparisons of cell-associated and secret-
ed disaturated phosphatidylcholine among the normal and transformed lung cell
cultures revealed that normal lung cultures possessed a 10-fold higher capa-
city to synthesize and secrete phospholipid although the percentage of se-
creted surfactant to total phospholipid was similar in both cell types (Table 4)
Ultrastructure of Newborn Hamster Lung Epithelial Cells
One of the characteristic properties of type II alveolar cells is the
existence of multilamellar bodies randomly distributed throughout the cyto-
plasm. These organelles serve as the source of surfactant, and alveolus
function depends on their formation and secretion into the alveolar space. in
250
-------
Table 3. Phospholipid Synthesis by Normal and Transformed
Newborn Hamster Lung Cells
Lung epithelium
Normal
Transformed
.„,
Passage no.
3
4
5
9
40
-4
cpm x 10 per
Lecithin
46.9
57.0
110.4
35.0
24.7
6
10 cells
DPC
6.1
11.6
16.0
4.2
4.7
Experimental conditions are as in legend of Table 2.
Table 4. Synthesis and Secretion of DPC by Newborn
Hamster Lung Cells
Lung epithelium
Normal
Transformed
1
Cell-associated
71
67
i DPCa
Cell-secreted
29
33
b
Total cpm
13.0
1 .4
3
24 hours exposure to choline [methyl- H] and processed as previously de-
scribed.
Total radioactivity represents the sum of DPC counts (x 10 ) in the
cells and culture media fractions after aluminum oxide chromatography. All
values are reported per 10 cells.
Figure 4a, a classical membrane-limited lamellar body is observed in close
association with Golgi vesicles, the latter probably being responsible for
the generation of the lamellar structures. A lamellar body with its highly
ordered structure appears at the cell surface (Fig. 4b) apparently after its
release from the cytoplasm by a specialized type of exocytosis (11). Trans-
formed lung cells derived from the hamster epithelial cultures also possessed
similar multilamellar structures which were readily secreted. It is of in-
terest that all other cell types (Table 2) examined by electron microscopy
demonstrated cytoplasmic multilamellar bodies similar to those of the hamster
lung epithelial cells.
251
-------
M
Figure 4. Transmission electron photomicrograph of newborn hamster lung epithelial cells during
in vitro passage. In thin section (a), a mature multilamellar body is shown. Note the close asso-
ciation of Golgi vesicles (X225,500). In (b) a small compact multilamellar structure is associated
with the cell surface, apparently originating via exocytosis (X280/500). Similar infrastructure
was observed in all hamster lung cell passages including transformed cell cultures.
-------
DISCUSSION
Limited information is available concerning the biochemistry and meta-
bolism of the mammalian respiratory tract which consists of many interacting
cell types and related organs. The cellular heterogeneity and biologic com-
plexity of the tracheobronchial epithelium have prevented the establishment
in culture of pure populations of normal differentiated cells for monitoring
respiratory function under various experimental conditions. Tissue slices,
organ explants, primary and organotypic cultures, and selected cell purifi-
cation procedures have offered useful models for examining pulmonary func-
tion. However, these systems do not provide homogeneous populations of
epithelial cells that replicate in vitro with retained biochemical and ul-
trastructural specialization.
In this report we define selected properties of hamster respiratory
cells readily passaged in vitro. Replicating monolayer cultures comprised
of homogeneous populations of epithelial cells were obtained from trachea
and lung tissues by a reliable technique previously described (6) and exa-
mined for their ability to synthesize and secrete mucus and surfactant, re-
spectively. Evidence is presented to support the biosynthetic proficiency
and uniqueness of these cell types.
Tracheal cell monolayers incorporated radioactive serine and N-acetyl-
D-galactosamine into macromolecules with a molecular weight of approximately
18,500 daltons based upon SDS-polyacrylamide gel electrophoresis. Exposure
of tracheal cell cultures to vitamin A stimulated cytoplasmic granule con-
tent and enhanced glycoprotein secretion. This information, along with
previously described biochemical and histochemical data, suggests the po-
tential application of this model system for investigating fundamental pro-
perties of mucus-secreting cells.
Characteristics of newborn lung epithelial cell monolayers described
here reinforce their metabolic specialization. Synthesis of phospholipid
and, more specifically, disaturated phosphatidylcholine, considered a major
component of lung surface-active material, was greatest in these cultures
when compared to a variety of other animal cell types. Electron microscopy
of newborn hamster lung cells passaged in monolayer for four to eight weeks
demonstrated the classical multilamellar bodies of type II alveolar cells.
The proximity of these organelles to Golgi vesicles and the intense phos-
phine 3R staining of the cells supported their biologic identity with type
II cells. The relationship between early passage lung cultures and late
transformed cell clones revealed significant differences in capacity to
synthesize surfactant. Transformed cells were much less biosynthetically
active in terms of phosphatidylcholine synthesis although prominent cyto-
plasmic multilamellar bodies were still visualized.
253
-------
Homogeneous cell populations of respiratory epithelium with retained
capacity to synthesize and secrete mucus or surfactant can be experimentally
manipulated, providing useful models for pulmonary research. Growth regu-
lation and cellular differentiation can be measured in the presence of various
stimuli or insults such as hormones and drugs, infectious agents, nutritional
deficiencies, carcinogens, and pollutants. Chemical and physical properties
of mucus glycoproteins and surfactant can be analyzed under a range of test
conditions. Such studies should clarify the biologic competence of the
tracheobronchial epithelium in maintaining pulmonary function and permit the
identification of factors that alter the apparent normalcy of these res-
piratory cells.
ACKNOWLEDGMENTS
This research was supported by Public Health Service Grant P50-HL 19171
and Research Career Development Award 1-K04-AI 00178 to J.B.B.
REFERENCES
K Baseman JB, Hayes NS: Differential effect of hormones on macromolecular
synthesis and mitosis in chick embryo cells. J Cell Biol 67:492-497,
1975
2. Bonanni F, Levinson SS, Wolf G, De Luca L: Glycoproteins from the ham-
ster respiratory tract and their response to vitamin A. Biochim Biophys
Acta 297:441-451, 1973
3. Breeze RG, Wheeldon EB: The cells of the pulmonary airways. Am Rev Reap
Dis 116:705-777, 1977
4. Douglas WHJ, Moorman GW, Teel RW: The formation of histotypic struc-
tures from monodisperse fetal rat lung cells cultured on a three-dimen-
sional substrate. In Vitro 12:373-381, 1976
5. Folch J, Lees M, Stanley GHS: A simple method for the isolation and
purification of total lipids from animal tissues. J Biol Chem 226:497-
509, 1957
6. Goldman WE, Baseman JB: Glycoprotein-secreting cells selectively cul-
tured from the hamster trachea: A model system for in vitro studies of
mucus synthesis. Submitted for publication
7. Kikkawa Y, Yoneda K: The type II epithelial cell of the lung. I.
Method of isolation. Lab Invest 30:76-84, 1974
8. Mason RJ, Nellenbogen J, Clements JA: Isolation of disaturated phos-
phatidylcholine with osmium tetroxide. J Lipid Res 17:281-284, 1976
9. Mason RJ, Williams MC, Greenleaf RD: Isolation of lung cells. In: Lung
Cells in Disease (Bouhuys A, ed). New York, North-Holland Publishing
Co., 1976, pp 39-49
254
-------
10. Mason RJ, Dobbs LG, Greenleaf RD, Williams MC: Alveolar type II cells.
Fed Proc 36:2697-2702, 1977
11. Ryan US, Ryan JW, Smith DS: Alveolar type II cells: Studies on the mode
of release of lamellar bodies. Tissue and Cell 7:587-599, 1975
12. Smith BT: Cell line A549: A model system for the study of alveolar type
II cell function. Am Rev Resp Dis 115:285-293, 1977
13. Stratton CJ: Multilamellar body formation in mammalian lung: An ultra-
structural study utilizing three lipid retention procedures. J Ultra-
struct Res 52:309-320, 1975
255
-------
DISCUSSION
DR. MOSSMAN: My question regards the tracheal epithelial cells. I wonder
whether you were able to clone these cells. In other words, were pure popul-
ations of epithelial cells used in your experiments? Secondly, you mention
in your abstract an actual transformation of these cells. What types of tumors
did they gave rise to in animals?
DR. BASEMAN: The first question concerns the ability to clone these cells.
Most of the data presented are not based on cloned cell populations. Because
the cloning process requires weeks before sufficient cell numbers are available
for experimentation, we do not routinely clone. By the time we complete the
cloning procedure, cells begin to senesce—i.e., the growth rate slows and
individual cells enlarge. However, our specific cell isolation technique
using a single hamster trachea results in the survival and growth of a small
yet selective population of respiratory epithelial cells that exhibit morpho-
logic homogeneity. When these cultured cells are compared to cloned cell
populations, all cells appear identical based upon structural, biochemical,
and histochemical criteria. Your second question concerns cell transformation.
We have not isolated "transformed" cells from the trachea, but the data in
the abstract refer to cells derived from hamster lung. In the latter case,
we routinely clone cells because a greater variety of lung cells survive the
cell isolation procedure in contrast to trachea-originated cultures. After
prolonged culture of lung cells, the majority of cells exhibit characteristics
of senescence. However, we observe small foci of cells that arise during the
late passages. These cells are considered transformed because they demonstrate
altered morphology, increased cell growth rate, and the property of continuous
passage (over 110 transfers during an 18-month period). Also, these cells have
a decreased capacity to synthesize surfactant although they do possess distinct
cytoplasmic multilamellar structures. We have no evidence that these cells are
neoplastic.
DR. MOSSMAN: The only reason I asked is that in our laboratory we isolated
cells from the hamster trachea, and we also found that cells senesced. At
this period of time, however, there were isolated populations which did
multiply, and we were able to clone the cells. This was recorded as an annual
meeting abstract for the Tissue Culture Association in In Vitro about four
years ago.
256
-------
DR. BASEMAN: I think that those cells differ from the tracheal cells that
we are describing. We are not certain as to the absolute identity of our
cells but believe that they possess specialized function in terms of mucus
synthesis and secretion. These cells may originate from an "intermediate"
cell which undergoes specific morphologic and physiologic changes in mono-
layer culture once removed from the environmental integrity of the hamster
trachea.
DR. HU: I understand. Some time ago you discussed with me the technique of
isolating respiratory cells.
DR. BASEMAN: Yes. After about a year of considerable effort, we consistently
generate these epithelial cell cultures from the hamster trachea. Why have
others been less successful? I believe that the cell isolation technique
that we have developed which allows the selective dissociation and survival
of specific tracheal cells prior to monolayer culture is the key.
DR. DAVIS: I agree with you that this is a useful method for studying respir-
atory cell physiology. In fact, we are investigating a similar area. What
I do not know is what the criteria should be for defining mucus-secreting
cells. Have you measured ion content of the cells?
DR. BASEMAN: These monolayer cultures of tracheal cells appear to synthesize
mucus selectively when compared to other available cell types* In terms of
ion flux, one of the problems is that we really do not know the exact criteria
for designating a cell "specialized." We think that when a cell selectively
synthesizes a macromolecule that appears distinct biochemically and histo-
chemlcally and that this function is under regulatory control as indicated
by vitamin A responsiveness while other cell types lack this biosynthetic
capability, then the cell should be classified as specialized.
257
-------
Retinoid Metabolism and Mode of Action
L. M. De Luca, W. Sasak, S. Adamo, P. V. Bhat,
I. Akalovsky, C. S. Silverman-Jones, and N. Maestri
National Cancer Institute
Bethesda, Maryland
Epidemiological studies indicate that the majority of human cancers are
caused by exposure to environmental carcinogens (13). These can be inhaled,
ingested with food, or absorbed through the skin and first come in contact
with the epithelial surfaces of the body, which have a greater metabolizing
activity than stromal fibroblasts. Related to this greater metabolic activity
for chemical carcinogens is the fact that approximately 80% of human cancer
originates in epithelial tissues (11).
Although exposure to carcinogens is essential for the neoplastic event
to occur, host factors are very important modulators of the oncogenic re-
sponse. These factors may be genetic or acquired. An example of genetic
factors is the levels of carcinogen-activating enzymes, which appear to vary
profoundly among different individuals (11). An example of acquired factors
is vitamin A.
The interest in vitamin A and its derivatives, the retinoids (22), has
derived from old and fundamental observations that the vitamin preserves the
integrity of epithelial tissue (for a review see ref. 7). Feeding a vitamin
A-deficient diet to hamsters causes replacement of the normal mucociliary
epithelium of the trachea by squamous metaplastic and keratinized cells (7,
22). A similar response may also be caused by chemical carcinogens or
mechanical injury. These conditions may be corrected by administering
vitamin A either to the whole animal or to the organ in culture (5, 14).
Because of these findings a flurry of activity in the area of prevention
259
-------
all-trans RETINOIC ACID
all-trans RETINOL
Figure 1. Structures of retinoic acid and retinol.
of chemical carcinogenesis by retinoids has occurred in recent years with the
scope of finding a potent and non-toxic retinoid, which might prevent neo-
plastic transformation in selected human populations at high risk of develop-
ing cancer.
Retinoids are also active in the alleviation of a variety of skin
diseases, including actinic keratosis and basal cell carcinomas (16, 19).
Inasmuch as derivatives of retinoic acid (Fig. 1) are not stored in the
liver and reach the target site in larger amounts than retinol (Fig* 1), they
have been used preferentially in preventive and therapeutic studies (7, 16,
22) .
The proposed concept is that, by increasing the concentration of the
retinoid in the target tissue, one enhances the potential of the tissue to
maintain the normal phenotype, thereby counteracting the sequel of tissue
alterations eventually leading to the neoplastic transformation caused by
carcinogens.
Although clearly such preventive studies do not require a full knowledge
of the biochemical mechanisms of action of the retinoids, such knowledge may
be crucial in understanding how the phenotypic expression of epithelial
tissues is maintained.
260
-------
RESULTS
The Tracheal Epithelium
The integrity of the tracheal epithelium is lost in vitamin A deficiency.
A study of the morphology of tracheal explants from vitamin A-depleted hamsters
and rats cultured in a vitamin A-depleted medium shows that the normal muco-
ciliary epithelium is replaced by a squamous metaplastic epithelium which kera-
tinizes and sheds keratin into the medium (5, 14).
As in other epithelial tissues, a major biochemical defect in vitamin
A deficiency in the respiratory mucosa occurs at the level of biosynthesis of
specific glycoproteins (for a review see ref. 6). Such alterations in glyco-
protein biosynthesis may have important consequences for the membrane. The
carbohydrate moieties of glycoproteins appear to act as "lectins" in that they
bind specifically to sites which recognize their terminal sugar at the non-
reducing end of the macromolecule (21).
Ashwell and Morrell and their collaborators (4) have shown that glyco-
proteins are readily removed from the blood stream of the rat when their
terminal sialic acid residues are removed and the penultimate sugar, galactose,
is exposed to allow recognition by a molecule at the surface of the hepatocyte
which sequesters the circulatory glycoprotein into the hepatocyte (4) and away
from circulation. Similar recognition properties have been suggested by Roseman
(20) to mediate intercellular interactions and the phenomenon of cellular
adhesion.
There is now a vast body of work to indicate that vitamin A is directly
involved in controlling the biosynthesis of specific glycoproteins in all
epithelial tissues so far studied (6). Most of this work has been conducted
in the whole animal, using radioactively labeled monosaccharides as precursors
of glycoproteins. More recent work has confirmed these findings in tissue
culture systems. Particularly, cultures of mouse epidermal cells have been
shown to respond to retinylpalmitate by a remarkable (5- to 11-fold) increase
in the incorporation of galactose, mannose, and glucosamine into epidermal
glycoproteins (3).
Similar results were obtained in organ cultures of corneal epithelial
cells from the eyes of rats. In this system it was shown that the corneal
epithelium responds to culturing in the presence of retinol or retinoic acid
by a selective increase in the incorporation of monosaccharides and without
effect on the incorporation of leucine (12). The incorporation of glucosamine
into high molecular weight glycoproteins (greater than 200,000 daltons) was
specifically enhanced by vitamin A, either in organ culture or in vivo (12).
Thus, vitamin A seems to control properties of biological membranes by con-
trolling the biosynthesis of specific glycoproteins of the membrane.
261
-------
Incorporation of [ H]benzo(a)pyrene into Normal and Vitamin A-deficient
Hamster Tracheas
A faulty membrane may allow a greater amount of environmental pollutants
inside the cell.
In a series of experiments we set out to measure the amount of [ Hjbenzo-
(a)pyrene incorporated into negatively charged lipids of hamster tracheas froa
normal and vitamin A-depleted animals.
Tracheas (10 per group) were incubated in culture medium L-15 for 30
minutes at 37°C. The medium contained either 5 yCi of [ H]3,4 benzo(a)pyrene
(specific radioactivity 11 Ci/mMole), 5 uCi of [15-3H]retinol (specific radio-
activity 1.25 yCi/mMole). or 5 uCi of [ Cjmannose (50 uCi/mMole) . Retinol and
mannose were used to study the specificity of the effect on benzo(a)pyrene.
The incorporation of these radioactive precursors into membrane phospholipids
was measured by chromatography of the lipidic extract (chloroform/methanol
2/1) on columns (0.75 x 45 cm) of DEAE-cellulose acetate. The free precursors
were eluted off the column with about 400 ml of 99% methanol. The radio-
activity associated with phospholipids was eluted with a gradient of 100 ml of
99% methanol to 100 ml of 100 mM ammonium acetate.
Table 1 shows that the amount of each of the three precursors incorporated
into negatively charged lipids of the tracheas from vitamin A-depleted hamsters
was several fold greater than in normal tracheas.
The vitamin A-deficient tracheas incorporated 3.9 times as much benzo(a)-
pyrene into phospholipids as their normal counterparts (Table 1). This find-
ing is in agreement with previous results describing a larger amount of radio-
active benzo(a)pyrene bound to the DNA of vitamin A-deficient tracheas (10).
Table 1. Incorporation of [3H]Benzo(a)pyrene, [-^H] Retinol and [14C]Mannose
into Negatively Charged Lipids of Normal and Vitamin A-
deficient Hamster Tracheas
Benzo ( a ) pyr ene
Retinol
Mannose
pMoles incorporated in 30 minutes at 37°C
Normal Deficient D/N
1.95 x 10~7 7.66 x 10~7 3.92
-7 -7
16.9 x 10 89.5 x 10 5.29
-5 -5
1.83 x 10 5.09 x 10 2.78
Conditions of incubation and processing of the lipid extracts are de-
scribed in the text. D/N stands for deficient/normal.
262
-------
However, retinol and the monosaccharide mannose were also incorporated in
larger amounts in the phospholipidic fraction of the deficient organs (Table
1), suggesting that general permeability characteristics of the deficient
epithelium are profoundly altered in vitamin A deficiency. It is therefore
reasonable to consider that the epithelial uptake of toxic substances from
the environment may be modulated by the nutritional status of the animal.
The Involvement of Retinoids in Adhesion
Vitamin A also affects the adhesive properties of cells. Glycoproteins,
(25), glycolipids, and glycosyltransferases (20) have been implicated as the
molecular determinants of adhesion. Inasmuch as retinol and retinoic acid
are essential constituents of membranes and modulators of the biosynthesis
of specific glycoproteins, it was of interest to determine whether they could
influence adhesion.
The system of choice was the spontaneously transformed mouse fibroblasts
(Balb/c 3T12-3 cells) because these cells display very poor adhesive properties
in culture and can be lifted from the culture dish surface in an EDTA-mediated
detachment assay (1). The cells appear round and display the typical morphology
of transformed cells with very poor adhesion to each other (Fig. 2A). However,
when cultured in the presence of 3.3 x 10 M retinol or retinoic acid, they ac-
quire morphological characteristics of "normal" cells (Pig. 2B). Moreover/ they
remain attached to the plate, when treated with EDTA, under conditions which '
lift the untreated cells. Eventually, retinoid-treated cells also come off the
plate, but only after prolonged EDTA treatment. Interestingly, saturation
density was not affected markedly by retinoid treatment and the more adhesive,
retinoid-treated cells had the same plating efficiency as the untreated 3T12
cells in dense (90%) and sparse (20%) culture conditions.
Inasmuch as this assay might be useful to measure retinoid-activity/ struc-
ture-activity relationship was investigated in this system. Retinol, retinoic
acid, and their 5,6-epoxyderivatives were the most active compounds (2, 8).
Derivatives of retinoic acid with activity in the hamster trachea differ-
entiation system (22) were also active in increasing adhesion. Derivatives
of retinol and retinoic acid, which were devoid of any vitamin A activity
in other systems, were also inactive in increasing adhesion (2, 8). The
free carboxyl group of retinoic acid was necessary for activity, since esterifi-
cation or amide formation led to inactive compounds.
In conclusion, it appears that this newly found biological activity of
retinoids in increasing adhesion of 3T12 cells correlates well with their
vitamin A activity in other biological systems; it also offers an easily
accessible and relatively fast assay for vitamin A activity.
263
-------
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Figure 2. Transmission electron microscopy of control (A) and retinoic
acid-treated (B) 3T12 cells (X3,500). Cells were seeded at 10,000 per cm2
and grown in the presence or absence of 3.3x10 M retinoic acid for six dav
264
-------
An investigation of the biosynthesis of glycoproteins in this system
of transformed cells is now under study. The incorporation of [2- HJmannose
into glycoprotein is greatly enhanced by retinoid treatment at the same time
as the increase in adhesion is observed. Particularly, the biosynthesis of
a mannose-containing glycoprotein of molecular weight 180,000 daltons (gp180)
was found to be highly stimulated by retinoids, whereas not much effect was
noticed on the biosynthesis or the iodination of the extracellular fibronec-
tins, which have been implicated in the phenomenon of intercellular and cell
to substrate adhesion (25).
In 1970 we first suggested that vitamin A controls membrane function by
its molecular involvement in the biosynthesis of glycoproteins (6). We pro-
posed that the phosphorylated form of vitamin A carries glycosyl residues
across the membrane bilayer to make them available for glycosylation of
glycoproteins. This hypothesis has received considerable attention after the
demonstration that phosphorylated vitamin A (retinylphosphate) is a component
of epithelial membranes (6). The biosynthesis of retinylphosphate (6) has
been demonstrated in vivo in rat liver and intestine (6, 15) and in vitro in
cultures of rat intestinal cells (9) and mouse epidermis (3).
Figure 3 illustrates the proposed scheme for the action of mannosyl-
retinylphosphate in the membrane. The molecule is shown with its hydrophobia
cyclohexene ring embedded in the lipid bilayer and the polar end in the hydro-
philic area in the all-trans configuration. The trans-9—cis isomerization
allows crossing of the bilayer with the mannosyl moiety now in the "inside"
of the membrane. Such trans-cis isomerization is postulated here to occur
via an isomerase enzyme. The 9-cis mannosylretinylphosphate would then
function as a donor of mannose to acceptor glycoproteins through the action
of glycosyl transferases.
