United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
xvEPA
Research and Development
EPA-600/S1-81-020 May 1981
Project Summary
Determining Effect of
Pollutants on the
Immune System
A. Zarkower, J. Davis, F. Ferguson, and D Stnckler
The purpose of this project was to
determine the effects of fly-ash inhala-
tion on the ability of animals to resist
infections, neoplastic growth, and the
development of hypersensitive re-
sponses. Mice were exposed to fly ash
from two sources, carbon black, and
filtered air only. Following various
exposure periods (days to months),
the immunologic competence of lymph-
cytes, neutrophiles, and macrophages
was assessed.
Fly-ash inhalation resulted in a
decreased response in the spleens and
mediastinal lymph nodes to Escherichia
coli antigen given by aerosol. This
suppression was much less severe
than that following inhalation of carbon
black and silica quartz. Fly ash had
little effect on the ability of T and B
lymphocytes to respond to mitogens
PHA (phytohemagglutin (PHA) and
lipopolysaccharide) and to be stimu-
lated for cytolytic response against
tumor cells. Both the in vitro response
of the (BCG)-BACILLE CALETTE GUER-
IN-sensitized micre to purified protein
derivative of tuberculin and in vivo
delayed-type hypersensitive reactions
were increased.
Exposure to fly ash had the following
more pronounced effects on macro-
phages: a decrease in the number of
pulmonary macrophages capable of
phagocytosis; a decrease in antibody-
dependent cell-mediated cytolysis
(ADCC), in contrast to enhancement
of ADCC after intratracheal injection
of fly ash and silica and inhalation of
silica; an increase in cytotoxie activity
against HBO tumor cells after intratra-
cheal injection of fly ash and a decrease
after inhalation of fly ash; and decreas-
ed ability to activate T cell mitogenesis
after fly-ash inhalation.
This Project Summary was develop-
ed by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC. to announce key findings of the
research project that is fully document
edd in a separate report of the same
title (see Project Report ordering in-
formation at back).
Introduction
The lung is the primary exposure site
for gases and for particles smaller than
2 /urn in diameter After inhalation, toxic
substances may interact with surface
proteins and cells or may enter the body
and be carried to such organs as the
lymphoid tissues, liver, and kidneys,
which are the principal organs of clear-
ance, detoxification, and excretion.
Although exposure to airborne sub-
stances can also result in absorption by
the gastrointestinal tract and the skin,
the lung accepts the greatest burden.
Inhalation of toxic substances can
cause disease either by direct damage
to lung tissues or by affecting other
organ systems. Direct lung damage
often follows exposure to high concen-
trations of toxic minerals (silica, asbes-
tos) or gases (ozone, nitrogen dioxide).
Substances that can have indirect
effects include lead, which can affect a
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variety of tissues (gastrointestinal tract,
nervous system, and blood); cadmium,
which can cause kidney damage and
osteomalacia; and mercury, which can
affect the central nervous system.
Gases and organic and inorganic parti-
culates have a variety of effects on
immunological responses in the body,
including suppression or enhancement
of antibody formation, cellular immuno-
responses, and phagocytic activities of
macrophages. Decreased antibody re-
sponses may increase susceptibility to
infectious diseases; increased stimula-
tion of specific types of antibodies may
increase resistance to infectious dis-
eases, but may also lead to immuno-
pathological conditions such as imme-
late hypersensitivity and immune com-
plex diseases. Decreased cellular im-
mune responses may decrease resis-
tance to certain infectious agents and
certain neoplastic processes, while
increased cellular immunocompetence
may manifest itself as delayed-type
hypersensitivity. Changes in neutrophile
and macrophage functions also can
affect resistance to both infectious and
neoplastic diseases
In the near future, expanded use of
coal for power generation and as stock
for liquid and gaseous fuels is expected
to yield a number of products (e g., SO?,
NOa, hydrocarbons, and particulates)
that could react with biological systems.
Fly ash will be a large component of the
se coal-derived air pollutants, and it is
composed of many elements that can
cause cell damage. Since inhaled parti-
culates will come into intimate contact
with cells of the immune system (macro-
phages and lymphocytes), changes in
immunological responses may occur,
leading ultimately to decreased ability to
cope with infectious agents and neopla-
stic cells. This project was designed to
test the effects of fly-ash inhalation on
cells involved in the immune response
and their functions.
Exposure Facilities
Female BALB/c mice were exposed
to fly ash (and in some cases, carbon) in
specially designed chambers. Com-
pressed air was pulsed into a dustbed,
and the particles entered an air stream
passing through the dust-generator
cavity. This air stream was mixed with a
stream of filtered air before it entered
the exposure chamber. Control cham-
bers received airstreams from the same
sources, though without the particles.