CONCLUSIONS
In addition to the role of vitamin A in the visual cycle as the chromo-
phore of rhodopsin (17, 24), evidence is now emerging for a new biochemical
function for the vitamin as a carrier of mannosyl residues for the bio-
synthesis of glycoproteins (6). Such a carrier role appears to be essential
for the biosynthesis of mannose-containing glycoproteins, as suggested by
studies in deficiency and excess vitamin A. There is in fact a 70% reduction
in the amount of mannose bound to glycoproteins of the hepatocyte membrane
in vitamin A deficiency and a 610% increase in the incorporation of mannose
in excess vitamin A.
It is thus suggested that, if such glycoproteins are necessary to regu-
late membrane functions, the diverse morphological and functional mani-
festations of the action of vitamin A [i.e., mucociliary function in respi-
265
-------
OUTSIDE
Figure 3. Working scheme for a trans-cis membrane-crossing action of vitamin A in glycosyl transfer
reactions. The lipid constituents of the membrane bilayer are shown with their polar head groups on
the exterior and the hydrophobic fatty acid chains in the interior of the bilayer. The trans to 9-cis
isomerization permits the crossing of about 40 angstroms by the mannosyl residue attached to retinyl
phosphate. The 9-cis molecule would then serve as a substrate for the mannosyl transferase.
-------
ratory epithelium (6), adhesive properties in fibroblasts (1, 2, 8), hormonal
responses in the reproductive systems (23), etc.] depend on the particular
tissue. Moreover, it appears that, in addition to retinol, a metabolite of
retinoic acid undergoes the same fate of phosphorylation and glycosylation
(6), thus functioning as vitamin A.
It is tempting to suggest that the function of the binding proteins
for retinol and retinoic acid is a protective one (18) against oxidation and
metabolism, prior to insertion of the vitamin in the hydrophobic environment
of the membrane bilayer, where it functions in the phosphorylated form.
Inasmuch as the vitamin maintains and pollutants perturb the normal
phenotype of the mucociliary epithelium, it would be of interest to study
whether toxic agents, such as ozone, interfere with the vitamin A-dependent
biochemical processes of epithelial tissues by destroying the retinol, retinyl
esters, and retinylphosphate present in these tissues. It would also be of
interest to measure whether an increased intake of nutritional factors such as
vitamin A would have a protective effect against the action of toxic substances
in the respiratory epithelium exposed to the toxic agent.
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4. Ashwell G, Worrell J: Membrane glycoproteins in recognition phenomena.
Trends in Biochemical Sciences 2:76-78, 1977
5. Clamon GH, Sporn MB, 3nith JM, Saffiotti U: Alpha- and beta-
retinylacetate reverse metaplasias of vitamin A deficiency in hamster
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6. De Luca LM: The direct involvement of vitamin A in glycosyl transfer
reactions of mammalian membranes. Vitam Hormon 35:1-57, 1977
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cultures. Lab Invest 33:451-460, 1975
15. Masushige S, Schreiber JB, Wolf G: Identification and characterization
of mannosylretinylphosphate occurring in rat liver and intestine in vivo.
J Lipid Res 19:619-627, 1978
16. Mayer H, Bollag W, Hanni R, Ruegg R: Retinoids, a new class of com-
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17. Morton RA: The chemistry of the visual pigments. In: Photochemistry
of Vision (Dartnall HJA, ed). New York, Springer-Verlag, 1972, p 33
18. Olson JA: Concluding remarks in "Vitamin and Carrier Functions of
Polyprenoids." Wld Rev Nutr Diet 31:232-242 (Cama HR, Sastry PS, eds).
Basel, Karger, 1978
19. Peck GL, Yoder FW: Treatment of lamellar ichthyosis and other keratiniz-
ing dermatoses with an oral synthetic retinoid. Lancet 2:1172, 1976
20. Roseman S: The synthesis of complex carbohydrates by multiglycosyltrans-
ferase systems and their potential function in intercellular adhesion.
Chemistry and Physics of Lipids 5:270-297, 1970
21. Sharon N: Lectins. Scientific American 236:108-119, 1977
22. Sporn MB, Dunlop MM, Newton DL: Prevention of chemical carcinogenesis
by vitamin A and its synthetic analogs (retinoids). Fed Proc 35:
1332-1338, 1976
23. Thompson WA, Howell JM, Thompson JN, Pitt GAJ: The retinol requirements
of rats for spermatogenesis and vision. Br J Nutr 23:619, 1969
268
-------
24. Wald G: The molecular basis of vis via 1 excitation. Nature (London)
219:800, 1968
25. Yamada KM, Olden K: Fibronectins-adhesive glycoproteins of cell surface
and blood. Nature 275:179-184, 1978
269
-------
DISCUSSION
PARTICIPANT: Is there any evidence that oxidation of the retinoic acid or the
retinol would decrease its activity?
DR. DE LUCA: Yes, definitely. Even the 5,6 epoxidation of retinol and retinoic
acid will produce compounds which _in vivo have been reported by Dr. Cama to be
less active than the retinoic acid or retinol. If you oxidize the chain, I
would say that, in general, oxidation leads to destruction or loss of activity.
DR. REID: What other sugars are carried, or is it only the mannose?
DR. DE LUCA: Yes, we have worked mostly with mannose. But Dr. Peterson and
Dr. Wolf have done some work in recent years with galactose. So, it appears
that both mannose and galactose are involved.
DR. NETTESHEIM: There are two reports from the literature that I am aware
of concerning the inhibition of in vitro transformation by retinoids with
x irradiation and with chemical carcinogens. Do you have any suggestions to
make? What the mechanism involved might be? IB it possible that since these
people are really looking at the phenotypic change, they are looking at a
similar phenomenon that's related to the adhesion phenomenon, or do you think
that it is really something that has to do with inhibition of neoplastic
transformation?
DR. DE LUCA: You're asking me, do we know what neoplasia is all about? I
don't know. It's a very difficult question.
270
-------
Session VI:
Pharmacological Modulation
-------
The Lung Mast Cell: Its Physiology and Potential
Relevance to Defense of the Lung
S..I. Wasserman*
Harvard Medical School
Boston, Massachusetts
The mast cell, with its specific immune-logic receptors, is positioned at
portals of entry of potentially toxic or noxious exogenous material• In the
lung the mast cell is located free in the bronchial lumen, in the bronchial
mucosa in intra-epithelial locations, as well as in deeper perivenular col-
lections (35, 37). As it is present prior to entry of noxious agents and is
thus freed from the requirements of mobilization and localization, the mast
cell may be the sentinel cell for induction of local inflammatory responses.
The development of inflammation by mast cells is consequent to the release
of its content of potent biologic mediators as well as by its capacity to
generate, de npvo, active biologic materials from the local microenvironment.
The knowledge that mediators are active in vivo follows from the in vitro
definition of their biologic activities which relate to the known patho-
physiology of inflammation. The inflammatory processes induced by mast cell
mediators are both acute and chronic and are subject to both positive and
negative feedback controls inherent in the properties of the mediators them-
selves. The purpose of this review is to delineate the physiology of the
mast cell, the mediators generated upon mast cell activation, and the regula-
tion of mast cell-dependent inflammatory events.
*Dr. Wasserman is a recipient of an Allergic Diseases Academic Award
(AI-00254) from the National Institutes of Health.
273
-------
M
Figure 1. Electron micrograph of isolated human lung mast cell.
(N), membrane microvilli (MV), granule (G).
Nucleus
MAST CELLS
g
Mast cells are present in human lung at concentrations of 1-7 x 10
cells/g lung tissue (30). Each mast cell possesses, in addition to the ubiqui-
tous subcellular organelles essential for all cell function, several hundred
metachromatically staining granules, each surrounded by a bilayer membrane.
Lung mast cell granules possess a definite subgranular architecture (Fig. 1)
of unknown functional significance. The mast cell membrane is ruffled and
possesses 50-300,000 receptors for IgE (15). IgE molecules belong to an
immunoglobulin class defined by its activity in mediating immediate hypersen-
sitivity reactions and by characteristic physicochemical properties. It is
comprised of two heavy and light chains linked by disulfide bridges with
a total molecular weight (m.w.) of 190,000. It is heat labile, but does not
form precipitates with specific antigen nor does it fix complement. The
serum concentration of IgE is under genetic control, being higher in atopic
than in normal individuals. Papain digests IgE into three fragments, two of
which (Fab) bind specific antigen and one (Fc) which binds to the specific
receptors on the mast cell surface. Receptors for the anaphylatoxins, C3a and
274
-------
C5a, fragments of the third and fifth components of complement, respectively,
have been attributed to the mast cell upon functional criteria. In addi-
tion, mast cells may be degranulated by non-immunologic stimuli. Thus enzymes
such as chymotrypsin, phospholipases A and C and sialidase, ionophores, poly-
cationic amines and proteins, radio-contrast media, and opiates may all
effect mast cell degranulation. While it is generally assumed that atopic
individuals exhibit their antigen-induced symptoms as a result of IgE-depen-
dent mast cell activation, the demonstration of non-IgE-mediated mechanisms
for mast cell mediator release does not diminish the primacy of IgE in allergy
but rather yields additional information on potential mechanisms for recruit-
ment of mediators.
ACTIVATION AND DEGRANULATION OF MAST CELLS
The IgE-dependent degranulation of mast cells is initiated by the bridging
of pairs of cell-bound IgE by specific antigen and terminates in two minutes.
Bridging results in an alteration of the cell membrane which probably acti-
vates a surface membrane esterase. The cell membrane perturbation thus initi-
ated is also associated with increased energy-dependent calcium entry into
the cell. The calcium requirement for degranulation is related to the acti-
vation of the membrane serine esterase, and perhaps also to requirements for
the expression of phospholipase activity, microfilament contraction, or mem-
brane fusion. Degranulation also requires glucose and an intact glycolytic
pathway. Finally, both ATP and calcium are required by a calcium-dependent
ATPase which probably activates contractile proteins. Following the ordered
completion of these processes, the perigranular membranes fuse with each
other and with the cell membrane, and the granules are extruded (Fig. 2).
Degranulation may be modulated at several steps in its sequence by endogenous
or exogenous agents. Thus, elevations in intracellular levels of cyclic
adenosine 3'5'-monophosphate (cAMP), which may be induced by prostaglandins,
histamine, and S-adrenergic agents, inhibit degranulation (16). Conversely,
a-adrenergic- or prostaglandin-induced falls in cAMP or rises in intracellular
cyclic guanosine 3'5'-monophosphate (cGMP) following interaction with cholin-
ergic stimuli or histamine may enhance degranulation (16). The opposing
actions of the cyclic nucleotides and the bidirectional control of degranu-
lation by cAMP are felt to represent competitive effects upon a putative
protein kinase which might regulate the state of cytoskeletal assembly. The
supposition that cytoskeletal elements are important in human mast cell degran-
ulation is supported by the inhibitory effects upon degranulation of colchi-
cine and by the augmentation induced by cytochalasin B (10, 27).
275
-------
Antigen
Co*
- Moior Defined Poth»oy
-- Hypothetical Agonilt
Hypothetical Inhibitoi
r
i
C9//J
Membrane ,
>
Receptor Receptor
Xr^--»
Proesterase
X I
\ Esterose^
\
\
\
I \
\ \
NS. \
* .!.
Glycolysis— ^AT
Microfiloments i
r i J
*
Contracted
Microfi laments
^ L
Inactive
~*-PhospholipaseA2 Phospholipid^
*" Active A2— "*"*Arachidonic Acid
Cyclo-oxygenose »-!
-* PGG2 "I
PGHj— ~^ 1
Thrombowne Az / rSSc2a
/ *^ J o/*r\
*^ •« 1 i i».y^^rf* 1 rv^T-
^ I • y^^K L.'^'i?
\ * 4 1 \
P> ' T f^ ,-._,;_ ^MO /-,„/,-. /?iy/3
Adenytate CyckiM 1 1
| 1
; Tubulin 1
• ' /
r "*
-LysophosprxjJipids
1
Membrane Fusion
I
i
i
;
GuanytattCyclow
DEGRANULATION
Figxire 2. Schematic representation of proposed metabolic pathways rele-
vant to antigen-induced degranulation of mast cells. [Modified from Lewis and
Wasserman: In: The Science and Practice of Clinical Medicine: Rheumatology
(Cohen A, ed). New York, Grune & Stratton (In press)].
MAST CELL-DEPENDENT MEDIATORS
Mast cells possess within their granules a variety of vasoactive, bron-
chospastic, and chemotactic mediators as well as a variety of active enzymes
and structural proteoglycans (Table 1). Mast cell activation leads to the
release of these preformed granular elements as well as to the generation of
potent bronchospastic, vasoactive/ and chemotactic substances. Although best
delineated in the case of the rat mast cell, it is likely that the human mast
cell will be found similar in these regards.
Bronchospastic and Vasoactive Mediators
Histamine. Histamina, the product of decarboxylation of the amino acid
histidine, is ionically bound to the proteoglycan-protein backbones of mast
cell granules and is displaced by sodium exchange in the extracellular fluid
(38). Histamine is catabolized by either oxidative deamination (histaminase)
or by combined methylation and oxidative deamination (histaminase plus
276
-------
Table 1. jtoat Cell-Dependent Mediators
Mediator
Structural
Characteristics
Function
Inhibition
Inactivation
I. BRONCHOSPASTIC AND VASOACTIVE ACTIVITY
Histamine
(preformed)
g-imidazolyl-
ethylamine
MW 111
Contraction of
smooth muscle,
increase of
vascular per-
meability ,
stimulation
of suppressor
T-lymphocytes,
generation of
prostaglandins,
enhancement
(H^ or inhi-
bition (H ) of
chemotaxis,
elevation of
CAMP (H2)
and cGMP (H.)
H. classical
thiourea
Histaminase
(diamine oxi-
dase)
or
Histamine
N-methyl
transferase
SRS-A
(newly
generated)
Acid hydro-
philic sulfur
containing
lipid (?)
MW v 400
Contraction of
smooth muscle,
increased
vascular
permeability,
synergistic
with histamine,
generation of
prostaglandins
FPL-55712
Arylsul-
fatases A
and 8
Serotonin
(preformed)
5-OH-trypt-
amine
MW 182
Contraction of
some smooth
muscle,
increased
vascular
permeability
Hydroxyzine Monoamine
cyproheptadine oxidase
Lysergic acid
277
-------
Table 1 Continued
Mediator
PAF(s)
(newly
generated)
Arachidonic
acid meta-
bolites
PGD, PGE,
PGI,
PGF2a'
HETE
Structural
Characteristics
Lipid-like
MW 400-1000
C2Q fatty
acids
(HHT C )
fatty acid)
Function
Release of
platelet amines/
platelet
aggregation,
sequestration of
platelets
Contract smooth
muscle ( PGD ,
PGF2a, TxA,
PGG2, PGH2)
Relax smooth
muscle (PGE.)
Elevate cyclic
Inhibition Inactivation
Unknown Phospho-
lipases
ETYA (cyclo- Several
oxygenase specific
lipoxygenase) prostaglandin
Nonsteroidal modifying
anti-inflamma- enzymes
tory agents
( eye looxygenase )
AMP (PGE, PGD,
PGI), elevate
cyclic GMP
(PGF. , PGG.)
2 oc 2
dose-dependent
chemotactic attrac-
tion of eosinophils
or neutrophils
(HETE or HHT)
II. CHEMOTACTIC ACTIVITY
ECF-A
(preformed)
Val/Ala-Gly-
Ser-Glu
MW 360-390
Chemotactic
attraction
and deacti-
vation of
eosinophils
and neutro-
phils
Gly-Ser-Glu
Val-Gly-Ser
Ala-Gly-Ser
Amino-
peptidase
Carboxy-
peptidase A
278
-------
Table 1 Continued
Mediator
Structural
Characteristics
Function
Inhibition
Inactivation
ECF-oligo-
peptides
(preformed)
Pepbides
MW 1300-2500
Chemotactic
attraction
and deacti-
vation of
eosinophils
and neutrc—
phils
Unknown
Unknown
NCF
(preformed)
Neutral
protein
MW > 750,000
Chemotactic
attraction
and deacti-
vation of
neutrophils
Unknown
Unknown
Lipid chemo-
tactic fac-
tors (newly
generated)
? HHT
? HETE
? other lipids
Chemotactic
attraction of
neutrophils
and eosino-
phils
Chemokinesis
of neutro-
phils
Deactivation
of neutrophils
Unknown
Unknown
Histamine
(preformed)
g-imidazolyl-
ethylamine
MW - 111
H . chemo-
tactic and
chemokinetic
activation of
eosinophils
H cherao-
H classical Histaminase
(diamine
oxidase)
or
H_ thiourea Histaminase
tactic and
chemokinetic
inhibition of
neutrophils
and eosino-
phils
N-methyl
transferase
279
-------
Table 1 Continued
Mediator
Structural
Characteristics
Function
Inhibition
Inactivation
III. STRUCTURAL COMPONENTS
Heparin
(preformed)
Proteoglycan
MW j* 60,000
Anticoagulation
Antithrombin
III interaction
Inhibition of
complement
activation
Protamine
Heparinase
Chondroitin
4 and 6 sul-
fate
(preformed)
Dermatan
sulfate
(preformed)
Proteoglycan
Proteoglycan
Platelet Unknown
factor IV
interaction
Unknown Unknown
Chondroiti-
nase AC
Chondroiti-
nase ABC
IV. ENZYMES
Chymase Protein
(preformed) MW = 29,000
Proteolysis
with chymo-
tryptic speci-
ficity
Serotonin
Heparin
Chymotrypsin
inhibitors
Unknown
Arylsulfa-
tase (pre-
formed)
N-acetyl-8-
D-glucos-
aminidase
(preformed)
Protein
MW » 100,000 (A)
60,000 (B)
Protein
MW = 158,000
Hydrolysis of
SRS-A and
various sul-
fate esters
Cleavage of
glucosamine
residues
*°A' SOA
4 4
Product,
substrate
Product
Unknown
Unknown
280
-------
Table 1 Continued
Mediator
Structural
Characteristics
Function
Inhibition
Inactivation
Basophil
(lung)
Kallikrein of
anaphylaxis
(preformed)
Protein
MW = 400,000
Proteolysis
with tryptic
specificity
Cleavage of
kinin 'from
kininogen
Cleavage of
Hageman factor
Trypsin
inhibitors
Unknown
(5-glucuroni- Protein
dase MW - 300,000
Cleavage of
glucuronide
conjugates
Product
Unknown
histamine N-methyl transferase) (42). The pulmonary effects of histamine are ex-
pressed as both direct and reflex constriction of both large and small airways
smooth muscle, thereby increasing airway resistance and decreasing compliance
(20). It also dilates small radicles of the pulmonary vascular tree and in-
creases the distance between endothelial cells of the venules, thereby in-
creasing the potential for transudation of serum and for extravasation of
leukocytes.
The biologic activities of histamine are expressed by its interaction
with either of two specific classes of receptors on target cells. Those re-
ceptors designated H predominate in skin and smooth muscle and are blocked
by classic antihistamines, while H receptors are selectively blocked by a
group of compounds, including the thiourea derivatives, buramiinide, metiamide,
and cimetidine (4). Pulmonary bronchoconstriction, vasodilation, and in-
creased cGMP are H., effects, while H. effects include inhibition of both human
lymphocyte-mediated cytotoxicity and IgE-mediated histamine release due to
elevation in cAMP content. Histamine also inhibits chemotaxis through HZ
receptors, presumably also by stimulating adenylate cyclase and increasing
cAMP.
Slow Reacting Substance of Anaphylaxis (SRS-A). Although SRS-A remains
structurally undefined, it is likely an unsaturated acidic sulfur-containing
lipid of 300-500 daltons (28). It is active as a constrictor of peripheral
airways to a much greater extent than of central airways (9) and causes vaso-
dilation. Human lung tissue, dispersed pulmonary cells, and nasal polyps
281
-------
MEMBRANE PHOSPHOLIPIDS
/\
Phospholipase A2
/ X
Arachidonic Acid Lysophosphatides
•PGI,
Lipoxygenase
/ Cyc/o-oxygenose
Prostacyclin
, Synthetase
12-andother-(HP)ETE
\
X
P6G2-
•PGH2
Other
Hydroxylated 12-(H)ETE
HETE
Products
Thromboxane
Synthetase
TxA,
HHT
TxB2
Figure 3. Pathways of generation of oxidative metabolites of arachi-
donic acid. Tx = thromboxane, PG = prostaglandin, HETE » hydroxy eico-
satetraenoic acid, HHT = 12-L-hydroxy-5,8,10 heptadecatrienoic acid. [Mod-
ified from Lewis and Wasserman: In: The Science and Practice of Clinical
Medicine: Rheumatology (Cohen A, ed). New York, Grune & Stratton (In press)]
generate SRS-A during IgE-dependent immunologic reactions. Decreasing
amounts of SRS-A are generated by increasingly pure human lung mast cell
preparations, suggesting that cell types other than mast cells alone are
involved in its generation (30).
Presently the only acceptable assay for SRS-A employs the anti-histamine-
treated guinea pig ileum, but it is not specific for this mediator. There-
fore, SRS-A must be further identified by its chromatographic properties, its
susceptibility to inactivation by arylsulfatases, and its decreased assay-
ability in the presence of a semispecific blocker, FPL55712.
Products of Arachidonic Acid Oxidation* In the human, products of
arachidonic acid metabolism constitute the vast majority of prostaglandins
and related compounds. Arachidonic acid mobilized from cell membrane phos-
pholipids by the action of phospholipase A is then either converted to pros-
taglandins and thromboxanes via a eyelooxygenase-dependent pathway or con-
verted by a lipooxygenase to 12-L-hydroxy-5,8,10,14 eicosatetraenoic acid
(HETE) and related compounds (Fig. 3). Several of these products have been
282
-------
described iji vitro subsequent to noncytolytic activation of lung tissue and
rat mast cells. Specifically, PGD , PGI , and HETE have been generated by
isolated rat mast cells, whereas PGF and PGE_ are present after IgE-dependent
A Q £•
activation of human lung tissue (31, 33). Guinea pig central airway smooth
muscle is constricted by PGF , thromboxane A., and both cyclic endoperoxides
PGG_. In the anesthetized dog, PGF. and PGD. both constrict central and
2 Z
-------
patients with cold urticaria (39) or asthma (1) following challenge with ice
water or antigen inhalation, respectively. HMW-NCF has been characterized
as a 750,000-dalton neutral protein which attracts and deactivates neutro-
phils ijn vitro. HMW-NCF appears to be a sensitive marker for mast cell acti-
vation as it is present in serum following challenge of allergic asthmatics
with doses of antigen insufficient to induce bronchospasm.
Histamina. In addition to its smooth muscle directed actions, histamine
is active in modulating the migration of inflammatory leukocytes. Thus, via
an H- mechanism, histamine enhances random and directed migration of eosino-
phils and neutrophils and is a weak chemotactic factor for human eosinophils.
By its H actions histamine inhibits both random and directed migration of
neutrophilic and eosinophilic polymorphonuclear leukocytes (7).