After varying lengths of time, the animals
were removed from the chambers, and a
variety of tests was done to determine
the immunologic competence of lym-
phocytes, neutrophiles, and macro-
phages.
Results
In thef irst series of experiments, mice
were exposed to carbon and fly ash
(from the Pennsylvania State University
(PSD) power plant) for 7, 21, 35, and 56
days. The total concentrations of the
generated particulates and the propor-
tions in the respirable size range «2.1
(im) are given in Table 1. Inhalation of
both fly ash and carbon decreased the
numbers of antibody-forming cells in
the spleen following stimulation of the
mice with Escherichia coli antigen
aerosols. The effect of carbon was much
greater than that of fly ash. Antibody-
forming activity in the mediastinal
lymph nodes was enhanced after 7 days
of exposure, followed by suppression
after 21 days and recovery after 35 and
56 days. Carbon and fly ash both caused
hypertrophy of the lymph nodes, with an
increase in the number of lymphocytes
at all times after exposure. Serum
agglutinating activity also decreased.
Little change occurred in the lympho-
cyte responses to T and B cell mitogens.
The recognition ac.tivity of the cells was
decreased after a 21-day exposure, as
was lymphocyte response to Con A. No
significant change was found in the
cytolytic capacity of T cells after any
period of exposure.
A nine-month exposure to fly ash at
somewhat higher concentrations re-
sulted in a highly significant decrease in
splenic antibody-forming cell response
in mice exposed to E. coli lipopolysac-
charide antigen by either aerosol or
intratracheal injection. Plaque-forming
cell responses in the mediastinal lymph
nodes were lower in both cases, though
not significantly.
In a second series of experiments,
mice were exposed to carbon and fly ash
provided by the U.S Environmental
Protection Agency (EPA), as well as that
from the PSU power plant. The total
concentrations of the particulates and
the proportions in the respirable size
range are given in Table 2.
The number of antibody-forming cells
in the spleens decreased after 7 and 24
days of exposure to fly ash, and de-
creased serum antibody activity was
found after 7, 21, and 56 days of ex-
posure. Carbon dust caused a very
pronounced and progressive reduction
in the number of antibody-forming cells
in the spleens after all times of ex-
Table 1. Paniculate Exposure Concentrations Expressed as fjg/m3 of Air
Mean Fly Ash ± Standard Error
Mean Carbon Black ± Standard Error
Experiment
Responses to
antigenic
stimulations
in vivo
Responses to
mitogens in
vitro and
recognitive
activity of
T cells
Cytolytic
activity of
stimulated
T cells
Exposure Period
7 days (7/28-8/4/77)
21 days (7/28-8/1 8/77)
35 days (7/28-9/2/77)
56 days (7/28-9/22/77)
7 days (8/8-8/1 5/77)
21 days (8/8-8/30/77)
35 days (8/8-9/13/77)
56 days (8/14-10/11/77)
7 days (9/2-9/9/77)
21 days (9/2-9/23/77)
35 days (9/2-10/7/77)
56 days (8/19-10/14/77)
Total
2667 ± 1417
3357 ± 798
3220 ± 512
2658 ± 463
4976
3686 ± 647
3281 ±440
2277 ± 350
2417
1535 ± 563
1 732 ± 444
21 37 ±380
< 2. 1 fjm
655 ± 348
959 ± 236
1009 ± 153
860 ± 140
1433
1214 ± 110
11 30 ±85
840 ± 135
909
562 ± 230
643 ± 182
798 ± 151
Total
4736 ± 1683
4987 ± 1334
4805 ± 855
4615 ± 700
2461
4879 ± 1644
4301 ± 1049
4956 ± 762
2148
4237 ± 1458
4448 ± 1053
4445 ± 670
< 2.1 (urn
947 ± 555
1387 ± 522
1561 ±349
1472 ± 269
859
1815 ± 560
1679 ±363
1642 ± 296
924
1291 ±485
1220 ± 350
1449 ± 267
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fable 2. Concentration of Particulates Expressed as /jg/m3 of Air
Mean Fly Ash ± Standard Error
Experiment
Mean Carbon Black ± Standard Error
Exposure Period
Total
Total
< 2.1 urn
In vivo
responses to
antigenic
stimulations
In vitro
responses to
mitogens.