Lipid Chemotactic Factors. The lipooxygenase product of arachidonic
acid, HETE, has an ECF-A-like spectrum of activity. Unlike ECF-A, however,
it increases eosinophil random migration and is a less potent chemotactic
deactivator than ECF-A (13). Equivalent effects on neutrophils have also
been described for the cyclooxygenase product 12-L-hydroxy-5,8,10-heptade-
catrienoic acid (HHT) (13). A possibly related factor is a nonpolar lipid
with chemotactic activity mainly for neutrophils which has been described in
anaphylactic diffusates of rat mast cell-rich peritoneal cell preparations.
Structural Proteoglycans
Heparin. The sulfated, metachromatic, mucopolysaccharide heparin has
been identified in human lung and localized to the mast cells isolated from
human lung tissue (26). Human lung heparin is a proteoglycan of approxi-
mately 60,000 m.w. comprised of a protein core to which are attached, by
xylosyl-seryl linkages, glycosaminoglycan side chains of average m.w. 20,000.
Human heparin interacts with human antithrombin III to accelerate anticoagu-
lation. Heparin from other sources has been shown to inhibit generation of
the convertase enzyme (C3bBb) responsible for amplification of cleavage of
the third component of complement (C3) (41), to inhibit binding of the C1q
fragment of complement to immune complexes (24), to inhibit action of the
activated first component of complement (C1s) upon its substrates, the
fourth (C4) and second (C2) components (24), and to prevent binding of C2 to
C4b (23). In addition, heparin has been shown to bind to platelet factor
IV and to liberate lipoprotein lipase.
Granule-Associated Enzymes
Chymase. Chymase has been isolated from the mast cell of the rat and
has been' identified histochemically in the human mast cell (14). This
enzyme is minimally active as a protease while stored in the granule, prob-
284
-------
ably due to masking of the active site by heparin, but once freed its
specific activity is comparable to that of pancreatic a-chymotrypain. Chymo-
trypsin inhibitors have been shown to inhibit some inflammatory consequences
of IgE-dependent activation in rabbit skin, adding relevance to the mast cell
location of this activity (18).
Lung Kallikrein of Anaphylaxis. A 400,000 m.w. preformed enzyme termed
lung kallikrein is released subsequent to antigen challenge of IgE-sensitized
human lung fragments (40). This activity can cleave kininogen to yield brady-
kinin and can activate Hagemen factor. The released kinins may contract
bronchial smooth muscle and increase vascular permeability. Kinins have
also been noted to induce bronchospasm when inhaled by asthmatic humans and
have been detected in the serum of a single patient sustaining an acute asthma
attack. Activation of Hageman factor with the induction of the clotting and
fibrinolytic cascades could also be reasonably expected to ensue upon release
of lung kallikrein*
Arylsulfatase. Arylsulfatase has been identified in human lung mast
cells (30). Arylsulfatase A and B enzymes can inactivate SRS-A and, further-
more, arylsulfatase A is immunologically released with the mast cell granule
in the rat (25).
Other Enzymes. B-glucuronidase and N-acetyl-B-D-glucosaminidaoe (hexo-
saminidase) have been identified in human lung and in rat mast cells and
both are released upon immunologic activation of the rat mast cell (34)*
These enzymes in concert with arylsulfatase, when obtained from non-mast cell
sources, have been demonstrated to degrade ground substance mucopolysaccha-
rides.
MEDIATOR INTERACTIONS
Although the mediators of immediate type hypersensitivity have been
identified and assessed for their effects as isolated factors, some under-
standing of their interactions is available* Thus the spasmogenic effects
of histamine and SRS-A are synergistic and, in addition, the presence of
either directly induces prostaglandin generation* The interactions of BCF-A,
histamine, and ECF-oligopeptides are not fully delineated, but it is known
that histamine in high concentrations inhibits and in low concentrations en-
hances ECF-A-induced eosinophil chemotaxis. In addition, the N and C ter-
minal tripeptides of ECF-A are reversible and irreversible inhibitors of
eosinophil chemotaxis, respectively (12). In addition, PGE enhances and
PGF. inhibits neutrophil chemotaxis, probably by their actions upon cyclic
nucleotide concentrations* However, given the number and complexity of mast
cell-dependent mediators other, potentially critical, biologic interactions
await elucidation.
285
-------
DETERMINANTS OF ACTIVITY OF MAST CELL MEDIATORS
Following activation of mast cells by IgE-dependent or other mechanisms,
the panoply of mediators generated and released and the myriad of their po-
tential interactions provide the substrate for induction of inflammatory
events. The putative role for the mast cell and its mediators in both the
homeostatic as well as pathophysiologic induction of inflammation in the
lung is strengthened by the location of the mast cell at the respiratory
surface. Thus the interaction of surface and intraluminal mast cells with
antigen or noxious agents could, by the action of locally released permea-
bility factors, lead to alterations of the respiratory epithelial barrier,
thereby permitting access to the large number of deeper situated mast cells.
Of more direct relevance has been the in vivo demonstration that solely mast
cell-dependent events can indeed cause both immediate and more persistent
inflammatory processes. The biphasic response of airways to inhaled antigen
may be comparable to the biphasic cutaneous response to IgE-dependent mast
cell activation (8, 36). In the cutaneous model a wheal-and-flare response
reflects altered vascular permeability at the site, whereas later inflamma-
tory events are accompanied by an intense cellular infiltration (36). Immuno-
chemical studies have not revealed the participation of immune complexes or
complement during this later inflammatory phase (36). Elicitation of both phases
by purely IgE-dependent mechanisms indicates that mast cell activation can con-
tribute to subacute and chronic as well as to acute pathobiologic processes.
This fact is central to according the mast cell an important role in asthma,
since this disease is characterized by acute exacerbations superimposed on
chronic hyperirritability of the airway. Whether continued alteration in the
threshold of airway response is due to mast cell mediators directly or to
cellular infiltration is not established, but mast cell-derived mediators could
be critical by either route alone or in combination.
The mechanism by which mast cell mediators might provoke such biphasic
inflammatory responses is depicted in Figure 4. The mast cell-dependent
generation of bronchospastic and vasoactive mediators establishes a local
vasodilatory or humoral phase of inflammation while the release of chemotactic
mediators provokes a cellular phase of inflammation. The humoral phase, ap-
parent within minutes, would lead to egress from the circulation of immuno-
globulin and complement as well as fibrinolytic, procoagulant, and kinin-
generating proteins. This response could be expected to be rapidly bene-
ficial to the host by aiding removal of invading microorganisms, by local-
izing and removing noxious agents, and by facilitating leukocyte migration
through venular disconnections. On the other hand, an unregulated humoral
phase might be expressed in disease as acute exacerbations of bronchospasm,
alterations in tracheo-bronchial mucus flow or consistency, or as upper air-
286
-------
NFUUUMATON
t (LIAtT
PERTURBATION j ^ i GRANULE
Bwtoal OiMow
(unccrtnuoa)
v TISSUE
^ OGGRAOATCN
AND REPAIR
ChymoM
HUMORAL
PHASE
Antibody
Conpivmnl
Figure 4. Schematic representation of mast cell-dependent humoral and
cellular inflammatory events. [Modified from Austen: Triangle 17:109,
1978]
way obstruction due to edema. The generation and release of chemotactic
factors would, in a period of several hours, be expected to call to the local
inflammatory focus both eosinophilic and neutrophilic polymorphonuclear
leukocytes. These cells could prove beneficial not only by their phagocytic
function but also by their ability to inactivate mediators (see below).
Conversely, if accumulation of leukocytes were uncontrolled, the pathobio-
logic consequences of tissue infiltration with leukocytes would ensue.
Clinically, such infiltration might be seen in the inflammatory bronchial
epithelium of asthmatic patients, in vasculitis, or perhaps, via the action
of leukocytic lysosomal enzymes, in the destruction of lung tissue and the
induction of fibrosis. By their capacities to deactivate leukocytes to
further chemotactic activation, mast cell chemotactic mediators might also
inhibit localization of leukocytes. In cold urticaria, following mediator
release by experimental challenge, circulating neutrophils are rendered un-
responsive to in vitro chemotactic stimulation. This effect is time limit-
ed but persists beyond the period in which measurable quantities of mediators
are noted ( 6 ) . Such an inhibitory action of chemotactic factors could prove
beneficial by blunting exuberant inflammation or, if prolonged or ill-timed,
might be harmful by preventing adequate host response to local insult. The
release of mast cell proteases and lysosomal enzymes may themselves lead to
alterations in ground substances and to activation of such protein inflamma-
tory cascades as complement, fibrinolysis, and coagulation. These processes
would amplify inflammatory events and might prove beneficial to the elimi-
287
-------
RELEASE
OF PRIMARY
ACTIVATION SECRETION OF GRANULES MEDIATORS
I 1
PERTURBATION — »- J^jJ
1
Histanine ^
/^, Irf MW ECF ^\^
** LUI A -^— ^^_
/\
FURTHER UNCOVERING
OF GRANULE ACTIVITY
/ \
Chymase Heparin
TARGET CELL ACTION BIOOEGRADATION
i i i
1
1
1
^ Histanfiinott
©•^\_^r- AryhulfataM B <
— "
\^~""~*- Phospholipase D
Phagocytosis j
^
GENERATION OF SECONDARY MEDIATORS
*•
bpid chentotactic SRS-A PAF
and chGfflokiretic *
factors L
Figure 5. Schematic representation of endogenous regulation of mediator
expression with particular emphasis on the role of the eosinophil. [Modified
from Austen: Triangle 17:109, 1978]
nation of microorganisms or toxic agents but might also/ if unregulated, lead
to tissue destruction, chronic inflammation, and fibrosis.
REGULATION OF MAST CELL MEDIATOR RELEASE AND ACTIVITY
The events which lead to the generation and release of mast cell media-
tors and thereby to the elicitation of mast cell-dependent inflammatory events
are subject to regulation at several critical points. Thus, activation of the
mast cell, the secretion of granules, the release of the preformed mediators
and generation of unstored mediators, the effect of mediators upon target
cells, and finally the persistence in tissue of the mediators are all under
biologic control (Fig. 5).
The extent and effect of activation of the mast cell are controlled by
regulation of access of the eliciting agent to this cell, the amount of
specific IgE antibody bound to the mast cell surface, and by the intensity
of the stimulus. The mast cell itself might alter local permeability allow-
ing increased antigen contact or, by the H action of histamine, may suppress
lymphocyte recognition of antigen and thereby prevent immune response to
288
-------
invaders. In addition, the ratio of specific mediator generated is depen-
dent on the intensity of the activating stimulus as limited stimulation of
the mast cell leads to intracellular accumulation of SRS-A without release
of SRS-A or histamine (22). Intracellular mast cell arylsulfatases may also
control mediator generation by degrading SRS-A prior to its release.
Subsequent to activation of the mast cell the secretion of granules is
under control of cyclic nucleotides as noted above. Of direct relevance is
the possibility that the mast cell itself, by the mediator histamine via an
H- action, and prostaglandins E , D , and I may directly elevate cAMP and
2 » * «
thereby inhibit granule secretion. Conversely, the H action of histamine to
elevate cGMP and that of PGF_ to lower cAMP levels might augment mediator
2o '
release.
Following secretion of the mast cell granule, the release of preformed
mediators and thus the full expression of their activity is dependent on the
independent solubilities of each of the mediators. Thus 6-glucuronidase,
arylsulfatase A, histamine, ECF-A and ECF-oligopeptides are fully soluble in
physiologic buffers, hexosaminidase requires 0.5 M NaCl for full elution from
the granule, and chymase-heparin complex requires 1.0 M NaCl for dissolution
(19, 25). The relevance of granule binding is emphasized by the marked in-
crease in proteolytic activity of the chymase following its elution from the
granule. Although the mechanism(s) by which the mast cell granule is solu-
bilized in vivo remains unknown, it is clearly an important step in regulating
the activity of mast cell inflammatory mediators.
Regulation of the action of mast cell mediators upon target cells may
derive from alterations in cyclic nucleotide concentrations, as noted above/
or by the interaction of several mediators or their metabolites with the same
target cell. Thus, histamine may be additive or inhibitory to ECP-A action
upon eosinophils depending on the ratio of the two activities present. In
addition, the constituent tripeptides of ECF-A are inhibitory to induction
to eosinophil chemotaxis by ECF-A. As noted above, mediators may also be
synergistic on smooth muscle as exemplified by SRS-A and histamine action.
The regulation of generation of unstored mediators has not been fully
elucidated, but data suggest that the local microenvironment is critical to
their generation. For example, SRS-A generation is maximal in mixed rather
than in pure mast cell populations (30), and SRS-A, PAF, and lipid chemo-
tactic factors can all be generated from mononuclear leukocyte populations.
The ratios of critical target cells thus may be crucial to the amount and
type of mediator generated.
Finally, the persistence of the effect of mediators is also regulated.
While this may reflect the limited availability of key mediators, probable
tachyphylaxis to some, and mediator clearance by excretion, it is also af-
fected by enzymatic inactivation of these biologic activities. As many mast
289
-------
cell-derived chemotactic mediators are eosinophilotactic, the eosinophil
content of mediator-inactivating enzymes may provide a feedback control of
mediator effects (Fig. 5). Notably, the eosinophil contains histaminase,
arylsulfatase B, and phospholipase D, which inactivate histamine, SRS-A, and
PAF, respectively. SRS-A is inactivated not only by extracted eosinophil
arylsulfatase/ but also by the enzyme-rich resting granulocyte, presumably
after cellular uptake of the mediator. Some other cells also contain mediator-
inactivating enzymes. Neutrophils contain histaminase, and at least one mono-
nuclear cell population is rich in histamine methyltransferase. Interest-
ingly/ both mast cells and basophils contain arylsulfatase.
Inactivation of arachidonic acid metabolites is complicated/ as some
intermediate metabolic products are biologically active. Thus PGD and
thromboxane A derived from the cyclic endoperoxides are potent bronchocon-
strictors. However, most stable catabolic end products of arachidonic acid
metabolism, such as thromboxane B. (from thromboxane A_) and PG-6-keto-F.
2 2 1o
(from PGI ), have little or no defined biologic activity.
CONCLUSION
The mast cell-derived preformed and newly generated mediators are re-
leased by IgE-dependent and independent mechanisms, and are biologically
available during inflammatory events. The relevance of these mediators to
allergic disease has been derived from studies of the pathophysiologic
alterations induced by the individual mediators, identification of the medi-
ators in tissue or biologic fluids of patients experiencing allergic re-
actions, and the known pathophysiology of the various atopic diseases.
Mediators of immediate hypersensitivity not only possess the ability to
induce immediate tissue responses such as a wheal and flare, anaphylaxis, or
rapid-onset brief-duration alterations in pulmonary function, but may also
mediate a prolonged inflammatory response. The fact that IgE and mast cells
are relevant to prolonged inflammatory events has been documented by passive
transfer with isolated IgE of delayed inflammatory responses in skin and by
the dependence on IgE antibody for similar delayed alterations in pulmonary
mechanics following inhalation of antigen. In lung, these delayed responses
are prevented by pretreatnient with disodium cromoglycate, which supports the
central role of the mast cell. Histopathologic assessment of delayed re-
sponses reveals an influx of neutrophils, eosinophils, basophils, lymphocytes,
and mononuclear leukocytes, the deposition of fibrin, and vascular abnor-
malities which may progress to frank vasculitis (36). Although some of the
mediators responsible for the early and later phases of the IgE-mast cell
reaction can be surmised from the kinetics of their in vitro effects, their
absolute identification and participation in disease require further defi-
nition.
290
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The postulated role of the mast cell and its mediators in inflammation
may provide insight not only into the clinical evolution of some allergic
disorders such as progression of seasonal to perennial asthma, but also into
the local homeostatic regulation of the lung environment and thereby the de-
fense of the lung. Although much remains to be clarified, the rapidly ex-
panding understanding of target cell activation together with the identifi-
cation of mast cell-derived mediators provides a framework for definition of
the complex processes that provide pulmonary defenses.
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10. Gillespie E: Cyclic AMP and microtubules. In: Cyclic AMP Cell Growth,
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1975
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14. Gomori G: Chloroacetyl esters as histochemical substrate* J Histochem
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24. Loos M, Volanakis JE, Stroud RM: Mode of interaction of different
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25. Lynch SM, Austen KF, Wasserman SI: Release of arylsulfatase A but
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26. Metcalfe DD, Lewis RA, Silbert JE, Rosenberg RD, Wasserman SI, Austen KF:
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27. Orange RP: Dissociation of the immunologic release of histamine and
' slow reacting substance of anaphylaxis from human lung using cytocha-
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28. Orange RP/ Murphy RC, Karnovsky ML, Austen KF: The physicochemical
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29. Oronsky AL, Buermann CW: Phagocytic release of human leukocyte neutral
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30. Paterson NAM, Wasserman SI, Said JW, Austen KF: Release of chemical
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117:1356-1362, 1976
31. Platshon LF, Kaliner MA: The effects of the immunological release of
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32. Raepple E, Hill H-U, Loos M: Mode of interaction of complement in the
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33. Roberts LJ, Lewis RA, Hansborough R, Austen KF, Oates JA: Biosynthesis
of prostaglandins, thromboxanes and 12-hydroxy-5,8,10,14 eicosatetraenoic
acid in rat mast cells. Fed Proc 37:384 (abst), 1978
34. Schwartz LB, Austen KF, Wasserman SI: Immunologic release of N-acetyl
g-D-glucosaminidase (hexosaminidase) and of 3-glucuronidase from purified
rat mast cells. Fed Proc (In press)
35. Selye H: The Mast Cell. Washington, Butterworths, 1965
36. Solley GO, Glcich GJ, Jordon RE, Schroeter AL: The late phase of the
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bodies. J Clin Invest 58:408-420, 1976
37. Tomita A, Patterson R, Suszko IM: Respiratory mast cells and basophiloid
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38. Uvnas B: Chemistry and storage function of mast cell granules* J Invest
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39. Wasserman SI, Soter NA, Center DM, Austen KF: Cold urticaria: recog-
nition and characterization of a neutrophil chemotactic factor which
appears in serum during experimental cold challenge. J Clin Invest
60:189-196, 1977
40. Webster ME, Hbrakova Z, Beaven MA, Takahashi H, Newball HH: Release of
arginine esterase and histamina from human lung passively sensitized
with ragweed antibody. Fed Proc 33:761, 1974
41. Weiler JM, Yurt RW, Fearon DT, Austen KF: Modulation of the formation
of the amplification convertase of complement, C3bBb, by native and
commercial heparin. J Exp Med 147:409-421, 1978
42. Zeiger RS, Yurdin DL, Colten HR: Histamine metabolism. II.
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Allergy Clin Immunol 58:172-182, 1976
294
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DISCUSSION
PARTICIPANT: Did I understand you to say that the rat mast cell makes PGI ?
DR. WASSERMAN: Yes, it does, with calciun ionophore. That is presented in
abstract form, and was published in Federation Proceedings/ 1978/ by Lewis in
collaboration with Roberts, Austen/ and Gates.
PARTICIPANT: To tie this a little bit to air pollution in particular, as you
mentioned at the end, the mast cells are sitting near the entry port* The paper
of H. B. Thomas, in the late 1960s, showed that 0.5 parts per million nitrogen
dioxide cause degranulation of submucosal mast cells in the rat lung. I think
it would be interesting to ask why that did occur; after all, the mast cells
are submucosal. How does the effect get down to the submucosa? Perhaps,
there is some sort of opening of tight junctions that would allow an autonomic
mediated response, or perhaps, antibody type material could even migrate down
there.
DR. WASSERMAN: Mast cells are not solely submucosal. One of the more important
recent observations is that they are both intraluminal and epithelial, and their
availability to noxious stimuli, antigens, and so forth is obviously facilitated.
It is presumed that their activation leads to increased access of materials to
deeper structures by the action of vasoactive materials.
DR. GOLDSTEIN: On your slide you mentioned a role for lysophospholipids
in degranulation, or in the membrane processes that occur. Is that something
that's hypothesized?
DR. WASSERMAN: Yes. Both diacylglycerol and lysophotidylcholine have been
postulated to enhance degranulation.
DR. GOLDSTEIN: Do you know it's an active compound, or do you have evidence
for it?
DR. WASSERMAN: Lysophospholipids are known to be fusigenic in other systems.
They cause lipid vesicles and platelets to fuse and they are very, very active
materials. However, in recent work, Sullivan has not been able to find
295
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lysophospholipids and has instead noted diacylglycerol, a product of phos-
pholipase C, in mast cells. This material accumulates within three seconds in
the mast cell, and it is also a very potent fusigen. It is possible that other
fusigens are available. It's also conceivable that lysophospholipids are so
active that when they're generated in mast'cells they are rapidly degraded
and thus are difficult to detect.
DR. BOUCHER: With respect to one of your last comments showing that mast
cells are close to the lumen at least below the junctions, a lot of eosino-
phils seem to be present, proportionately, with them. Is there any data that
suggest that these mast cells are, in fact, activated at that site?
DR. WASSERMAN: The association of the eosinophils and mast cells has been
known for a long time. Somebody with a lot more time and patience than I have
once ground up a cow, and in every tissue noted mast cells and eosinophils to
occur together and generally at portals of entry. They are found throughout
the upper and lower respiratory tract, gastrointestinal tract, urinary tract,
and skin, although the eosinophils are not as prevalent in the skin. It's my
prejudice that the eosinophil-mast cell axis that we described here is probably
much more effective as a homeostatic regulator of low-grade chronic mast cell
activation (which obviously must be ongoing at all times) than as an ana-
phylaxic control. In fact, in many allergic reactions, the eosinophilic
response is a marker for continued activation of the mast cells and for con-
tinued disease activity. It would be my prejudice that the eosinophils are
noted near mast cells because of previous mast cell activation and the generation
and release of specific eosinophil chemotactic factors.
296
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Angiotensin Converting Enzyme:
I. New Strategies for Assay
J. W. Ryan, A. Chung, and U. S. Ryan
University of Miami
Miami, Florida
Angiotensin converting enzyme appears to occur in association with endo-
thelial cells of virtually all vascular beds {for review see 22). The enzyme
also occurs in association with brush border of renal proximal tubule (2, 30)
and small intestine (31). However, bulk conversion of angiotensin I into
angiotensin II occurs, apparently unaided by blood enzymes, within the pulmo-
nary vascular space. Angiotensin converting enzyme is situated on the lumi-
nal surface of pulmonary endothelial cells (19, 23, 26) and is strategically
placed for bulk processing, not only of angiotensin I but also bradykinin
and perhaps other oligopeptides (neurotensin, enkephalins) having adequate
affinity for the enzyme. The lungs have an enormous vascular bed through which
flows the entire cardiac output* Further, because of the peculiar situation
of the lungs within the general circulation, their venous effluent empties
directly into the arterial circulation. Hence, the activity or inactivity of
lung angiotensin converting enzyme may matter greatly to the quality of arterial
blood and to the function of distant organs. Whether angiotensin converting
enzyme is important to intrinsic lung function remains to be determined.
A major barrier to Improved understanding of the pathophysiologic role of
lung angiotensin converting enzyme is that of adequate techniques for assay.
In principle, one should be able to measure modulations of enzyme activity
by examining for variations in the pulmonary arteriovenous gradients of angio-
tensin I, angiotensin II, and bradykinin. However, these substances occur
in blood at concentrations of less than 10 M. Radioimmunoassays can be
used, but the precision of such assays is not great and their specificities,
297
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especially for bradykinin, are not proved. The assay problem is not sub-
stantially improved by following the changes of activity of intravenously
administered bradykinin or angiotensin I, either by radioimmunoassay or the
dynamic, on-line bioassay system of Vane (29). Furthermore, the bioassay is
not readily adaptable for use with human subjects*
In an effort to develop simpler and more precise means of measuring
angiotensin converting enzyme, we have synthesized a series of acylated
tripeptides, each bearing a radioisotope in the moiety used for acylation.
Most have been prepared to contain a H-atom, but gamma-emitting isotopes,
e.g., I, can be used as well. Angiotensin converting enzyme functions
as a dipeptidyl carboxypeptidase and acts on acylated tripeptides to yield
a dipeptide and an acyl-amino acid (for reviews see 1, 27). By careful
selection of amino acid residues one can vary the affinity of substrate for
enzyme. As has been shown for benzoyl-Gly-His-Leu (hippuryl-His-Leu; refer-
ences 4, 5), the acyl-amino acid product can be separated from the acyl-
tripeptide substrate by partitioning between acid-aqueous solvent and an
organic solvent such as ethyl acetate or toluene. The efficiency of the
extraction technique is a function of amino acid residues and their sequence*
Thus an aromatic or aliphatic amino acid is advantageous for the H-labeled
product and a basic amino acid is useful when placed in the dipeptide leaving
group.
Six radiolabeled acylated tripeptides have been prepared, and four have
been characterized in detail as substrates for angiotensin converting
enzyme. Three of the latter compounds can be used to measure converting
enzyme of lungs perfused with artificial salt solutions. We believe that
these three substrates can be used to measure lung converting enzyme in vivo.
MATERIALS AND METHODS
Synthetic bradykinin and its higher homologs, angiotensin I and its lower
homologs, and BPP and its lower homologs were prepared by the solid phase
peptide synthesis technique (28). The completed peptides were purified by
two or more of the following techniques: counter current distribution, molec-
ular sieve chromatography (Bio-Gel P-2, Sephadex G-25), and partition chroma-
tography (LH-20 within 6% butanol in HO; Sephadex G-25 with butanol, acetic
acid, HO; 4:1:5). The peptides were synthesized by J. M. Stewart, University
of Colorado Medical Center, and G. H. Fisher, University of Miami (12).
The compounds, p-I-benzoyl-Gly-Gly-Gly, p-I-benzoyl-Pro-Phe-Arg,
p-I-benzoyl-Gly-His-Leu, p-I-benzoyl-Phe-Ala-Pro, p-I-benzoyl-Phe-His-Leu,
and p-I-benzoyl-Phe-Ser-Pro, were synthesized by step-wise solution methods
(17, 18). Purifications were accomplished with one or more of the following
298
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techniques: molecular sieve chromatography (Sephadex G-10), ion-exchange
chromatography (CM-Sephadex or DEAE-Sephadex with ammonium acetate buffers).
The p- H-benzoyl-derivatives were prepared by New England Nuclear Corp. by
3 2
dehalogenation in H. gas at 39 Ib./in. for 1 hour over 5% rhodium on calcium
*• 3
carbonate in dimethyl-formamide and HO, 1:1 (vol:vol). Each of the H-labeled
acyl-tripeptides was obtained at specific radioactivities of >20 Ci/mmol. The
H-labeled compounds were purified on Sephadex G-10, and each behaved as a
pure substance in two or more thin layer chromatography systems (18) and on
paper electrophoresis at pH 2.0 and 5.0 (20)* The H-labeled compounds,
stored at 1 mCi/ml of ethanol at -28°C, did not undergo detectable radiolysis
over a period of 12 ifcnths.
Our studies used angiotensin converting enzyme partially purified from
guinea pig lung or urine and human urine (18). Pure rabbit lung angiotensin
converting enzyme was provided by R. L. Soffer, Cornell University Medical
College (27).
The experiments using anesthetized rats or perfused isolated rat lung
were performed as described by Roblero et al^. (16) and Ryan et al. (24).
For in vitro assays, we used the protocol described by Ryan et al. (17). In
brief, reactions were stopped by adding 0.1N HC1 to a pH <2. The H-labeled
acyl-amino acid product was extracted into an equal volume of organic solvent.
Ethyl acetate was used for all substrates except H-benzoyl-Phe-Ala-Pro.
Toluene was used for extraction of reaction mixtures containing the latter
substrate (18).
RESULTS.AND DISCUSSION
Recognition Sites of Angiotensin Converting Enzyme
In the early 1970s, it was recognized that angiotensin converting enzyme
is misnamed. The enzyme acts as a dipeptidyl carboxypeptidase capable of
removing the C-terminal dipeptide of a variety of oligopeptides (1, 9, 11).
In fact, bradykinin is a better substrate (lower K ) than angiotensin I, and
m
the enzyme is sometimes known as kininase II. In terms of K , the venom
m _e
peptide, BPP_ (13), is the best of the known substrates (K <10 M).
3a HI
Clearly, the enzyme is not specific, but there is reason to believe that
the enzyme is highly selective. Further, it is evident that the enzyme
possesses a number of recognition sites, knowledge of which is useful for
design of synthetic substrates.
In our early studies on the metabolism of kinins by intact lungs, we
noted that bradykinin is inactivated more rapidly than any of its N-extended
higher homologs (16, 21). When pure lung angiotensin converting enzyme
became available (8), it was found that much of the selectivity of processing
299
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of kinins by intact lungs could be explained in terms of one enzyme (see
Table 1). Clearly, the enzyme is sensitive to amino acid residues at a dis-
tance from the cleavage site. Chain length and charge of the substrates
appear to be important factors.
Angiotensin I and its lower homologs are also treated differently by
converting enzyme• As shown in Table 2, des-Asp -angiotensin I is a somewhat
better substrate than angiotensin I itself (lower K , higher V /K ). in
m max m
terms of V /K , the C-terminal pentapeptide is a better substrate than
max m
angiotensin I. In comparison with some of the acylated tripeptide substrates
described below, the C-terminal hexapeptide is a surprisingly poor substrate*
Nonetheless, TabIs 2 emphasizes the points that converting enzyme is sensitive
to amino acid residues distant from the cleavage site and that chain length
is an important factor.
Greater insight into the importance of enzyme recognition sites has come
from studies of two inhibitors of converting enzyme, BPP (
-------
Table 2. Reaction of Pig Lung Angiotensin Converting
Enzyme with Angiotensin I and Its C-terminal Lower Homologs
As p- Arg-Val -Tyr- 1 1 e-Hi s-Pr o-Phe-Hi s-Leu
Arg-Val-Tyr-Ile-His-Pro-Phe-Hi s-Leu
Val-Tyr-Ile-His-Pro-Phe-His-Leu
Tyr-Ile- Hi s-Pro-Phe-Hi s-Leu
Ile-His-Pro-Phe-Hi s-Leu
Km (wM>
33
11
170
100
1,000
max
1.98
0.99
7.94
8.83
37.71
V /K
max m
0.060
0.090
0.047
0.088
0.038
For experimental details see reference 3. Reaction rates were measured
in terms of the appearance of the fluorophor formed by reacting His-Leu with
o-phthaidialdehyde. V is expressed in terms of ymol/min/mg of enzyme
protein.
Both studies have shown that Trp of BPP interacts with an important recogni-
7cl
tion site/ apparently distant from the catalytic site of the enzyme. However,
the distant binding site may not be fixed or stationary, as the N-terminal
homolog of BPPga,
-------
Table 3. Inhibition of Angiotensin Converting Enzyme by BPP
and Some of Its Lower Homologs (12)
Structure I (nM)
20 Ci/mmole. Further, each compound was labeled specifically in
position 4 of the benzoyl ring.
Compound I, after tritation, is [ H]hippuryl-Gly-Gly. Hip-Gly-Gly is
commonly used as a substrate for converting enzyme (10) and appears to be
remarkably resistant to hydrolysis by other enzymes. However, (shown in
Table 4 ) , Hip-Gly-Gly has relatively little affinity for converting enzyme ,
and its H-derivative cannot be used to obtain a sensitive assay.
Compound II, the acylated C-terminal tripeptide of bradykinin, yields a
much more sensitive assay, although its V /K suggests that one or both of
in
the products is a poor leaving group. Under some conditions, II is hydrolyzed
by a carboxypeptidase B-like enzyme and is not specific. The encouraging
results obtained with III, Hip-His-Leu, deserve comment, as the magnitude of
its K is heavily dependent on selection of buffer and salts. Cushman and
302
-------
Table 4. Characteristics of the Reactions of
Acylated Tripeptides with Angiotensin Converting Enzyme (18)
I.
II.
III.
IV.
Substrate
benzoyl-Gly-Gly-Gly
b enzoyl -Pr o-Phe- Ar g
b enzoyl-Gl y-Hi s-Leu
b enzoyl -Phe- Al a-Pr o
V
max
( uM/sdn)
83.3
1.4
5.6
0.091
K
m
(UM)
5000
172
177
12.5
V
max
K
m
0.0167
0.0081
0.0316
0.0073
Each of the H-labeled substrates, with varying concentrations of
unlabeled carrier, was reacted with guinea pig urinary angiotensin converting
enzyme such that no more than 10% of initial substrate was used. Similar
results are obtained when rabbit lung converting enzyme is used.
Cheung (4, 5) were the first to use Hip-His-Leu as a substrate for converting
enzyme, and under their conditions of assay (0.1M phosphate buffer, pH 8.3,
plus 0.3M NaCl), Hip-His-Leu has a relatively low affinity for the enzyme
(K >2000 uM) . Quite by accident, we found that its affinity is greatly in-
m
creased by Na SO at 0.75M (10). Phosphate is inhibitory; thus the most
sensitive assay uses [ H ] Hip-Hi s-Leu in 0.05M HEPES buffer, pH 8.0, plus 0. 1M
NaCl and 0.75M Na SO . As yet, we have no explanation for the dramatic
effects of SO4. Nonetheless, our work and that of Dorer et al. (10) indicate
that it will be important to check for effects of organ perfusion solutions
(e.g., Krebs-Henseleit solution) on the interactions of a given substrate with
angiotensin converting enzyme. Plasma and blood are likely to present their
own problems.
Compound IV, an acylated analog of the C-terminal tripeptide of B*p5a» can
be used in assays 400 times more sensitive than those using Hip-Gly-Gly (I).
Neither II nor IV requires Cl~, and each is apparently insensitive to added
Na,S04. The Km for IV is intermediate between that for angiotensin I (33
and that for bradykinin (0.85 yM) (9, 27).
CONCLUDING REMARKS
Depending on the goals of the experiment, compound I, III, or IV can be
used to measure angiotensin converting enzyme of plasma, tissue homogenates,
cells in culture, or isolated, perfused lungs. Compound IV may well be suitable
fur use with lungs of intact animals. Because of the wide range of affinities
of the substrates for converting enzyme and their high specific radioactivities,
303
-------
it should be possible to examine for modulations of converting enzyme activity
using a wide range of substrate concentrations. Each of the H-labeled sub-
strates, without carrier, is labeled such that 1 nM solutions contain easily
detectable radioactivity. Ill us the range of concentrations readily tested
extends from 1 nM to greater than twice K . So far as is known, none of the
m
substrates or products is biologically active or toxic. The acyl-tripeptides
are relatively inexpensive and can be made in quantity.
Again, according to the goals of a given experiment, an acyl-tripeptide
substrate can be selected to give <10% to >90% hydrolysis during one transit
through the lungs. Thus, it should be possible to define conditions to assess
easily effects of variables such as perfusion rate, pressure, toxic inhalants,
and oxygenation.
ACKNOWLEDGMENTS
This work was supported in part by the Council for Tobacco Research—
U.S.A. Inc., the John A. Hartford Foundation, Inc., and the United States
Public Health Service (HL-22086, HL-21568, and HL-22896).
REFERENCES
1. Bakhle YS: Converting enzyme in vitro measurement and properties.
In: Handbook Exp Pharmac (Page IH, Bumpus FM, eds). Berlin, Springer-
Verlag, 1974, vol 37, pp 41-80
2. Caldwell PRB, Seegal BC, Hsu KC, Das M, Soffer RL: Angiotensin-converting
enzyme: Vascular endothelial localization. Science 191:1050-1051, 1975
3. Chiu AT, Ryan JW, Stewart JM, Dorer FE: Formation of angiotensin III
by angiotensin-converting enzyme. Biochem J 155:189-192, 1976
4. Cushman DW, Cheung HS: A simple substrate for assay of dog lung angio-
tensin converting enzyme. Fed Proc 28:799, 1969
5. Cushman DW, Cheung HS: Spectrophotometric assay and properties of the
angiotensin-converting enzyme of rabbit lung. Biochem Pharmac 20:
1637-1648, 1971
6. Cushman DW, Cheung HS, Sabo EF, Ondetti MA: Design of potent competitive
inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercap-
to alkanoyl amino acids. Biochemistry 16:5484-5491, 1977
7. Cushman DW, Pluscec J, Williams NJ, Weaver ER, Sabo EF, Kocy O, Cheung HS,
Ondetti MA: Inhibition of angiotensin-converting enzyme by analogs of
peptides from Bothrops jararaca venom. Experientia 29:1032-1035, 1973
8. Dorer FE, Kahn JR, Lentz KE, Levine M, Skeggs LT: Purification and
properties of angiotensin-converting enzyme from hog lung. Circ Res 31;
356-366, 1972
304
-------
9. Dorer FE, Kahn JR, Lentz KE, Levine M, Skeggs LT: Hydrolysis of brady-
kinin by angiotensin-converting enzyme. Circ Res 34:824-827, 1974
10. Dorer FE, Kahn JR, Lentz KE, Levine M, Skeggs LT: Kinetic properties
of pulmonary angiotensin-converting enzyme. Hydrolysis of hippuryl-
glycylglycine. Biochim Biophys Acta 429:220-228, 1976
11. Dorer FE, Ryan JW, Stewart JM: Hydrolysis of bradykinin and its higher
homologues by angiotensin-converting enzyme. Biochem J 141:915-917,
1974
12. Fisher GH, Ryan JW, Martin LC, Pena GA: Structure-activity relationships
for kininase II inhibition by lower homologs of the bradykinin potentiating
peptide BPP . Adv Exp Med Biol (In press)
9a
13. Greene LF, Stewart JM, Ferreira SH: Bradykinin-potentiating peptides from
the venom of Bothrops jararaca. Adv Exp Med Biol 8:81-87, 1970
14. Ondetti MA, Rubin B, Cushman DW: Design of specific inhibitors of angio-
tensin-converting enzyme: Mew class of orally active antihypertensive
agents. Science 196:441-444, 1977
15. Ondetti MA, Williams NJ, Sabo EF, Pluscec J, Weaver ER, Kocy 0:
Angiotensin-converting enzyme inhibitors from the venom of Bothrops
jararaca* Isolation, elucidation of structure, and synthesis.
Biochemistry 10:4033-4039, 1971
16. Roblero J, Ryan JW, Stewart JM: Assay of kinins by their effects on
blood pressure. Res Commun Chem Path Pharm 6:207-212, 1973
17. Ryan JW, Chung A, Ammons C, Carlton ML: A simple radioassay for
angiotensin converting enzyme. Biochem J 167:501-504, 1977
18. Ryan JW, Chung A, Martin LC, Ryan US: New substrates for the radio-
assay of angiotensin converting enzyme of endothelial cells in culture.
Tissue & Cell 10:555-562, 1978
19. Ryan JW, Day AR, Ryan US, Chung A, Marlborough DI, Dorer FE: Localiza-
tion of angiotensin converting enzyme (kininase II). I. Preparation
of antibody-heme-actapeptide conjugates. Tissue & Cell 8:111-124, 1976
20. Ryan JW, Niemeyer RS, Goodwin DW, Smith U, Stewart JM: Metabolism of
14
(8-L-[ Clphenylalanine)-angiotensin I in the pulmonary circulation.
Biochem J 125:921-923, 1971
21. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in rat lung.
Adv Exp Med Biol 8:263-272, 1970
22. Ryan JW, Ryan US: Pulmonary endothelial cells. Fed Proc 36:2683-2691,
1977
23. Ryan JW, Ryan US, Schultz DR, Whitaker C, Chung A, Dorer FE: Subcellu-
lar localization of pulmonary angiotensin converting enzyme (kininase II).
Biochem J 146:497-499, 1975
305
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24. Ryan JW, Smith U, Niemeyer RS: Angiotensin I: Metabolism by plasma mem-
brane of lung. Science 176:64-66, 1972
25. Ryan US, Clements E, Habliston D, Ryan JW: Isolation and culture of
pulmonary artery endothelial cells. Tissue & Cell 10:535-554, 1978
26. Ryan US, Ryan JW, Whitaker C, Chiu A: Localization of angiotensin
converting enzyme (kininase II). II. Immunocytochemistry and immuno-
fluorescence. Tissue & Cell 8:125-146, 1976
27. Soffer RL: Angiotensin-converting enzyme and the regulation of vaso-
active peptides. Ann Rev Biochem, pp 73-94, 1976
28. Stewart JM/ Young JD: Solid Phase Peptide Synthesis. San Francisco/
W. H. Freeman Co, 1969, p 103
29. Vane JR: The use of isolated organs for detecting active substances in
the circulating blood. Brit J Pharmacol 23:360-373, 1964
30. Ward PE, Erdb's EG, Gedney CD, Dowben RM, Reynolds RC: Isolation of
membrane-bound renal enzymes that metabolize kinins and angiotensins.
Biochem J 157:643-650, 1976
31. Wigger HJ, Stalcup SA: Distribution and development of angiotensin con-
verting enzyme in the fetal and newborn rabbit. An immunofluorescence study.
Lab Invest 38:581-585, 1978
306
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Angiotensin Converting Enzyme:
II. Pulmonary Endothelial Cells in Culture
U. S. Ryan and J. W. Ryan
University of Miami
Miami, Florida
One of the technical limitations to progress in understanding specific
functions of a particular cell type is the availability of that cell type
in pure culture. Over the past 12 years, a number of different specific
metabolic activities have been attributed to pulmonary endothelial cells*
The cells can selectively process adenine nucleotides and biogenic amines
(for review see 7 and 8). In addition, endothelial cells are capable of form-
ing prostaglandins such as PGE (19, 20, 24) and PGI. (15, 32)• Endothelial
cells appear to have a great capacity for inactivating kinins and for con-
verting angiotensin I into angiotensin II (for review see 19 and 25) and, in
this sense, endothelial cells of the pulmonary vascular bed can regulate the
hormonal composition of systemic arterial blood. In addition, endothelial
cells appear to possess a number of hemostatic factors including a -macro-
globulin (1), plasminogen activator (31), and factor VIII antigen (11).
By and large, metabolic activities attributed to pulmonary endotheliun
were based on conclusions drawn from indirect evidence. Perhaps the most
direct method to examine for unique or characteristic properties of a given
line of endothelial cells is to use isolated cells (17, 22, 30) or cells in
culture* Others have developed methods for isolating and culturing endothelial
cells from human umbilical vein (9, 12, 13, 16). Similar techniques have been
used to isolate and culture endothelial cells from portal vein and aorta
(2, 3, 14, 28, 29). Therefore, to Improve our ability to examine directly
for specific metabolic activities of pulmonary endothelium, we have begun a
program to obtain pulmonary endothelial cells in culture. Two methods have
307
-------
been developed: 1) cells can be obtained from pulmonary artery and vein of
large animals (cow and pig) and 2) cells can be obtained from the microvas-
culature of small animals (rats, guinea pig, and rabbit) commonly used in
studies of "nonventilatory functions" of the lungs. The latter technique can
also be used to obtain cells from a lobe of lung from large animals and may
be adaptable for use with human tissue obtained by open biopsy.
MATERIALS AND METHODS
Isolation and Culture of Pulmonary Artery Endothelial Cells
Pulmonary arteries from newborn or unweaned calf (veal) are obtained
within 30 minutes of slaughter. Calf hearts with attached great vessels are
transported at 48C to the laboratory where they are rinsed with Puck's saline.
Each pulmonary artery is separated from the aorta and trimmed of excess fat
and connective tissue. The pulmonary artery is tied as close to the heart
as possible and the vessel is removed. Each vessel is washed by immersion in
three changes of Puck's saline containing 3x strength antibiotics (Biostat
100, 3 ml per liter). When all of the vessels have been trimmed and washed,
they are placed in a beaker of Puck's saline with 3x antibiotics and stored
at 4°C for about 90 minutes. This storage step appears to reduce the like-
lihood of subsequent bacterial contamination. The vessel segments are then
wanned to 37°C in a shaking water bath. Each pulmonary artery segment is
drained of excess saline and filled with a solution of 0.25% collagenase
(Worthington type CLS II) in Puck's saline containing normal strength anti-
biotics (Biostat 100, 1 ml per liter). The free end of each artery is then
clamped with a large hemostat. The arteries are suspended in a 2-liter
beaker of phosphate buffered saline and incubated at 37»C in a shaking water
bath for 25 minutes. The arteries are then removed and the exteriors blotted
dry on sterile tissue. Each artery is then suspended by the ligature over a
small beaker or centrifuge tube and the hemostat is released. The collagen-
ase mixture containing cells is decanted into 15-ml tubes and centrifuged
at 750 x ^ for 10 minutes at 4°C. The supernatant is discarded and the
pellets washed by resuspending and repelleting twice with cold medium 199
(without fetal calf serum). The final pellets are resuspended in 1 ml of
2
medium 199 and seeded into 25-cm tissue culture flasks containing 4 ml of
medium 199 with 20% fetal calf serum and normal strength (1 ml per liter)
Biostat 100. The flasks are capped loosely and placed in a CO (5%) incubator
at 37°C. The cells show the greatest plating efficiency when seeded as small
clumps rather than as monodispersions. Hence, hemocytometer counts may not
be accurate. However, cells seeded at densities of approximately 1.5-3 x 10
308
-------
per flask reach confluency within five days. At confluence, the cells reach
a density of about 4 x 10 per cm (10 per flask). The medium is changed
every two days.
The cells can be subcultured as follows: Flasks are washed for about
10 minutes with 5 ml of Ca and Mg -free Puck's saline at 37°C. The saline
wash is aspirated off and the cells are incubated with 0.05% trypsin with
with 0.02% EDTA in Ca and Mg -free Puck's saline (5 ml at 37°C). The
cells are monitored on an inverted microscope to determine the optimal length
of time of exposure to the trypsin-EDTA solution. We have found that after
3 minutes the small, polygonal endothelial cells begin to lift off while the
larger, flatter smooth muscle cells still adhere to the flask. With a minimum
of agitation, the enzyme mixture containing cells is transferred, using a 5-ml
pipet, to 15-ml conical centrifuge tubes. The cells are centrifuged at
750 x _3 for 10 minutes. The trypsin solution is removed by aspiration and
the pellet is resuspended in a small quantity of medium 199 without fetal
calf serum. The flasks are usually split two for one. Each new flask is
seeded with approximately 10 cells. The doubling time for bovine pulmonary
artery endothelial cells is about 24 hours. Flasks which are predominantly
endothelial (less than 5% contamination with smooth muscle cells) can be
purified by successive subculture, taking advantage of the difference in
attachment and detachment rates of the two cell types. Trypsin preferentially
detaches bovine endothelial cells; thus, short periods (2 to 3 minutes) of
exposure to trypsin prior to transferring cells tend to leave smooth muscle
cells adherent to the flasks. Furthermore, endothelial cells are the first
to reattach. Hence, cells are seeded into new flasks and incubated for one
hour at 37°C. Medium and any free-floating cells are removed by aspiration
and replaced with fresh medium containing fetal calf serum and antibiotics.
Isolation and Culture of Endothelial Cells from the Lungs of Small Animals
The techniques described above for the isolation and culture of endo-
thelial cells from bovine pulmonary artery have provided useful information
on specific metabolic activities of pulmonary endothelium (for review see 19).
But the techniques are not suitable for obtaining endothelial cells from the
vessels of small animals or from the microvasculature. We have, therefore,
developed a technique to obtain endothelial cells from the lungs by vascular
perfusion with collagenase in a HEPES buffered salt solution. The surgical
procedure and techniques for collecting cells are similar using rats, rabbits,
or guinea pigs of either sex (see Figure 1).
The following describes the perfusion system used with a female New Zealand
white rabbit weighing 2.16 kilograms: A tracheostomy was performed and a
309
-------
Catheter into
pulmonary vein
Catheter into
^m
pulmonary artery
•
Figure 1. Perfusion system for (a) rendering lungs free of blood and
(b) treating the microvasculature with collagenase to allow collection of
endothelial cells. The perfusion system is described in the text. Small
gauge catheters are inserted and tied into the pulmonary artery and pulmonary
vein sinus. Saline is perfused in a forward direction (artery to vein) until
the effluent is free of blood. Perfusion of enzyme is from the venous to the
arterial side, a maneuver accomplished by connecting the drain cannula (in
the pulmonary vein) to the enzyme stopcock and by disconnecting the cannula
to the pulmonary artery from the saline stopcock. The free end of the arter-
ial cannula is then directed to a collection tube.
310
-------
tracheal tube was inserted, secured with a ligature, and attached to a Harvard
respirator (tidal volume 18 ml, 45 strokes per minute, inspiration to expira-
tion ratio of 0.45). Heparin (5,000 units) was injected intravenously.
Ligatures were placed around the pulmonary artery and the left atrium to
create a venous sinus into which all pulmonary veins drained. The saline
perfusion system was opened to the arterial catheter. The pulmonary artery
was opened and the catheter inserted and secured with a ligature. A hole was
immediately cut in the right ventricle for drainage; a hole was also cut in
the left ventricle and a drain catheter inserted through the mitral valve and
into the atrium and secured with a ligature. The perfusion solution was
pumped for 5 to 10 minutes, at which time the effluent was clear and colorless.
When the lungs became white and their venous effluent was free of blood, the
collagenase stopcock was opened, the respirator was turned off, and the
trachea was clamped. The free end of the pulmonary vein catheter was attached
to the collagenase stopcock. The pulmonary artery catheter was detached from
the saline perfusion system and directed to a cell collection tube. Thus,
retrograde perfusion of the lungs was established. The free end of the pul-
monary artery catheter was directed into a sterile 15-tnl centrifuge tube to
collect the cell-enzyme suspension. Perfusion with collagenase was continued
until the lungs became edematous and flow stopped spontaneously. The collec-
tion tubes were centrifuged for 10 minutes at 750 x 3. The supernatant was
removed by aspiration and each pellet was resuspended in 10 ml of 0.01 HEPES
buffered saline, pH 7.4. The tubes were centrifuged again and the super-
natant discarded. The pellets were resuspended in 1 ml of medium 199 using
a Vortex mixer. Corning tissue culture flasks, 25 cm , were filled with 4 ml
of medium 199 containing 20% fetal calf serum and antibiotics. Each culture
flask was seeded with 1 ml of suspension and the flasks were incubated at 37°C
in 100% humidity in a 5% CQ^ atmosphere. The number of cells seeded per flask
is approximately 2.8 x 10 . However, the seeding density cannot be determined
accurately since the cells are removed as clumps or sheets, not as a monodis-
persion, and because blood cells are present (and beneficial) in the original
isolate. Those flasks containing more than 95% endothelial cells (as judged
by phase contrast microscopy) are retained for culture, and the lines are
purified over the next few passages using differential adherence procedures
as described above.
RESULTS AND DISCUSSION
Identification of Endothelial Cells
Cells obtained by either method are readily identifiable as endothelial
according to morphological and functional criteria. When examined in the
311
-------
inverted microscope by phase contrast microscopy, the cells grow as mono-
layers with a cobblestone appearance characteristic of endothelium (Figs. 2
and 3). When examined in the electron microscope, they contain all of the
cellular organelles expected of pulmonary artery endothelial cells in situ,
including Weibel-Palade bodies (Figs, 4 and 5). The cells are routinely
examined by electron microscopy of thin sections of monolayers still attached
to the culture flasks (23, 28). However, we have developed means of examining
the cells in culture without removal from the culture flasks by additional
techniques including freeze-fracture of monolayers, scanning electron micro-
scopy, and examination of surface replicas (10). Thus, we are now able to
recognize a variety of views of endothelial cells. In addition, our cultures
possess functional aspects characteristic of endothelial cells. The cells
125 8
possess converting enzyme activity as demonstrated by using [ I]Tyr -brady—
kinin as substrate (4, 26, 28) and a variety of synthetic substrates (see paper
by Ryan _et al. in this volume). In addition, the cells are reactive with
antibodies to angiotensin converting enzyme as can be shown with immuno—
fluorescence microscopy and imiminocytochemistry (28). Using the latter
technique, we were able to localize angiotensin converting enzyme on the
plasma membrane and caveolae of endothelial cells in culture, a result in
accord with our previous demonstration of angiotensin converting enzyme on
the luminal surface of pulmonary endothelial cells in situ. Bovine endo-
thelial cells also react with antibodies to human factor VIII and a -macro-
globulin, and this we have demonstrated by imnuinofluorescence (23).
Synthesis of Prostaglandins by Pulmonary Sndothelial Cells
Results of our studies show that bovine pulmonary artery endothelial
cells are capable of forming prostaglandins and related substances from
14 3
1- C-arachidonate. Using EM autoradiography of cells incubated with [ H]-
acetyl salicylate (a specific inhibitor of prostaglandin endoperoxide synthase),
we were able to show that the enzyme is situated on the endoplasmic reticulum
(20, 24, 27). PGE is the major metabolite. A substance which co-chroma-
tographs with 6-keto-PGF , a breakdown product of PGI , is also formed, but
in highly variable quantities. Synthesis of PGI by endothelial cells has
been recently described by a number of laboratories (e.g., see 32). It is
likely that part or all of the PGI formed by lungs arises from endothelial
cells. Synthesis of PGI by pulmonary endothelial cells, along with their
ability to degrade ADP (see below), may account in large part for the capacity
of endothelial cells to prevent or inhibit platelet aggregation. We were
surprised to find that PGB occurs as a major product. Conceivably, PGB is
formed from PGE during extraction. If so, the rate of PGE synthesis
312
-------
% * <
• 4
r * -" M
,,'Z- ..
* -
* » *
*t «\ f %
-? _^ * »
Figure 2. Phase contrast micrograph of cow pulmonary artery endothelial
cells after 21 hours in culture. The cells form a cluster of approximately
100 cells per cluster. X150.
4
Figure 3. Confluent monolayer (approximately 4 x 10 cells/cm ) of cow
pulmonary artery endothelial cells. Phase contrast. X750.
313
-------
01:
V-^
• -. ' IT I1
•':,' "•*•-. 3
V -ir\
v 4H>v S/ii
>*•• £
- > •
Figure 4. Electron micrographs of calf pulmonary artery endothelium
sectioned transversely, including a portion of the plastic culture flask on
which the cells were growing (F). The boundary between the flask and the cell
is delineated by a fine dense line indicated by an arrow (probably formed
from components of PCS) (see also Figure 5). (a) The overall appearance
of a monolayer in transverse section and the contact inhibition between neigh-
boring cells. X"\,68Q. (b) The main body of the cell containing rough (*)
and smooth ER. Golgi complexes (G) are frequently found around the nucleus
(N). X8,800. (c) The thick portion of the cell in the region of the
nucleus and the slender peripheral extensions. These extensions do not con-
tain abundant endoplasmic reticulum but are characterized by numerous micro-
tubules and microfilaments. Caveolae (arrowheads) occur on both the peripheral
and central portions of the cells. X5,500. Reproduced with permission from
Tissue & Cell 10:535-554, 1978.
314
-------
I • * .
*,.?>
-''-..*v, it •.; •'AtelTA ^
Figure 5. Thin section of endothelial cell showing Weibel-Palade bodies
(arrows). X32,000.
approached 0.5 nm/yg of cell protein per hour. The possibility should be
considered that pulmonary artery endothelial cells have an unusually large
capacity for synthesizing PGE , a vasodilator thought to prevent or lessen
hypoxic pulmonary vasoconstriction. Our results indicate that pulmonary
endothelial cells can account for part of the efflux of prostaglandin-like
substances from embolized or anaphylactic lungs. However, our results pro-
vide no information on the cellular origins of thromboxane A , likely to be
a primary mediator of anaphylaxis, or of substances such as 15-keto-dehydro-
metabolites of PGE and PGF , all compounds which may occur in lung venous
effluent in much higher concentrations than those of PGE., and PGF . Pre-
2 2a
viously, we have shown that endothelial cells in culture cannot degrade PGF
and E , excellent substrates for 15-OH-dehydrogenase (19).
Endothelial Cells and the Degradation of ADP
Intact blood vessel walls are known to have anti-thrombotic properties,
thought to be associated with the endothelial cell lining layer. As men-
tioned above, it has recently been shown that endothelial cells in culture
can synthesize prostacyclin (PGI2), a substance which can prevent platelet
aggregation and also disaggregate platelet clumps. The lungs can also de-
grade the potent, platelet aggregating agent adenosine-5'diphosphate (ADP)
(5). Thus, in conjunction with our studies on prostaglandins and related
substances, we have examined pulmonary artery endothelial cells in culture
for ADPase activity. Approximately 1O6 cells were incubated with [14C]-
or [ H]ADP (with or without carrier) at 37«C in 5 ml of culture medium. At
timed intervals, the incubation medium was examined by thin-layer
315
-------
chromatography. ADP at concentrations of 3x10 M to 3x10 M was found to
have a half-life of about 3.5 minutes. The first product formed was 5'-AMP,
which was itself degraded to yield adenosine and inosine. Adenosine did not
accumulate in the medium but was taken up by the cells and incorporated into
ADP and then into ATP. In a second series of experiments/ endothelial cells
were removed from their flasks with a rubber policeman and homogenized. Sub-
cellular fractions were prepared by differential centrifugation. ADPase
activity occurred most abundantly in the microsomal fraction, enriched in
terms of angiotensin converting enzyme (ACE). ACE is known to occur as a com-
ponent of the plasma membrane of endothelial cells (cf. 23). Thus, pulmonary
endothelial cells can prevent intravascular thrombosis by degrading ADP. The
ADPase activity is probably a component of the cell membrane.
Synthesis of Angiotensin Converting Enzyme by Endothelial Cells in Culture
Recently we undertook a study to determine whether endothelial cells in
culture synthesize angiotensin converting enzyme, or whether they merely
provide highly specific receptors for the enzyme. Previously, we have shown
that endothelial cells from cow, pig, rabbit, and rat possess angiotensin
converting enzyme, and that the enzyme occurs along the plasma membrane and
associated caveolae (21, 26, 28). Further, we have shown that the angiotensin
converting enzyme activity persists through more than 50 passages (19).
Initially, we took these data to mean that endothelial cells in culture are
capable of synthesizing angiotensin converting enzyme. However, all of our
previous studies used cells grown in medium containing fetal calf serum (FCS)
at 20 to 30% v/v. FCS is a rich source of angiotensin converting enzyme;
hence, it seemed possible that endothelial cells provide receptors for, but
do not synthesize, angiotensin converting enzyme. To resolve this problem,
we obtained cultures of bovine pulmonary endothelial cells in FCS previously
treated by heating at 56°C, a maneuver reported to inactivate angiotensin
converting enzyme (6). The primary cultures were propagated through four
passages, such that the average progeny in the fourth generation of each cell
in the original isolate was 6,400. The cells were assayed using [ H]benzoyl-
Phe-Ala-Pro as substrate (18). The specificity of the assay was tested by
—5 —6
using SQ 14,225 (10 M and 10 M), a specific inhibitor of angiotensin convert-
ing enzyme.
Enzyme activity per cell was remarkably constant, although there was a
tendency for cell size to increase along with, in the fourth generation, an
increase of enzyme activity per cell. Under the conditions of these experi-
ments, there was a 12,428-fold increase in angiotensin converting enzyme
activity. Thus, these results support the concept that bovine pulmonary artery
316
-------
endothelial cells in culture are capable of specific protein synthesis.
Electron micrographs of grazing sections of the cells in culture suggest that
the cells appear to have the subcellular machinery (extensive rough endoplasmic
reticulum and polyribosomes [Fig. 6]) to synthesize a complex glycoprotein such
as angiotensin converting enzyme (23).
CONCLUDING REMARKS
As described above, techniques for the isolation and culture of endo-
thelial cells from bovine pulmonary artery have been established. These cul-
tures have provided useful information on specific metabolic activities of pul-
monary endothelium, and have the advantage that large quantities of cells can
be obtained in original isolates. In addition, antigenic sites of bovine cells
appear to cross-react with antibodies to human antisera. However, the tech-
niques are not suitable for obtaining endothelial cells from small animals.
Moreover, endothelial cells of pulmonary artery and those of the microvascular
bed of the lungs in situ differ in terms of structure, in addition, the small
caliber and large extent of surface area of the pulmonary microcirculation make
it likely that processing of blood-borne substrates such as angiotensin, brady-
kinin, adenine nucleotides and amines takes place at the level of the smallest
vessels. We have, therefore, developed a technique for obtaining endothelial
cells from the lungs tty vascular perfusion with collagenase (500 units/ml) in
HEPES buffered salt solution.
Although one can readily isolate and culture endothelial cells from
pulmonary artery of, for example, cow (23, 26, 28), and one can readily isolate
and culture endothelial cells from human umbilical vein (12, 19), we believe
that our newly developed techniques for pbtaining endothelium from the lungs of
small animals by vascular perfusion may have at least two effects that will
accelerate the pace and broaden the scope of studies on specific metabolic
functions of the lungs. First, the vast preponderance of studies on the pos-
sible metabolic functions of the lungs have been performed with small animals,
in which it is virtually Impossible to obtain an adequate number of cells from
main-stem pulmonary artery. Second, although we and others have shown that
cells from main-stem pulmonary artery are capable of metabolizing bradykinin,
angiotensin, ADP, adenosine, and biogenic amines, the general assumption is
that the processing of such substances in vivo occurs most prominently at the
level of the microcirculation. Our new techniques of cell isolation allow use
of small animals and therefore allow a more direct comparison with previous
functional studies in which small animals, such as rats and rabbits, have been
used.
317
-------
&*-?*
fe^-vJK* ;t >'< ••'••-7' ' -• '-
ff**>.»W'*^.>isj-,.*•*, *•• *• ••—'•*•< *
S
Figure 6. En face section of calf pulmonary endothelial cell spanning the
cell from the culture flask (F) to the nucleus. The plane of section has grazed
the nuclear envelope revealing nuclear pores (arrowheads) and also grazed the
plasma membrane revealing abundant caveolae (*). In addition to elongated
mitochondria (M), the cytoplasm contains large numbers of whorled polyribosomes
(arrows) attached to the cisternae of the endoplasmic reticulum. The insert
shows an enlargement of a portion of the field showing the spiral arrangement
of ribosome complexes. X22,000; insert X75,000. Reproduced with permission
from Tissue & Cell 10:535-554, 1978.
318
-------
ACKNOWLEDGMENTS
This work was supported by grants from the USPHS (HL21568, HL22087, and
HL22896), the John A. Hartford Foundation, Inc., and the Council for Tobacco
Research—U.S.A., Inc.
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1. Becker CG, Harpel PC: a -Macroglobulin on human vascular endothelium.
J Exp Med 144:1-9, 1976
2. Blose SH, Chacko S: In vitro behavior of guinea pig arterial and venous
endothelial cells. Dev Growth Differ 17:153-165, 1975
3. Booyse FM, Sedlak BJ, Rafelson ME: Culture of arterial endothelial cells:
Characterization and growth of bovine aortic cells. Thromb Diath Hemorrh
34:825, 1975
4. Chiu AT, Ryan JW, Ryan US, Dorer FE: A sensitive radiochemical assay for
angiotensin-converting enzyme (kininase II). Biochem J 149:297-300, 1975
5. Crutchley DJ, Eling TE, Anderson MW: ADPase activity of isolated perfused
rat lung. Life Sci 22:1413-1420, 1978
6. Das M, Soffer RL: Pulmonary angiotensin-converting enzyme antienzyme anti-
body. Biochemistry 15:5088-5094, 1976
7. Fishman AP, Pietra GG: Handling of bioactive materials by the lungs.
New Engl J Med 291:Part I, 884-890, 1974
8. Fishman AP, Pietra CG: Handling of bioactive materials by the lungs. New
Engl J Med 291:Part II, 953-959, 1974
9. Gimbrone MA, Cotran RS, Folkman J: Human vascular endothelial cells in
culture. J Cell Biol 60:673-684, 1974
10. Hart MA, Ryan US: Surface replicas of pulmonary artery cells in culture.
Tissue & Cell 10:441-449, 1978
11. Jaffe EA, Hoyer DW, Nachman RL: Synthesis of antihemophilic factor antigen
by cultured human endothelial cells. J Clin Invest 52:2757-2764, 1973
12. Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial
cells derived from umbilical veins. J Clin Invest 52:2745-2756, 1973
13. Lewis LJ, Hoak JC, Maca RD, Fry GL: Replication of human endothelial cells
in culture. Science 181:453-454, 1973
14. Macarak EJ, Howard BV, Kefalides NA: Properties of calf endothelial cells
in culture. Lab Invest 36:62-67, 1977
15. Maclntyre DE, Pearson JD, Gordon JL: Localisation and stimulation of pros-
tacyclin production in vascular cells. Nature 271:549-551, 1978
16. Maruyama Y: The human endothelial cell tissue culture. Z Zellforsch
Mikrosk Anat 60:69, 1963
17. Pugatch EMJ, Saunders AM: A new technique for making Hautchen preparations
of unfixed aortic endothelium. J Atheroscler Res 8:735-738, 1968
319
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18. Ryan JW, Chung A, Martin LC, Ryan US: New substrates for the radioassay
of angiotensin converting enzyme of endothelial cells in culture. Tissue
& Cell 10:555-562, 1978
19. Ryan JW, Ryan US: Pulmonary endothelial cells. Fed Proc 36:2683-2691, 1977
20. Ryan JW, Ryan US, Habliston D, Martin L: Synthesis of prostaglandins by
pulmonary endothelial cells. Trans Assoc Amer Physns 91:343-350, 1978
21. Ryan JW, Ryan US, Schultz DR, Whitaker c, Chung A, Dorer FE: Subcellular
localization of pulmonary angiotensin converting enzyme (kininase II).
Biochem J 146:497-499, 1975
22. Ryan JW, Smith U: The metabolism of angiotensin I by endothelial cells.
In: Protides of the Biological Fluids (Peeters H, ed). Oxford, Pergamon
Press, 1973, pp 379-384
23. Ryan US, Clements E, Habliston D, Ryan JW: Isolation and culture of pul-
monary artery endothelial cells. Tissue & Cell 10:535-554, 1978
24. Ryan US, Habliston D, Martin L, Ryan JW: Pulmonary endothelial cells and
prostaglandin synthesis. Circulation 55 & 56 (Suppl III):III-123, 1977
(Abstr 473)
25. Ryan US, Ryan JW: Correlations between the fine structure of the alveolar-
capillary unit and its metabolic activities. In: Metabolic Functions of
the Lung (Bakhle YS, Vane JR, eds), Vol 4 of Lung Biology in Health and
Disease (Lenfant c, ed). New York, Marcel Dekker, 1977, pp 197-232
26. Ryan US, Ryan JW, Chiu A: Kininase II (angiotensin converting enzyme) and
endothelial cells in culture. Adv Exp Med Biol 70:217-227, 1976
27. Ryan US, Ryan JW, Whitaker C: Localization of prostaglandin synthetase in
lung cells. J Cell Biol 75:179a, 1977 (Abstr HM042)
28. Ryan US, Ryan JW, Whitaker C, Chiu A: Localization of angiotensin con-
verting enzyme (kininase II). II. Immunocytochemistry and immuno-
fluorescence. Tissue & Cell 8:125-146, 1976
29. Shepro D, Batbouta JC, Carson MP, Robblee L, Belamarich FA: Serotonin
transport by cultured bovine aortic endothelium. Circ Res 36:799-806, 1975
30. Smith U, Ryan JW: Electron microscopy of endothelial and epithelial com-
ponents of the lungs: Correlations of structure and function. Fed Proc
32:1957-1966, 1973
31. Todd AS: Some topographical observations on fibrinolysis. J Clin Path
17:324-327, 1964
32. Weksler BB, Marcus AJ, Jaffe EA: Synthesis of prostaglandin I (prosta-
cyclin)by cultured human and bovine endothelial cells. Proc Natl Acad
Sci USA 74:3922-3926, 1977
320
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DISCUSSION
PARTICIPANT: Could you tell me the evidence that the endothelial cells in
vivo synthesize angiotensin converting enzyme, rather than picking it up from
the blood?
OR* U* RYAN: The best evidence is that they're actually synthesizing* Of
course, the serum enzymes have got to come from somewhere* James probably
can be a little more certain on this. The servm enzymes may well have come
from the lungs*
DR. J* RYAN: That's somewhat suggestive, but what is pretty clear is that the
serum enzyme differs molecularly from the lung enzyme. It has extra sugars.
That's not the result you'd expect that the enzyme is part of the serun.
PARTICIPANT: The other possibility is that the synthesis is in the liver, and
that the sugars are split off into the pulmonary endothelial cell membrane,
and then the enzyme is absorbed. But that doesn't seem to be the case*
DR. J* RYAN: The cells in culture certainly make the enzyme; there's no
question about that. Liver has a remarkably little angiotensin converting
enzyme, but that's not quite the same as saying that it might possibly make
it very fast. Our prejudice is otherwise.
DR. BROMBERG: Where does the enzyme come from in sarcoidosis?
DR. U. RYAN: I don't know. I don't think the evidence is that good that it
comes from macrophages. I don't see why it couldn't be endothelial cells, but
we don't know.
DR. J* RYAN: Let's ask that question of you. What percentage of these
patients have vasculitis?
DR. BROMBERG) According to the International Congress at the New York Academy
about three years ago, it was listed in three papers as appearing in severe
sarcoidosis, but I guess that there's some dissent*
321
-------
DR. U. RYAN: We began a study on that, but the biopsy material was very hard
to analyze. If you think about these pictures/ that's an incredibly fine
localization which requires extremely good fixation.
322
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Divergent Effects of Prostaglandins
on the Pulmonary Vascular Bed
P. J. Kadowitz, E. W. Spannhake, and A. L Hyman
Tulane University
New Orleans, Louisiana
In most organ systems including the lung, arachidonic acid (5,8,11,14-
licosatetraenoic acid) is converted into the endoperoxide intermediates PCG.
and PGH by a microsomal cyclooxygenase (7, 8, 22). The endoperoxide inter-
mediates (PGG and PGH ) are then converted by terminal enzymes into primary
prostaglandins (PG), thromboxane A (TXA ) , or prostacyclin, £G1 (1, 5, 6, 8,
9, 21-23). The distribution and activity of terminal enzymes determine the
pattern of products formed from endoperoxide intermediates in an organ (10, 11,
23). Many reports indicate that the endoperoxide intermediate, endoperoxide
analogs, PGE , PGF and PGD all increase pulmonary vascular resistance in
a variety of species (10, 12, 14-17, 27). In contrast, PGE- has dilator
activity in the pulmonary circulation of fetal and neonatal animals (3). The
pulmonary vascular effects of TXA are uncertain, but this labile substance
has potent smooth muscle stimulating and platelet aggregating activity and its
breakdown product, TXB . has modest pressor activity in the pulmonary vascular
bed (9, 18). In contrast to the effects of PGH-, PGE_, PGD-, PGF.. and TXB.,
222 201 2
the newly discovered metabolite of arachidonic acid metabolism, PGI , has
pulmonary vasodilator activity (9, 18). However, the effects of arachidonic
acid on the pulmonary vascular bed are unclear. Arachidonic acid has been
shown to increase pulmonary vascular resistance in a number of species when
injected as a bolus and to contract isolated segments of pulmonary artery (4,
10, 12, 14, 25, 28). These responses are blocked by indomethacin, suggesting
that in the lung arachidonate, when administered as a bolus, is converted to
substances that have pulmonary vasoconstrictor and bronchoconstrictor activity
323
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(10, 12, 14, 25, 28). It has, however, recently been reported that arachidonic
acid has depressor activity in the pulmonary circulation when the prostaglandin
precursor is infused (12). It has also been recently shown that the newly dis-
covered bicyclic prostaglandin, PGI , dilates feline and canine pulmonary vascu-
lar beds (13, 19). The purpose of the present report is to compare responses
to primary prostaglandins (PGE , PGF and PGD ), arachidonic acid, and prosta-
cyclin in the canine pulmonary vascular bed and the airways.
METHODS
The pulmonary vascular effects of primary prostaglandins, arachidonic
acid, and prostacyclin were investigated in mongrel dogs unselected as to sex
and weighing 14.2-24.5 kg. The dogs were anesthetized with pentobarbital
sodium (30 mg/kg, iv) and strapped to a Philips heart table in the supine
position. A 6F Edslab thermal dilution catheter was passed into the main pul-
monary artery from the external jugular vein under fluoroscopic guidance
(Philips image intensifier). Pulmonary arterial pressure was measured from
the distal port on the Edslab catheter. A 7F Teflon catheter was passed into
the left atrium transseptally and large bore catheters were positioned in the
aorta from a femoral artery and in a femoral vein. Cardiac output was de-
termined with an Edwards thermal dilution computer, model 9500, after injection
of 5 ml of 5% dextrose solution (cooled to 0°C) into the superior vena cava
(proximal port on the Edslab catheter). Values for cardiac output averaged
120 ml/kg per minute and compared favorably with cardiac outputs determined
by the indicator-dilution technique in this laboratory. The dogs breathed
room air or room air enriched with 0 spontaneously through a cuffed endc—
tracheal tube.
In dogs in which constant flow perfusion of the left lower lobe was em-
ployed, a specially designed 20F double-lumen balloon catheter was introduced
through a jugular vein into the arterial branch of the left lower lung lobe
under fluoroscopic guidance. A 1.5 mm Teflon catheter with its tip positioned
about 2 cm distal to the tip of the perfusion catheter was used to monitor
perfusion pressure in the lobar artery. Catheters with side holes near the
tip were passed into the main pulmonary artery and femoral artery and trans-
septally into a small intrapulmonary vein and the left atrium. Precautions
were taken to ensure that pressure measurements were made without wedging in
veins 2-3 mm in diameter. Briefly, a 0.9 mm Teflon catheter with two side
holes near the tip was passed through a 3 mm Teflon catheter that previously
had been wedged in a small intrapulmonary vein. The 0.9 mm catheter was then
withdrawn 1-3 cm from the wedge position until pressure dropped abruptly. The
0.9 mm catheter was fixed in place with a Cope adaptor after the larger catheter
had been withdrawn to the left atrium. These methods have been described in
324
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PUMP
OBARt ARTERIAL
PERFUSION CATHETER
PULMONARY
VEIN CATHETER
Figure 1. Diagram showing catheterization procedure in the dog. A
specially designed 20F balloon catheter is passed into the artery of the lower
left lobe and the lung is autoperfused with the blood withdrawn from the right
atrium. Vascular pressures are measured in the perfused lobar artery, a small
pulmonary lobar vein, and the left atrium and in the main pulmonary artery and
the aorta. The Carlen's endobronchial catheter permits the left lower and
right lungs to be ventilated separately.
detail previously, and a diagram of the catheterization procedure is shown in
Figure 1 (12, 15). All vascular pressures were measured with Statham P23D
transducers zeroed at the level of the middle of the right atrium, and mean
pressures were recorded on an oscilloscopic recorder (model DR-12, Electronics
for Medicine). After all catheters had been positioned and the dogs heparinized
(500 U/kg, iv) the balloon on the perfusion catheter was distended with 2-4 ml
of 50% sodium diatrizoate (Hypaque Winthrop) until pressure in the lobar artery
and small vein decreased to near left atrial pressure. Hie vascularly isolated
left lower lobe then was perfused with a Sarns roller pump (model 3500) with
blood withdrawn from the right atrium. The pumping rate was adjusted so that
325
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mean pressure in the perfused lobar artery approximated mean pressure in the
main pulmonary artery and thereafter was not changed during the experiment.
The pumping rate averaged 260 ml per minute. These dogs spontaneously breathed
room air or room air enriched with O through a cuffed endotracheal tube.
In experiments in which the effects of the analog on airway pressure
were evaluated, the dogs were intubated with a Carlen's endobronchial divider
(No. 39) and the left lower lobe and right lungs were ventilated separately
with a Harvard dual-cylinder respirator (model 618) at a rate of 20 cycles
per minute with stroke volumes of 110 ml per minute for the left lower lobe
and 280 ml per minute for the right lungs. The dogs received succinylcholine
chloride (Anectine, Burroughs Wellcome), 2.5 mg/kg, iv, to paralyze ventilation.
Translobar airway pressure was measured with a Statham differential transducer
(PM5) bridged between the left side of the endobronchial divider and the
pleural space and was recorded on the Electronics for Medicine recorder.
For studies on lung function, mongrel dogs, unselected as to sex and
weighing from 14.2 to 18.5 kg, were anesthetized with chloralose (50 mg/kg)
and urethane (500 mg/kg) administered intravenously. Polyethylene catheters
were advanced from the femoral artery and vein for the recording of aortic
pressure (P ) and the administration of drugs, respectively. A 6F Edslab
double lumen thermodilution catheter (Edwards Laboratories) was passed from
the external jugular vein into the main pulmonary artery under fluoroscopic
guidance. Pulmonary arterial pressure (Pp.) was measured from the distal port
of this catheter. In some experiments, left ventricular end-diastolic pressure
was measured through a Cordis pig-tail catheter advanced from a femoral artery
or, alternately, left atrial pressure was measured directly through a 7F Teflon
catheter positioned transseptally in the left atrium. Cardiac output was de-
termined with an Edwards Laboratories Thermal Dilution Computer, Model 9500A.
All vascular pressures were measured with Statham P23BB or P23AC transducers
zeroed at atrial level. Mean pressures were obtained from the pulsatile signal
by electrical averaging.
The dogs were ventilated with a Harvard ventilator through a short tracheal
cannula (2-3 cm diameter) introduced by tracheostomy. Transpulmonary pressure
(P ) was measured by a Statham PM5E differential transducer interposed between
the tracheal cannula and a Harvard pleural cannula inserted through the chest
wall at the 4th or 5th intercostal space. Pneumothorax was adjusted to 10-20
ml immediately after introduction of the pleural cannula. Air flow, (V), was
measured with a Fleisch No. 1 pneumotachograph heated above body temperature
and coupled to a Grass PT5A differential transducer. P and V signals were
processed by a Hewlett-Packard 8816A Respiratory Analyzer which provided, on
line, volume (V), dynamic compliance (C. ), and resistance of the lung (R_),
C was computed between points of zero flow by dividing the volume of each
breath by the difference between end-inspiratory and end-expiratory P . R
326
-------
Polygraph
Prissun
Transducer
Pleura/ Cannuto
INTACT
DOG CHEST)
•ssj^—fPressur*
fin—I Transducer
JL ^fVentilaHi
Tracneal Cannula •
Pneumotachograph
COf Analyzer
wspiratory
Anafynr
Figure 2. Schematic diagram of the procedure used to evaluate the effects
of the prostanoids on lung function in a dog with an intact chest*
was computed at early expiration by the method of Mead and Whittenberger (25}
in which instantaneous resistive pressure was divided by instantaneous flow.
Resistive pressure is derived by subtracting the ratio of volume to computed
compliance from P,
TP
Percent CO in end-tidal air was monitored periodically
with a Sectarian LB-2 medical gas analyzer connected to a tap between the respi-
rator and the pneumotachograph. P__, V, V, R. , c. and vascular pressures
TP L ayn
were recorded on a Grass model 7C 8-channel recorder. The preparation used
to investigate the effects of the prostaglandins on lung function in a dog with
an intact chest is illustrated in Figure 2, and this preparation has been de-
scribed previously (25). The animals were heparinized with sodium he par in
(Upjohn), 500 units/kg iv, and spontaneous breathing was arrested with succinyl-
choline chloride (Anectine, Burroughs Wellcome), 30-40 mg iv, repeated as needed.
After neuromuscular blockade, increments of anesthetic were given as needed.
Minute volume was set with tidal volume at 12 ml/kg and the rate sufficient
to maintain end-tidal CO. near 5% and blood gases in a normal range. A lung
volume history was established by hyperinflating the dog to 3 times the tidal
volume 3 minutes prior to each administration of test agent into the superior
vena cava (SVC).
Arachidonic acid (NuChek), 99% pure, and indomethacin (Merck) were freshly
prepared as sodium salts: arachidonic acid in 10% ethanol in 100 mM sodium
carbonate and indomethacin in 100 mM sodium carbonate in normal saline. Prosta-
glandins D , E , and F , and the PGH analog (Upjohn) were dissolved in ab-
solute ethanol and stored at -20"C. Working solutions were freshly prepared in
saline. PGI2 was prepared in 20 mM Tris buffer, pH 8.5, and was stored in a
freezer.
All values are expressed as the mean + standard error of the mean unless
327
-------
otherwise indicated. Tests of significance for group and paired comparisons
were done according to standard statistical methods (24). A P value less than
0.05 was considered significant.
RESULTS
The pathway for synthesis of primary prostaglandins (PGD , PGE , and PGP ),
2 2 2 a
thromboxane A (TXA ), and prostacyclin (PGI ) in the lung is illustrated in
Figure 3. The fatty acid precursor, arachidonic acid, is converted into
endoperoxide intermediates by a microsomal cyclooxygenase. The endoperoxide inter-
mediates are then converted by terminal enzymes into prostaglandins, TXA , or
prostacyclin. Alternatively, the endoperoxides may be broken down to 12-hydroxy-
heptadecatrienoic acid (HHT) and malondialdehyde (MDA) which have little reported
biologic activity. Although the endogenous precursor is derived from the break-
down of phospholipids in cell membranes, exogenous arachidonic acid can also serve
as substrate for the cyclooxygenase. The effects of exogenously administered
arachidonic acid on the pulmonary vascular bed perfused at constant flow are
illustrated in Figure 4. Injection of 3 mg arachidonic acid, as a rapid bolus
into the perfused lobar artery, produced a rapid increase in lobar arterial and
small vein pressures but had no effect on left atrial pressure. The effects of
arachidonic acid on lobar arterial and venous pressure were dose-related in the
COOH
V
IMDAI
Figure 3. Diagram showing the pathway for biosynthesis of PGD , PGP ,
2 2a
PGE , thromboxane A , and PGI in the lung.
328
-------
3 mg | Arachidonic Acid
mmHg 30
lOOr- 20
50
- 10 -
PUL. A.
LOBAR A.
AORTA
SMALL V.
L. ATRIUM
I 20 SEC |
Figure 4. Records from an experiment, in a dog with an intact chest:, showing
the effects of arachidonic acid (3 mg) injected into the lobar artery on mean
pressures in the main pulmonary artery (Pul. A.), the lobar artery (Lobar A*)/
the aorta, the small intrapulmonary vein (Small V.). and the left atrium
(L. Atrium). Blood flow to the left lower lobe was maintained constant with
a pump*
range of doses of 0.1-3 mg, and at doses of 1-3 mg aortic pressure was decreased.
The pressor activities of arachidonic acid, PGE_, PGF_ , and a stable
2 2d
endoper oxide (PGH.) analog in the pulmonary vascular bed under conditions of
controlled flow are compared in Figure 5. PGF_ is about 10-fold more potent
2d
than PGE which in turn is about 10-20 times more active than arachidonic acid.
The stable endoperoxide analog' is about 10-fold more active than PGP2 . Al-
though not shown in Figure 5, the pressor activities of PGF, and PGD are
2a 2
very similar. In addition to increasing lobar arterial and venous pressures,
arachidonic acid increases translobar airway pressure, indicating that the
prostaglandin precursor is converted into substances that have bronchomotor
activity in the dog (Fig. 6). The airway effects of arachidonic acid were
studied further using the procedure illustrated in Figure 2. The effects of
arachidonic acid on the airways are depicted in Figure 7. Rapid bolus in-
jection of 10 mg of the prostaglandin precursor into the superior vena cava
increased transpulmonary pressure to 200% of control value. The response at-
tained a peak within 20 seconds, and transpulmonary pressure gradually returned
toward control value. The rise in transpulmonary pressure was accompanied by
329
-------
mmHg
3(h
V)
-------
(CM HjO)
16 r
8
0
0 10 mg ARACHIDONIC ACID
Figure 7.
acid (10 mg) on transpulmonary pressure (P )
Record from an experiment illustrating the effects of archidonic
(inspiration up), air flow (V)
(inspiration upward from zero, expiration down), tidal volume (V), lung resistance
(RT), dynamic compliance ( C, ) in a dog with an intact chest. Mean pulmonary
ij ayn
arterial pressure (P_,) was increased and mean aortic pressure (P. ) (not shown
PA c Ao
in this figure) was decreased. Injection was made as a bolus into the superior
vena cava at the point Indicated.
a transient rise in lung resistance and a greater and more sustained decrease
in dynamic compliance* The effects of arachidonic acid on lung function were
dose-related, and hyperinflation of the lung to total lung capacity 10-15
minutes after administration of the prostaglandin precursor usually returned
lung resistance and dynamic compliance to control levels. The effects of
arachidonic acid, PGD., and PGF_ on lung function in the intact dog are com-
2 20t
pared in Figure 8. All three substances increased pulmonary arterial pressure
and lung resistance and decreased dynamic compliance in a dose-dependent manner.
PCaD2 was three tiroes more active than PGF in increasing lung resistance and
decreasing lung compliance, whereas both prostaglandins were more than 1000-
fold more active than arachidonic acid. PGE had only small airways effects
4to
331
-------
MM HG
25
"
}Q
5
u/
3 6 10
AA(mg)
10
10 30
PGF2a(jjg)
Figure 8. Dose-response curves comparing effects of arachidonic acid (AA),
PGD , and PGF_ on lung resistance (R_), dynamic compliance (C, ), and pul-
2 2ct L ayn
monary arterial pressure (P.,,) in a dog with an intact chest.
Jif\
in the range of doses of 1-30 yg. The stable endoperoxide analog and PGD had
similar effects on the airways in the intact dog.
•Die effects of indonethacin on pulmonary vascular, airway/ and systemic
vascular responses to arachidonic acid are illustrated in Figure 9. The in-
creases in transpulmonary pressure, pulmonary arterial pressure, and aortic
pressure were abolished 5-10 minutes after administration of indomethacin,
2.5-5 mg/kg iv. The pulmonary vascular effects of the newly discovered bi-
cyclic prostaglandin, prostacyclin (PGI ), are summarized in Table 1. PG1
produced a small but significant reduction in lobar arterial and small vein
pressures when injected as a bolus in doses of 1-10 ug into the perfused lobar
artery. Although responses to PGI were small under resting conditions when
4*
tone in the pulmonary vascular bed was minimal, decreases in lobar arterial
and venous pressures were substantial when pulmonary vascular resistance was
actively increased (Table 1). In preliminary studies PGI had little effect
332
-------
CONTROL
AFTER INDOMETHACIN
Cardiac Output
(L/min)
P
fmmHgJ
4.06
1.27
1.46
fmmHgJ
10
6mg Arachidonic acid
6mg Arachidonic acid
Figure 9. Records from an experiment illustrating the effects of arachi-
donic acid (6 mg) on transpulmonary pressure (P-,_) » mean pulmonary arterial
pressure (P ), and mean aortic pressure (P ) before (panel A) and 20 minutes
JrA AO
after (panel B) a slow intravenous infusion of indcmethacin, 2.5 mg/kg. At
the arrows, arachidonic acid was injected as a bolus into the superior vena
cava. Cardiac output in L/min was measured by the thermal dilution technique
at the peak of the response.
on the airways in the cat and dog under resting conditions but decreased
transpulmonary pressure when bronchomotor tone was enhanced.
DISCUSSION
Results of the present study show that when injected as a bolus the prosta-
glandin precursor, arachidonic acid, increases lobar arterial and small vein
pressures in the intact dog. Inasmuch as pulmonary blood flow was held con-
stant and left atrial pressure was unchanged, the rise in lobar arterial pres-
sure reflects an increase in pulmonary vascular resistance. The rise in small
vein pressure and in pressure gradient from the lobar artery to the small vein
suggests that arachidonic acid increases pulmonary vascular resistance by con-
stricting small intrapulmonary veins and upstream segments believed to be small
arteriec (12). The primary prostaglandins (PGE., PDG0, and PGP, ) as well as
22 2a
333
-------
Table 1. Pulmonary Vasodilator Effects on PGI Under Resting Conditions
and When Pulmonary Vascular Resistance Is Enhanced by Infusion of an
Endoperoxide Analog
Pressure (mm Hg +_ SE)
Control
PGI2/ 1-10 ug
Control
PGI , 1-10 wg
n = 6
Lobar artery
15.9 + 0.8
14.0 + 1.0*
Infusion of
31.5 + 1.3
25.1 + 2.6*
Small vein Left atrium
10.5 + 0.6 1.8 + 0.4
9.2 + 0.6* 1.8 + 0.4
11,9 PGH. Analog 1-5 ug/min
2
21.4 + 1.0 1.7 + 0.2
17. 1 + 1.3* 1.6 + 0.2
Aorta
120+5
99 + 8*
125 + 1.5
106 + 5.2*
*P<0.05 when compared to corresponding control (paired comparison).
PGH and a stable PGH analog all increase pulmonary vascular resistance in the
dog and cat (10, 12, 14-17, 27, 28). Arachidonic acid also increased transpul-
nionary pressure when the lung was ventilated at constant volume with a positive
pressure ventilator (12). In experiments in which the effects of arachidonic
acid on lung function ware further investigated, the increase in transpulmonary
pressure was associated with a transient rise in lung resistance and a sustained
fall in dynamic compliance (25). The endoperoxide analog, PGP , and PGD all
increased lung resistance and decreased dynamic compliance and in this regard
were similar to but much more potent than arachidonic acid (25-27). The effects
of PGE on the pulmonary vascular bed and on the airways were modest when this
substance was injected in the same doses as PGD or PGF^ (12, 14, 17, 25). Pul-
2 2a
monary vasoconstrictor, bronchoconstrictor, and systemic vasodepressor responses
to arachidonic acid were blocked after administration of indomethacin, a cyclooxy-
genase inhibitor (10-12, 14, 25). These data suggest that the effects of arachi-
donic acid on the airways and pulmonary and peripheral vascular beds are due to
the conversion of the substrate into vasoactive and bronchoactive metabolites
in the cyclooxygenase pathway. The pulmonary vasoconstrictor response to arachi-
donic acid is associated with increased synthesis of E and F "like" prosta-
glandins; however, it is not known if PGD or TXB is produced by the lung in
the intact dog (12). The effects of arachidonic acid on the pulmonary vascular
bed were not dependent on the presence of platelets or other formed elements,
in that the response to arachidonic acid was not diminished 'when the lung was
perfused with a dextran solution (12). These findings suggest that the sub-
strate is converted to vasoactive substances by the lung itself and that plate-
334
-------
let aggregation or release of vasoactive products from the platelets plays
little or no role in this response (12).
In contrast to the pressor effects of PGF_ , PGD-, and PGE- or the PGH,,
Zot i i 2
analog which may mimic the effects of TXA on the pulmonary vascular bed/ the
newly discovered bicyclic prostaglandin, PGI , had vasodilator activity in the
pulmonary vascular bed (13, 19). The pulmonary vasodilator effects of PGI
were modest under resting conditions but .were greatly enhanced when pulmonary
vascular resistance was increased actively by infusion of a vasoconstrictor
substance (13). Although PGI had vasodilator activity, arachidonic acid, when
administered as a bolus, consistently increased pulmonary vascular resistance
in the intact dog and cat and in the isolated dog lung (10, 12, 14, 25, 28)•
These results suggest that under physiologic conditions in the intact state,
the predominant products formed in the lungs when arachidonate is injected as
a bolus are vasoconstrictor in nature. Alternatively, it is possible that both
vasoconstrictor and vasodilator metabolites are formed but that the activity of
the vasoconstrictors overshadows the action of any simultaneously formed PGI,
"like" substances, it has been reported that PGI is the predominant metabolite
formed from arachidonic acid and endoperoxide intermediates in vascular tissue
(2, 5, 21). Indeed, we have observed in some animals that slow infusions of
arachidonic acid decrease pulmonary vascular resistance in the intact dog and
cat. The explanations for the divergent responses to rapidly injected arachi-
donic acid and slowly infused arachidonic acid are uncertain at the present
time. It is, however, possible that when excessive amounts of substrate are
converted to PGH , the endothelial prostacyclin synthetase may be overwhelmed
and the endoperoxide may isomerize to PGD_ and PGE- or be reduced to PGF_ •
2 2 la
We have reported that administration of cyclooxygenase inhibitors such
as indomethacin and melofenanate results in a slow gradual increase in pul-
monary vascular resistance in the intact dog (20). It has been shown that a
PGI. "like" substance is continually released by the lung (6). We have, there-
fore, suggested that under resting conditions, the pulmonary vascular bed is
maintained in a dilated state by production of a vasodilator product in the
cyclooxygenase pathway (20). Recent evidence suggests that this vasodilator
product in the cyclooxygenase pathway is a PGI, "like" substance (6, 13, 19).
It would therefore be of interest to ascertain if industrial pollutants such
as NO. or SO have adverse effects on enzymes such as cyclooxygenase or prosta-
cyclin synthetase which function to synthesize PGI- which serves to maintain
the pulmonary vascular bed in a dilated state under resting conditions.
335
-------
REFERENCES
1. Anggard E, Samuelsson B: Prosynthesis of prostaglandins from arachidonic
acid in guinea pig lung. J Biol Chem 240:3518-3521, 1965
2. Bunting S, Gryglewski R, Moncada S, Vane JR: Arterial walls generate from
prostaglandin endoperoxides a substance (prostaglandin X) which relaxes
strips of mesenteric and coeliac arteries and inhibits platelet aggregation.
Prostaglandins 12:897-913, 1976
3. Cassin S, Tyler T, Leffler C, Wallis R: Pulmonary and systemic vascular
response of perinatal goals to prostaglandins E and E . Amer J Physiol
(In press)
4. Gruetter CA, MeNamara DB, Hyman AL, Kadowitz PJ: Contractile responses of
intrapulmonary vessels from three species to arachidonic acid and an epoxy-
methano analog of PGH-. Can J Physiol Pharmacol 56:206-215, 1978
5. Gryglewski RJ, Bunting S, Moncada S, Flower RJ, Vane JR: Arterial walls
are protected against deposition of platelet thrombi by a substance
(prostaglandin X) which they make from prostaglandin endoperoxides.
Prostaglandins 12:685-713, 1976
6. Gryglewski RJ, Korbut R, Ocetkiewicz A, Splawinski J, Wojtaszek B, Swies J:
Lungs as a generator of prostacyclin. Hypothesis on physiological signifi-
cance. Naunyn-Schmiedeberg's Arch Pharmacol 304:45-50, 1978
7. Hamberg M, Svensson J, Wakabayshi T, Samuelsson B: Isolation and structure
of two prostaglandin endoperoxides that cause platelet aggregation* Proc
Natl Acad Sci US 71:345-349, 1974
8. Hamberg M, Samuelsson B: Prostaglandin endoperoxides. VII. Novel trans-
formations of arachidonic acid in guinea pig lung. Biochem Biophya Res
Commun 61:942-949, 1974
9. Hamberg M, Svensson J/ Samuelsson B: Thromboxanes: A new group of biolog-
ically active compounds derived from prostaglandin endoperoxides. Proc
Natl Acad Sci US 72:2994-2998, 1975
10. Hyman AL, Chapnick BM, Kadowitz PJ, Lands WEM, Crawford CG, Pried J,
Barton J: Unusual pulmonary vasodilator activity of a novel prostacyclin
analog: Comparison with endoperoxides and other prostanoids. Proc Natl
Acad Sci USA 12:5711-5715, 1977
11. Hyman AL, Kadowitz PJ, Lands WEM, Crawford CG, Fried J, Barton J: Coro-
nary vasodilator activity of 13,14-dehydroprostacyclin methyl ester: Com-
parison with PGI2 and other prostanoids. Proc Natl Acad Sci USA 75:3522-
3526, 1978
12. Hyman AL, Mathe AA, Matthews CC, Bennett JT, Spannhake EW, Kadowitz PJs
Modification of pulmonary vascular responses to arachidonic acid by alter-
ations in physiologic state. J Pharmacol Exp Ther 207:388-401, 1978
13. Hyman AL, Kadowitz PJ: Pulmonary vasodilator activity of prostacyclin
(PGI ) in the intact cat. Arc Res (In press)
336
-------
14. Kadowitz PJ, Spannhake EW, Peigen LP, Greenberg S, Hyman AL: Compara-
tive effects of arachidonic acid, bisenoic prostaglandins and an endoper-
oxide analog on the canine pulmonary vascular bed. Can J Physiol Pharmacol
55:1369-1377, 1977
15. Kadowitz PJ, Hyman AL: Influences of a prostaglandin endoperoxide analog
on the canine pulmonary vascular bed. GLrc Res 40:282-287, 1977
16. Kadowitz PJ, Gueretter CA, McNamara DB, Gorman RR, Spannhake EW, Hyman AL:
Comparative effects of the endoperoxide PGH_ and an analog on the pulmonary
vascular bed. J Appl Physiol 42:953-958, 1977
17. Kadowitz PJ, Joiner PD, Hyman AL: Effects of prostaglandins E_ on pulmonary
vascular resistance in intact dog, swine and lamb. Europ J Pharmacol 31:
72-80, 1975
18. Kadowitz PJ, Hyman AL: Vasoconstrictor effects of thromboxane B on the
canine and feline pulmonary vascular bed. Submitted.
19. Kadowitz PJ, Chapnick BM, Peigen LP, Hyman AL, Nelson PK, Spannhake EW:
Pulmonary and systemic vasodilator effects of the newly-discovered prosta-
glandin PGI . J Appl Physiol 45:408-413, 1978
20. Kadowitz PJ, Chapnick BM, Joiner PD, Hyman AL: Influences of inhibitors
of prostaglandin synthesis on the canine pulmonary vascular bed. Amer J
Physiol 299:941-946, 1975
21. Moncada R, Gryglewski R, Bunting S, Vane JR: An enzyme isolated from
arteries transforms prostaglandin endoperoxides to an unstable substance
that inhibits platelet aggregation. Nature (Lond) 263:663-665, 1976
22. Migteren DH, Hazelhof E: Isolation and properties of intermediates in
prostaglandin biosynthesis. Biochim Biophys Acta 326:448-461, 1973
23. Pace-Asciak CR: Qxidative biotransformations of arachidonic acid. Prosta-
glandins 13:811-817, 1977
24. Snedecor CW, Qochran WG: Statistical Methods. Ames, Iowa, Iowa State
University Press, 1967, 6th Edition, pp 91-119
25* Spannhake EW, Lemen PJ, Wegmann MJ, Hyman AL, Kadowitz PJ: Effects of
arachidonic acid and prostaglandins on lung function in the intact dog.
J Appl Physiol 44:397-495, 1978
26. Spannhake EW, Lemen RJ, Wegmann MJ, Hyman AL, Kadowitz PJ: Analysis of
the airway effects of a PGH analog in the anesthetized dog. J Appl
Physiol 44:406-415, 1978
27. Wasserman MA, DuCharme EW, Griffin RL, DeGraaf GL, tobinson FG: Broncho-
pulmonary and cardiovascular effects of prostaglandin D in the dog.
Prostaglandins 13:255-269, 1977
28. Wicks TC, Rose JC, Johnson M, Ramwell PW, Kot PA: Vascular response to
arachidonic acid in the perfused canine lung. Arc Res 38:167-171, 1976
337
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DISCUSSION
DR. WASSERMAN: Have you looked at the effects of PGI on compliance and re-
sistance changes as well?
OR. KADOWITZ: Yes, the dog is a poor model even when you have a marked in-
crease in bronchomotor tone; the infusion of aerosol of PGI doesn't seem to
have much effect. The way we can see the effect of PGI- is by administering
the substance before the bronchial constrictive challenge* If you administer
PGI and then follow with a histamine aerosol, the bronchial constrictive
response will be markedly attenuated. But, when you administer it when the
bronchial motor tone is elevated, you don1t see the rapid kind of reversal
that we see in the pulmonary vascular bed, and this same kind of thing has
been observed by several other people.
338
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Environmental Influences on Uptake of
Serotonin and Other Amines
A. B. Fisher, *E. J. Block, and G. G. Pietra
University of Pennsylvania
Philadelphia, Pennsylvania
"University of Florida
Gainesville, Florida
CLEARANCE OF SEROTONIN AND OTHER AMINES BY THE LUNG
It is well established that the lungs of mammalian species remove 5-hydro-
xytryptamine (5-HT or serotonin) from the pulmonary circulation and metabolize
it to 5-hydroxyindoleacetic acid (5-HIAA) (2, 14, 15, 19, 20, 24). This pro-
cess of uptake and metabolism serves to inactivate circulating serotonin, and
may be important in regulating the systemic arterial concentration of this
powerful vasoconstrictor. Although other organs may also remove serotonin
from the perfusate, the role of the lung may be of major importance because
this organ consists of a vast capillary bed which receives the entire cardiac
output. The site of serotonin uptake in the pulmonary capillaries has been
localized to the pulmonary endothelial cell (17, 25).
Several lines of evidence indicate that capillary endothelial uptake of
serotonin is carrier-mediated, and is very likely an active transport process.
First, measurement of 5-HT uptake as a function of perfusate concentration
suggests a saturable process (17, 19, 22). Second, uptake requires the pre-
sence of Na in the perfusate (19) and is inhibited by ouabain (19, 21), suggest-
ing involvement of the Na -K activated ATPase. Third, uptake is inhibited by
hypothermia (15, 17, 19), anoxia (19, 24), cyanide (24), and absence of a meta-
bolizable substrate (24), indicating the requirement for metabolically generated
energy. The use of 2-deoxy-glucose illustrates the requirement for metabolic
energy. This agent, which serves as an ATP trap and inhibitor of glucose
metabolism, leads to depression of serotonin uptake that can be reversed by
339
-------
adding a metabolizable substrate (13). Finally, uptake of serotonin can be
blocked by inhibitors of amine transport such as imipramine, chlorpromazine,
and cocaine (16, 19, 24). Definitive proof that transport occurs by active
transport requires demonstration of uptake against a concentration gradient,
but this has not yet been accomplished because of the difficulty of demonstrating
free, i.e., unbound, intracellular serotonin.
The rate-limiting step for serotonin clearance from the perfuaate is the
uptake process rather than the subsequent metabolism of 5-HT to 5-HIAA. Thus
the presence of a monoamine oxidase (MAO) inhibitor such as iproniazide does not
affect the rate of uptake of 5-HT although conversion to 5-HIAA is markedly
depressed (2, 14, 15, 19, 24). On the other hand, a possible effect of pro-
longed MAO inhibition on serotonin uptake has not been evaluated.
Other amines are also removed from the circulation by lung endothelium al-
though uptake mechanisms may vary. Norepinephrine, like serotonin, is removed
by a carrier-mediated process of pulmonary endothelium (14, 17, 21) that can
be differentiated pharmacologically from the 5-HT carrier (16). Imipramine,
which is not transported intracellularly and does not undergo metabolic trans-
formation, is removed through specific binding to the cell membrane (18).
Studies of serotonin uptake from this laboratory with isolated guinea pig
lungs are shown in Figures 1 and 2. Similar results were obtained using
isolated rat lungs although the normal rates of serotonin clearance are slightly
higher with this latter species. Rats were vised for studies of oxygen toxicity
since the reaction of their lungs to oxygen is better defined and their lungs
are easier to perfuse.
PULMONARY OXYGEN TOXICITY
Exposure of animals to oxygen partial pressures above 0.5 atmospheres
absolute (ata), i.e., approximately 350 mm Hg, results in their death with a
time course that is a hyperbolic function of inspired oxygen (3, 9). With 0
partial pressures up to approximately 2.5 ata, the lungs are the predominant
site of injury. Electron microscopic studies of oxygen-damaged lungs have
shown lung cell injury with destruction most marked in the pulmonary endothelium
(1, 10, 20). This information provided the background to investigate the in-
fluence of hyperoxia on the subsequent ability of lungs to remove serotonin and
other amines from the pulmonary circulation. The goal was to define the early
manifestations of hyperoxic damage to pulmonary endothelium.
EFFECTS OF OXYGEN EXPOSURE ON CLEARANCE OF SEROTONIN AND OTHER AMINES
Experimental Procedures
Specific pathogen-free rats weighing 250-300 g were exposed to O for varying
340
-------
0.2r
UJ —
o e
z =
o
cr
10 20 30
TIME (min)
Figure 1. Recirculating perfusion of isolated guinea pig lung with [ C]-
serotonin. The concentrations of serotonin (solid lines) and 5-hydroxyindole-
acetic acid (dashed lines) are plotted versus time of perfusion. Experiments
were carried out in the absence (solid dots) or presence (open dots) of 0.1 mM
iproniazide. Results are mean + SE for 3 experiments under each condition.
durations at either 1 ata in an environmental chamber or 4 ata in a hyperbaric
pressure chamber. The use of specific pathogen-free rats is important to mini-
mize the complication of possible respiratory tract infections. Sexually mature
animals were chosen for study because of the known decreased susceptibility of
immature rats to the toxic effects of 0 (26). Ambient CO concentration in
the chamber was maintained below 3 mm Hg with CO absorbent. Since rats ex-
posed to O do not maintain food intake, food was withheld from both control
and experimental animals to minimize the influence of this variable. Drinking
water was provided ad libitum. Some animals were maintained on a vitamin E-
deficient diet for approximately 6 weeks before 0 exposure. Vitamin E deficiency
in these rats was confirmed with the dialuric acid erythrocyte hemolysis test
(6). Rats maintained on the vitamin E-deficient diet gained weight normally.
Following exposure of rats, lungs were removed and evaluated in an isolated
lung perfusion system for their ability to remove serotonin, norepinephrine, or
341
-------
o
V>
0.60-1
0.5O-
0.4O-
e
o
9
o
o
o
o
o
!•
«
V)
•-' 0.30-
o
\
\ 0.20
O
2
Z
0.1O-
0.09-
0.07.
IMIPRAMINE
CYANIDE
IOOOACETATE
OUABAIN
CONTROL
i
O
I
10
I
IS
I
20
I
25
I
30
TIME
Figure 2. Semi-log
-------
imipramine from the perfusate. Serotonin uptake was evaluated in a recirculatr
ing system (12). The first order rate constant was calculated from the semi-
logarithmic decrease of perfusate serotonin concentration; the fractional clear-
ance was calculated from the rate constant and the average recirculation time
(24). Uptake of norepinephrine and imipramine was evaluated using "once-through"
perfusion. Clearance of these amines was calculated from the arterial-venous
difference in concentration and the rate of perfusion (5). Assay for each of
the amines in the perfusate was done by radiolabel counting following separation
of the amine from its metabolic products.
Effects of Oxygen Exposure on Serotonin Clearance
Normal Rats. Fractional clearance of serotonin in control animals that
were not exposed to oxygen was approximately 0.80, indicating that approximately
80% of the serotonin presented to the lung was cleared during a single passage
through the pulmonary circulation. Mean serotonin clearance in lungs from rats
exposed to oxygen was decreased by 5% after 4 hours O-, 12% after 12 hours 0 ,
25% after 18 hours 0 , and 35% after 48 hours 0 (Pig. 3). The effect of <>2 on
serotonin clearance was an approximate hyperbolic function of exposure time
with an estimated plateau value of 40-50% depression of clearance. These re-
sults suggest that the ability of the lungs to remove serotonin from the per-
fusate was depressed within the initial 12 hours of oxygen exposure.
Hyperbaric oxygen at 4 ata greatly accelerated the depression of serotonin
clearance. After 1 hour of hyperbaric exposure, clearance was depressed by 30%
(Fig. 4) which was approximately equivalent to the effect of 1 ata 0. for 24
hours.
Vitamin E-deficient Rats. Vitamin E-deficient rats demonstrated increased
susceptibility to the effects of oxygen on pulmonary clearance of serotonin.
With 0 at 1 ata, 5-HT clearance was depressed by 45% after 12 hours of ex-
posure. This exceeded the effect of 48 hours 0 exposure on normal animals
(Fig. 5). The increased susceptibility to 0 of vitamin E-deficient rats was
also seen with hyperbaric exposure. Serotonin clearance in vitamin E-deficient
rats was depressed by approximately 30% after 45 minutes and 45% after 60 minutes
of exposure (Fig. 4). Rats repleted with vitamin E by intraperitoneal injection
(1 mg/kg twice weekly) or supplementation of drinking water responded to 0 at
£
1 ata similarly to normal diet rats. There was no apparent protection against
hyperoxia by these pharmacologic doses of vitamin E (6).
Protection Against the Effects of Oxygen* The use of hyperbaric exposure
provided a convenient model for evaluating the effect of possible protective
agents on the pulmonary response to hyperoxia since only short exposures were
required. Rats were treated with a single dose of possible protective agents
intraperitoneally 45 minutes prior to exposure (8). GSH (reduced glutathione)
343
-------
I.Or
LU
O
LU
•
— 1
O
1 0.6
§
oc.
#0.4
_J
lo.2
0
n
M
-
M»
(44)
A.
(7)
_
(7)
T
(9)
T
[
(8)
T
1
(5)
04 12 18 24 48
02 EXPOSURE AT I ATA (Mrs)
Figure 3. Serotonin clearance by lungs from normal rats exposed to oxygen
at 1 atmosphere absolute (ata). Results are the mean + SE for the number of
experiments indicated at the bottom of each block*
(16 mmol/kg body weight) or sodium succinate (12 mmol/kg body weight) had no
effect on subsequent depression of serotonin clearance by hyperbaric oxygen ex-
posure (Fig. 4). It should be noted that these rats were fed prior to adminis-
tration of succinate although fasting is required to prolong survival by this
T>
agent (23). Pretreatment of animals with superoxide dismustase (Palosein ,
Diagnostic Data Inc., Mountain view, CA) (60 nmol/kg body weight) partially pre-
vented the subsequent effects of hyperbaric oxygen on serotonin clearance (Fig. 4)
In five animals pretreated with superoxide dismutase, mean serotonin clearance
was depressed by only 11% compared with 30% decrease in saline-injected control
animals.
344
-------
1.0
LJ
(J
z
1 0.8
UJ
O
z 0.6
z
0
6
5 0.4
en
<
1 0.2
o
cr
ll» ^"
Vit. E
NORMAL DIET DEFICIENT
i I t
•
T
1
gj
0
O
116)
It
-
CM
O
I
O
(0
(5)
T
1
X
CO
•*•
14)
i_
H-
g
U
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14)
T
x
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eft
15)
£_
CVJ
z
ID
(4)
T
i
^.
CM
2
I
0
(O
>*r
(5)
02 EXPOSURE AT 4 ATA
Figure 4. Serotonin clearance by lungs of rats exposed to O at 4 ata.
Control animals were maintained in room air. Animals maintained on a normal
diet were exposed to O for 1 hour. Vitamin E-deficient animals were exposed
to 0 for 45 or 60 minutes as indicated, individual groups of animals were
pretreated by intraperitoneal injection of GSH (reduced glutathione), succinate,
or superoxide dismutase.
Recovery from Hyperoxic Lung Damage* The hyperbaric exposure model was
also used to study recovery from oxygen-induced depression of serotonin clear-
ance (7). Following O exposure at 4 ata, animals breathed room air for vary-
ing periods before measurement of serotonin clearance. In animals fed a normal
diet, serotonin clearance was partially restored to control values after 1.5 hours
of air breathing and was approximately 90% of control after 3 hours. On the other
hand/ recovery of vitamin E-deficient animals from 0 toxicity was much delayed.
Vitamin E-deficient animals showed essentially no recovery of serotonin clearance
by 3 hours after O exposure and even 24 hours post-exposure of air breathing,
serotonin clearance was 30% below control values. These data suggest that vitamin
E not only protects against hyperoxic depression of serotonin clearance but is
also required for recovery from the toxic effects of 0 .
345
-------
UJ
U
Z
-------
Table 1. Effect of o Exposure on Lung Clearance of Amines
Norepinephrine
nmol/min/g dry
%*
Imipramine
nmol/min/g dry
%*
Control
0 at 1 ata
2.76 + 0.07
(18)
100
7.52 + 0.05
(12)
100
12 hr
24 hr
48 hr
2.59 + 0.22
(I)
1.69 + 0.14
(8)
1.35 + 0.21
(6)
94
61
49
7.55 + 0.10
(?)
7.53 + 0.14
(6)
7.49 + 0.07
(6)
100
100
100
Values are mean + SE for ( ) experiments*
Percent of mean control.
1 ata for up to 48 hours or 4 ata for 1 hour had no signs of respiratory dis-
tress and their lungs appeared grossly normal. The hyperbaric oxygen exposure
was just below the convulsive threshold but an occasional rat did develop
generalized seizures and was not used for further studies. Vitamin E-deficient
rats developed respiratory distress after 18 hours exposure to 1 ata or 1 hour
exposure to 4 ata of oxygen.
Physiologic Changes in Lungs from O -exposed Rats
£
Perfusion pressure at constant flow rate and ventilation pressure at con-
stant tidal volume were measured in the isolated lung during lung perfusion to
evaluate lung mechanical and vascular properties. Similar values for these
pressure measurements were obtained for control and oxygen-exposed normal or
vitamin E-deficient animals. To evaluate the distribution of perfusate to the
85
lung/ Sr-labeled microspheres (15 y diameter) were infused into the pulmonary
artery (6). The distribution of radioactivity per unit tissue was slightly
greater in the lower compared to upper lobes, but was similar in control and
0 -exposed animals. The ratio of lung dry to wet weight and the end of per-
fusion was 0.17-0.18 for both control and oxygen-exposed animals. These are
normal values for dry to wet weight ratio of rat lungs perfused with electrolyte
solutions/ indicating the absence of significant fluid accumulation*
The results suggest that derangement of lung mechanics, altered pulmonary
perfusion, or lung edema was not associated with the decreased serotonin clear-
ance that occurred with 0 exposure.
347
-------
Metabolic Effects of Hyperoxia
Lung metabolism as a function of oxygen exposure was evaluated by measuring
lung tissue adenine nucleotides and the rates of lung lactate and pyruvate pro-
duction (11). After one hour of perfusion, lung ATP and ATP/ADP were maintained
at a high level with no significant difference between control and O -exposed rats
(Table 2). The rates of production of lactate and pyruvate by the isolated per-
fused lung and the lactate to pyruvate ratio were unaffected by exposure of rats
to oxygen for 18 or 24 hours (11). However, lungs from rats exposed to oxygen
for 48 hours demonstrated 60% increase in lactate production with an approximate
doubling of the lactate to pyruvate ratio. These studies indicate that, at
least through 24 hours of O exposure, there was no alteration of lung glycolysis
or energy balance, suggesting that interference with energy generation metabolism
was not responsible for altered amine clearance. On the other hand, the possi-
bility of metabolic changes specifically localized to lung endothelial cells can-
not be excluded.
Table 2. Effect of 0 Exposure on Adenine Nucleotide Content of Rat Lungs
ATP ATP/ADP
umol/g dry pmol/g dry
Control (3) 12.8 + 0.1 7.2 + 0.7
0 at 1 ata
2
24 hr (3) 12.2 +1.0 8. 1 + 0.4
48hr(3) 11.8+0.6 7.6+1.1
Control (7) 10.5 + 0.4 8.6 _+ 0.7
at 4 ata
1hr(4) 9.8+0.4 8.0+0.5
O at 4 ata
Results are mean +_ SE for ( ) experiments.
Table 3. H Localization in Autoradiographs of Pat Lung Following Perfusion
with [ H]-5-Hydroxytryptamine
Grains/n Observed/expected
Endot helium Other Endothelium Other
0 , 4 ata x 1 hr
0 , 4 ata x 1 hr
0 , 1 ata x 48 hr
0.09
0.11
0.13
0.02 1.7 0.3
0.03 1.6 0.4
0.03 1.6 0.4
Results are mean values based on 10-15 micrographs.
348
-------
ALV
Figure 6. Electron microscope autoradiogram of the lung from a rat exposed
to 4 ata 0 for 1 hour and subsequently perfused with [ H]-5HT in the presence
of iproniazide. Developed silver grains have accumulated predominantly over the
capillary endothelium (EN). There are no anatomic alterations in the constituents
of the alveolar-capillary membrane attributable to oxygen exposure. Tubular
myelin and amorphous material probably representing surfactant are present at
the interface between alveolar air and alveolar epithelium. Sections were
stained with uranyl acetate-lead hydroxide. ALV, alveolar space; C, capillary
space. (Original magnification X6,300).
Electron Microscopy and Autoradiography
In order to study the effect of O on localization of 5-HT, the lungs from
two rats exposed to CL at 4 ata for 1 hour and one rat exposed to cr at 1 ata for
2 3 2
48 hours were perfused with [ H]-serotonin in the presence of iproniazide to
prevent 5-HT metabolism. Examination of these lungs with the electron micro-
scope failed to reveal ultrastructural abnormalities at the level of the alveolar
septum. On autoradiography of the O -exposed lungs, silver grains were localized
predominately to the endothelial cells (Fig. 6 and Table 3). These results in-
dicate that this degree of hyperoxia did not cause ultrastructural damage to
the lung and that the accumulation of serotonin even with oxygen-exposed animals
was still predominately in the endothelial compartment.
349
-------
MECHANISMS OF DEPRESSION OF AMINE UPTAKE
Clearance of serotonin and norepinephrine was inhibited at an early stage
in the development of pulmonary oxygen toxicity. The evidence that 0 toxicity
was at an early stage was the failure to demonstrate pulmonary derangement by
morphologic, physiologic, or metabolic criteria. On the other hand, removal of
imipramine was not depressed at this stage of oxygen exposure. This differential
effect on clearance can be explained by the differences in normal handling of the
amines. Serotonin and norepinephrine are probably transported actively by the
pulmonary endothelial cell while imipramine is cleared by passive binding.
Therefore, we conclude that oxygen may exert its effect on the pulmonary endothe-
lial cell membrane and thereby interfere with the active transport process for
circulating amines. Since uptake of both serotonin and norepinephrine was affected,
the toxic effect of 0 either involved more than one carrier or some basic
mechanism common to the transport of both amines* The results with metabolic
studies suggest that the common mechanism was not alteration of energy supply.
The answer to this problem awaits further definition of the precise mechanisms
involved in amine transport.
SUMMARY AND CONCLUSIONS
Exposure to elevated partial pressure of oxygen results in early and re-
versible depression of active amine (serotonin and norepinephrine) transport by
the rat lung. A possible mechanism is that O exerts a toxic effect on the pul-
monary endothelial cell membrane. The findings may provide a convenient meta-
bolic marker for the early toxic effects of oxygen on the lung. The significance
of decreased amine clearance in the pathogenesis of the systemic manifestations
of oxygen poisoning remains to be evaluated.
REFERENCES
1. Adamson IVR, Bowden DH, Wyatt JP: Oxygen poisoning in mice; ultrastructural
and surfactant studies during exposure and recovery. Arch Pathol 90:463-
472, 1970
2. Alabaster VA, Bakhle YS: Removal of 5-hydroxytryptamine in the pulmonary
circulation of rat isolated lungs. Br J Pharmacol 40:486-492, 1970
3. Bean JW: Effects of oxygen at increased pressure* Physiol Rev 25:1-147,
1947
4. Block ER, Cannon JK: Effects of hyperoxia on imipramine uptake and meta-
bolism by the isolated, perfused rat lung. Res Comm Chem Path Pharm 22:621-
624, 1978
5. Block ER, Cannon JK: Effect of oxygen exposure on lung clearance of amines.
Lung 155:287-295, 1978
350
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6. Block ER, Fisher AB: Depression of serotonin clearance by rat lungs
during oxygen exposure. J Appl Physiol 42:33-38, 1977
7. Block ER, Fisher AB: Effect of hyperbaric oxygen exposure on pulmonary
clearance of 5-hydroxytryptamine. J Appl Physiol 43s254-257, 1977
8. Block ER, Fisher AB: Prevention of hyperoxic-induced depression of pul-
monary serotonin clearance by pretreatment with superoxide dismutase.
Am Rev Resp Dis 116:441-447, 1977
9. Clark J, Lambertsen CJ: Pulmonary oxygen toxicity: A review* Pharmacol
Rev 23:36-133, 1971
10. Crapo JD, Marsh-Salin J, Ingram P, Pratt PC: Tolerance and cross-tolerance
using NO and 0 . II. Pulmonary morphology and morphemetry. J Appl Physiol
44:370-379, 1978
11. Fisher AB: Energy status of the rat lung after exposure to elevated P «
J Appl Physiol 45:56-59, 1978 2
12. Fisher AB, Steinberg H, Bassett D: Energy utilization by the lung. Am J
Med 57:437-466, 1974
13. Fisher AB, Steinberg H, Dodia C: Reversal of 2-deoxyglucose inhibition of
serotonin uptake in isolated guinea pig lung. J Appl Physiol 46:447-450,
1979
14. Gillis CN, Iwasawa Y: Technique for measurement of norepinephrine and
5-hydroxytryptamine uptake by rabbit lung. J Appl Physiol 33:404-408, 1972
15. Qruby LA, Rowlands C, Varley BQ, Wyllie JH: The fate of 5-hydroxytryptamine
in the lungs. Brit J Surg 58:525-532, 1971
16. Iwasawa Y, Gillis CN: Pharmacological analysis of norepinephrine and 5-
hydroxytryptamine removal from the pulmonary circulation: Differentiation
of uptake sites for each amine. J Pharm Exp Therap 183:341-355, 1973
17. Iwasawa Y, Gillis CN, Aghajanian G: Hypothermia inhibition of 5-hydroxy-
tryptamine and noradrenaline uptake by lung: Cellular localization of
amines after uptake. J Pharm Exp Therap 183:341-355, 1972
18. Junod AF: Accumulation of C-imipramine in isolated perfused rat lungs*
J Pharm Exp Therap 183:182-187, 1972
19. Junod AF: Uptake, metabolism and efflux of C-5-hydroxytryptamine in iso-
lated perfused rat lungs. J Pharm Exp Therap 183:341-355, 1973
20. Kistler GS, Caldwell PRB, Weibel ER: Development of fine structural damage
of alveolar and capillary lining cells in oxygen-poisoned rat lungs. J
Cell Biol 32:605-628, 1967
21. Nicholas TE, Strum JM, Angelo LS, Junod AF: Site and mechanism of uptake
of H-1-norepinephrine by isolated perfused rat lungs. Circ Res 35:670-680,
1974
351
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22. Pickett RD, Anderson MW, Qrton TC, Eling TE: The pharmacodynamics of
5-hydroxytryptamine uptake and metabolism by the isolated perfused rabbit
lung. J Pharm Exp Therap 194:545-553, 1975
23. Sanders AP, Hall IH, Woodhall B: Succinate: Protective agent against
hyperbaric oxygen toxicity. Science 150:1830, 1965
24. Steinberg H, Bassett DJP, Fisher AB: Depression of pulmonary 5-hydroxy-
tryptamine uptake by metabolic inhibitors. Am J Physiol 228:1298-1303,
1975
25. Strum JM, Junod AF: Radioautographic demonstration of 5-hydroxytryptamine-
H uptake by pulmonary endothelial cells. J Cell Biol 54:456-467, 1972
26. Tierney DF, Ayers L, Kasuyama RS: Altered sensitivity to oxygen toxicity.
Am Rev Reap Dis 115 (Suppl 2):59-65, 1977
352
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DISCUSSION
PARTICIPANT: la there any speculation as to why the process for serotonin is
so different from that for prostaglandin? As you reiterated, for prostaglandins,
the rate-limiting step is not uptake but: metabolism by the dehydrogenase.
DR. FISHER: Why is uptake rate-limiting for serotonin but metabolism rate-
limiting for prostaglandin? Is that right? I suppose that it relates to
the respective rate constants for the processes* The serotonin uptake process
is high capacity (high V ) with a relatively low K compared to the MAO.
max m
The reverse, I suppose, is true for the prostaglandins. In addition, there's
probably intracellular binding of serotonin, because it doesn't leak out.
Once it's transported in, it will stay there for a long time. For prosta-
glandins, that apparently is not true from what Tom Eling said. In the absence
of metabolism, prostaglandin leaks out. That may also play a role.
PARTICIPANT: Have you and, say, Tom Eling ever exchanged transport inhibitors?
DR. FISHER: I have not used his.
PARTICIPANT: I wonder if you've ever given thought to the hypothesis that has
been advanced that cell death is responsible for the altered lung function.
DR. FISHER: I suppose that if there were cell death in oxygen toxicity, then
that would lead to functional alterations. The I'D for rats in oxygen
is about 72 hours. Morphologic and physiologic changes probably take roughly
48 hours to reach the stage where you might see cell death. We think that we
were looking at an earlier stage. In addition, the effects were rapidly
reversible. I don't think that cell death accounts for what we found.
We think that these effects precede morphologic or physiologic change and
represent an early stage. Certainly, if the oxygen exposure ware continued,
then you* d begin to see things like cell death as well as pulmonary edema
which then might cause nonspecific alterations in nonrespiratory lung functions.
353
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Session VII:
Summary and Future Research Needs
-------
Summary and Future Research Needs
P. A. Brpmberg and *D. B. Menzel
University of North Carolina
Chapel Hill, North Carolina
*Duke University
Durham, North Carolina
DR. BROMBERG: It will be hard to summarize this conference in the 15 minutes
that we each have, and I don't think either of us is going to make a formal
attempt to do so. First, I can speak for all of us in thanking the EPA for
arranging this symposiun and for inviting a very distinguished panel of arti-
culate and ingenious scientists. Secondly, I want to point out that the EPA
has remarkable powers. Having put out the lure of a meeting at Hilton Head
Island in the middle of the cold winter, and having gotten everyone down here
on a beautiful Sunday, they proceeded to change the weather to keep everyone
indoors and in perfect attendance. It would be awfully nice if they could
call upstairs and switch it all back again, so that some of us, or all of us
perhaps, could get home.
This is a remarkable meeting in that, with very few exceptions, almost
no mention was made of pulmonary mechanics and of gas exchange. That doesn' t
happen very often in meetings devoted primarily to lung research, but perhaps
has been happening more frequently in the recent past and it may become a trend
perhaps for the future. Another interesting note was that almost everyone is
working or collaborating with an ultrastructuralist, or would like to have
access to ultrastructural techniques. Dr. Reid, among others, gave us a very
nice demonstration of how ultrastructural techniques can be combined with
biochemical techniques in experimental studies of the reaction of the lung to
insults. Dr. Satir presented most elegant ultrastructural biochemical data
on the mechanism of ciliary motion.
The organization of the meeting, as I perceive it, looks at the lung as
having three segments. There was a large area of attention to the epithelium,
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and at the end, there was a large area of attention to the endotheliun. In the
middle, I suppose appropriately/ there were a number of speakers who talked
about cells that more or less fit between the epitheliun and the endotheliun.
Notably missing, or in large measure missing, were discussions of smooth mus-
cle and of neural function at more than a passing level. The keynote of the
epithelial sections was set by Dr. Boucher and Dr« Gatzy, who pointed to the
barrier function of the epitheliun and the attempts to quantitate the nature
and the site of this barrier* Ihey paid special attention to the intercellular
junctions and the so-called tight junctions of the epitheliun and defined their
properties by electrical measurements and by using a variety of non-charged
probe molecules*
Of particular Importance are the macromolecules which may gain access to
important cells residing deep in the epitheliun. These cells were discussed
by Dr. Bienenstock, Dr. Brain, and Dr. Wasserman. I might parenthetically add
that there are a few lucky M.D.s who have picked up a Ph.D. at this meeting,
and that's another sign of the power of the Environmental Protection Agency.
Dr. Wasserman is among them, and I think Dr. Fisher also is a beneficiary. In
any event, these powerful cells—Dr. Spitznagel1s cell, the poly, the mast cell
of Dr. Wasserman, and the "big Mac" of Dr. Brain—have an enormous armamentarium.
It's important to consider how materials deposited in the airway may or may not
be able to gain access to these cells, and how environmental pollutants and
toxic agents might alter the barrier function of the epitheliun and permit more
ready access of other inhaled materials to these critical cells, which when
turned on have the potential not only for protective action, but apparently for
very damaging actions. We were shown by Dr. Wasserman in particular, and other
speakers as well, the remarkable balance between active effects and mechanisms
which repair or inhibit these effects.
This underlies a big problem that the EPA faces in trying to develop its
research program. It may be relatively easy to show effects of one sort or
another, but at what point do these effects go from the normal range or the
range that can be dealt with by intrinsic mechanisms to the point where you
have disease. To do this, one must bear in mind the intrinsic variability of
the hunan subject. The fact that some of us have less hair than others and
some of us have brown eyes while others have blue eyes is obvious. But there
are many other differences of which we're perhaps less aware that make some of
us more susceptible to certain insults than others, because our compensatory
mechanisms are not as effective. A stress that can be easily tolerated by one
individual may become a stress that produces overt disease in another. Even
though the EPA is a regulatory agency and will continue to look on its role in
research as developing data that directly abut on its regulatory function/ the
Agency must bear in mind that it is important to understand mechanisms to the
extent that one can predict idiosyncrasies and unusual reactions that some
358
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"
people in the population will have to a given stress. To do this "blindly
using empirical protocols is impossible. There are too many agents to be test-
ed and too many possible protocols* To do well-directed, intelligent, pertinent
experiments, one has to have some reasonable conceptions of mechanism.
Another problem that the EPA in particular has to face is that the pay-
off is hxznan effects. Yet, the experiments that can be performed in man are
limited. In the past few days scientists have described to us systems that are
very complex and far removed from the intact human being. But there are other
techniques that are either directly applicable or potentially applicable to
intact human subjects. We should make efforts to develop techniques that can
be used in human subjects, and to apply them. For example, in studying tracheal
tissue one can look at rats and hamsters, and so forth, but it is also possible
with a fiberoptic bronchoscope to obtain samples of tracheal epithelium. Such
epithelium can be cultured successfully. Dr. Collier has done so by using
specimens that we have provided for him during ordinary bronchoscopies in
clinical practice. He has been able to show by using such materials that these
specimens can be maintained for many days/ that they retain ciliary activity,
that they can be infected by a variety of specific infectious agents, that
characteristic morphologic pictures can be produced, and that these pictures
are different from what is seen when one uses the same infectious agents in
animal models. It's going to be important to try as much as possible to use
human tissue, even for in vitro experiments. Dr. Boucher has shown us techniques
that can be applied to intact animals, and a few steps have been made to apply
them to intact humans, in whom one can measure one of the bioelectric parameters
of the airways epitheliixa, and one can even induce local changes by the local
application of very minute quantities of drugs. Other examples could be given.
We always need to bear in mind the potential application of our techniques to
human studies and the use of human tissue as much as possible in our experiments.
This is a very exciting era in lung biology; people with all kinds of
background and training have been stimulated in a variety of ways to look at the
lung as more than an organ that simply deals with shuttling air in and out, and
transferring oxygen to the blood, and CO out of the blood. I foresee that this
kind of research is going to be of increasing importance to all aspects of
government activity, to the NIH, NIEHS, and also to the EPA. It will take some
ingenuity to do the right kind of experiments certainly, but it is going to take
an attitude on the part of the EPA, in particular, not to disregard mechanism in
favor of what may seem to be the short-term payoff of defining levels that are
or are not "toxic." To do the latter job well will require some very clever
application of the knowledge of mechanisms , and that is the plea that I would
like to leave with you. Thank you.
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DR. MENZEL: We've seen here the beginnings of a new era in pulmonary research.
It is directed to the definition of the normal physiologic state of the lung/
emphasizing both respiratory and nonrespiratory metabolism. In defining the
pathophysiologic state/ mechanisms of action must be known so that alterations
in particular values may be recognized as abnormal. Since biochemical mea-
surements often are more sensitive than morphologic changes, connections be-
tween altered morphology and altered physiology must be sought. Unfortunately/
the biology of the lung is but poorly understood. Much effort, then, is being
expended on basic or baseline data to increase our surety that the measured
effect on exposure to an environmental pollutant is indeed abnormal and hence
toxic. To these ends our tools are still blunt and need to be honed to as
fine an edge as possible.
Controversy surrounds the use of animal data in assessing human toxicity.
Much of this discord results from the pioneer state of lung research. To be
sure, there are differences between man and animals/ especially in the morphology
of the lung. But there are far fewer differences in the basic physiologic
processes as seen here for ion transport, metabolism of xenobiotic compounds,
and the uptake and metabolism of prostaglandins, angiotensin, and biogenic
amines. On this scale the difference between animals and man is one of dose.
Morphology is highly important, as evidenced by the heavy collaboration
between physiologists/ pharmacologists, and morphologists. One approach has
been to eliminate morphology by studying pure systems. Here the biochemistries
of the hepatic and pulmonary systems of cytochrome VAt-nr for example, are
remarkably similar. Tissue specificity is expressed in the molecular forms
of cytochrome P.50 present in the lung and the differences in indueibility
by environmental agents. The uptake mechanisms unique to the lung are likely,
however/ to encourage recycling and higher concentrations of reactive inter-
mediaries than in the liver. This may make the lung more susceptible to both
environmental carcinogens and toxicants acting through activated intermediaries
which bind covalently to cell macromolecules. The biochemistry and phar-
macology of the lung has been aided by the isolation and culture of both organs
and specific cells. Much more work is needed here to identify susceptible
lung cells and to develop specific markers, enzymatic or glycoprotein, indicative
of pulmonary damage. Release of marker enzymes by the liver and heart has been
established as a valuable tool for estimating hepatic damage by toxicants and
myocardial damage by ischemia in the clinical assessment of man. Similar
techniques are likely for pulmonary damage.
The development of ex vivo and J.n vivo perfused lung preparations has been
accomplished in the last five years. It is now possible to study the transient
events of prostaglandin metabolism which had been obscured by competing factors
and compensatory mechanisms in intact preparations. Of particular note axe
360
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the studies relating SO5 to bronchitis and the consequences of aberrant prosta-
glandin metabolism in asthmatics. Perhaps here we will understand some of the
more subtle effects seen in man on lifetime exposure to polluted air*
We cannot lose sight of the fact that regulation of pollution is the
ultimate gjm to which these studies will be applied* Therefore, it is im-
portant to define the point at which an effect ceases to become a normal
response and becomes pathophysiologic. It is also necessary to attempt to
extrapolate these measurements made in animals to the hvroan condition in a
manner which is defensible and at the same time sensitive* Certainly the
question of the variability of man in regard to his response is particularly
important* This integration is represented by the organization of this meeting.
It represents the philosophy of those within the Health Effects Research
Laboratory with whom I have had the pleasure to work, starting with Dr. Gordon
Hueter, Director of HERL, and Dr. William Durham who are not here today but
are represented most immediately, in our presence, by Dr. D. E« Gardner and
Dr. Ed Hu and their colleagues. Certainly, they recognize the need for the
development of methods that are sensitive, the application of new methods, and
the integration of these to toxic exposures which are relevant to the exposure
of man in the environment and ultimately to the prevention of disease. This
particular mission is deserving of the finest research that we can bring
to the issue. I am very pleased to have been able to associate with you in
demonstrating that it is possible to bring together high quality, thesis-
directed research in the application to inhalation toxicology. Thank you.
361
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TECHNICAL fiEPORT DATA-
!Plecae read Inarvctiora on the reverse before completing)
1 . DEPORT NO.
EPA-60Q/9-79-Q22
12.
3. RECIPIENT'S ACCESSION>NO.
a. TiTLs AND SUBTITLE
Experimental Models for Pulmonary Research
5. REPORT=OATE '
June;, 1979
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS-"
Environmental Toxicology-, Division:
Health Effects Research Laboratory
U.S. Environmental"Protection Agency
Research Triangle-Park,,NC• 27711;
10. PROGRAM ELEMENT NO.
1AA816
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY'NAME.ANO^ADOHESS'j:
Health Effects.Research Laboratory
Office of Research and'Development
U.S. Environmental Protection-Agency
Research Triangle-Parki NG:27711
RTP, NC
13.:TYPS-'OF'REPORT AND PERIOD COVERED1
14. SPONSORING AGENCY CODE
EPA 600/11
IS. SUPPLEMENTARY NOTES
16. ABSTRACT
This report is the-proceedings.of a:symposium convened:at Hyatt on Hilton
Head Island,;South Carolinai,February 5-7, 1979;. This, volumecconsists of
23 formal presentations, that-cover, the .-five major areas -discussed, during the
symposium: permeabil ity of respiratory ep.ithe!iurn;and .pulmonary phagocytes;
respiratory tract immunity: model systems-of respiratory infectious diseases;
regulation of mucus secretion and;cellularidifferentiation; and pharmacological
modulation in the-lung; The: purpose: of-this symposium:is:, to. exchange research
approaches and techniques with experts in the scientific community and to
explore the possible-application-of these:new model system^and techniques for
studying environmental effects on pulmonary health.
17.
KEY WORDS AND DOCUMENT ANALYSIS
OESCniPTORS'
b.lOENTIFieRS/OP5N ENDED TERMS- |c. COSATI Fieid/Group
Model system.
Pulmonary Health
Pharmacological Modulation
Infectious Di seases
Mucus Membrane
Air Pollutants
M^del systems"for
pulmonary research
06F,M,0,T
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
i 19.-SEGUHITY CLASS (ThisRepon)
UNCLAS-SIFIED.
21. NO. OF PAGES
374
20. SECURITY CLASS'(Thispage I
r UNCLASSIFIED
22. PRICE
EPA Form-2220O (9-73)
362"
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