recognitive
activity of
T cells and
phagocytosis
by macrophages
Cyto/yt/c
activity of
stimulated
T cells
7 days
21 days
21 days
35 days
56 days
148 days
7 days
21 days
35 days
56 days
148 days
7 days
35 days
56 days
154 days
(11/16-11/23/77)
(11/18-12/9/77)
(11/23-12/14/77J
(12/8/77-1/12/78)
(12/15/77-2/9/78)
(12/22/77-5/19/78)
(12/8-12/15/77)
(11/15-12/6/77)
(11/16-12/21/77)
(1/10-3/7/78)
(12/13/77-5/9/78)
(1/13/77-1/20/78)
(11/29/77-1/3/78)
(1/11-3/8/78)
(12/20-5/23/78)
2232
2039
2143
2059
*1628
1502
*2634
1459
*7S37
2262
*1159
2042
2115
1334
*2674
1297
1879
1905
*3129
2113
*2819
1134
*2674
1459
*1831
±256
±343
±286
±289
±375
±285
±430
± 140
±221
±298
±228
±343
±235
±237
±97
± 131
±240
±387
+ 1281
±384
±839
±237
±429
± 140
±221
933
785
764
741
433
543
615
535
461
794
366
809
804
502
623
509
465
661
691
703
676
502
623
535
461
±77
± 139
± 130
±86
±77
± 100
±98
±50
±50
± 104
±72
± 136
±90
±86
±97
±48
±55
± 134
±283
+ 777
± 794
±86
±96
±50
±50
4492
4707
3608
2511
2934
3509
3180
5399
4027
2984
3520
3758
3172
2984
3509
±
±
±
+
±
±
±
±
+
+
+
±
±
±
±
1917
1192
355
346
459
312
550
1372
910
512
342
1933
526
512
312
1538 ±
1472 ±
1104±
857 ±
841 ±
1178±
932 ±
1407 ±
1248 +
885 ±
1162 ±
996 ±
909 ±
885 ±
1178 ±
1029
386
125
157
125
119
161
429
292
139
130
512
142
139
119
* Exposure data for fly ash supplied by EPA.
posure, with almost complete suppres-
sion after 148 days of exposure. The
effect of fly ash on the mediastinal
lymph nodes varied from an increase in
the number of antibody-forming cells
after 7 and 21 days of exposure to no
effect or a small decrease of response
after 56 days. The cellular immune
reactions were not affected, except for
suppression of cytolytic activity by PSD
fly ash after 35 days. Both fly ash and
carbon caused a progressive decrease in
phagocytic activity by lung-derived macro-
phages from 21 to 146 days of exposure.
A third series of experiments exam-
ined the effects of fly ash on various
functions of the alveolar macrophage.
Exposure of mice to PSU fly ash at
concentrations of 742 /ug/m3 of air of
particles <2.1 /urn for up to four weeks
decreased the proportions of macro-
phages capable of phagocytosis and the
proportion of very active macrophages
(seven or more bacteria phagocytized).
For practical reasons, golden hamsters
were used instead of mice in studies of
antibody-dependent cell-mediated cyto-
toxicity by alveolar macrophages. Intra-
tracheal injection of 2 mg silica into
golden hamsters significantly enhanced
the ADCC 1, 7,14,42, and 70 days after
injection; intratracheal injection of 2 mg
fly ash resulted in enhancement at 14,
42, and 70 days. Inhalation exposure of
golden hamsters to respirable-sized
silica at an average concentration of
3102 /ug/m3 of air enhanced ADCC
function slightly after 7 days and signi-
ficantly after 14, 42, and 70 days.
Inhalation of fly ash (5886 A
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particles < 2.1 /nm in diameter and
68.36 ±5.56% < 4.7//m. Differences in
viability of macrophages from the fly-
ash-exposed and control mice, as deter-
mined with trypan blue absorption,
were not statistically significant. Inhala-
tion exposure of mice to fly ash for two,
three, and four weeks significantly
reduced the number of alveolar macro-
phages that phagocytized Staphylococ-
cus aureus in in vitro culture.
The elements present in respirable-
sized fly ash. particles used in this
project were determined by energy
dispersive X-ray analysis (EDXA). This
type of analysis in both the scanning
and transmission electron microscopy
modes was used to determine the
elements present in alveolar macro-
phages from mice exposed and not
exposed to fly ash.
Examination of control alveolar
macrophages in the transmission mode
revealed so much variation in ultrastruc-
ture that characteristic alveolar macro-
phages could not be ascertained. Varia-
tions were observed in the amounts of
mitochondria, endoplasmic reticulum,
phagosomes, lysosomes, secondary
lysosomes, and myelin figures and in
the shape of nuclei. Scanning electron
microscopy also revealed much variation
among control cells, including differ-
ences in cell shape and size, membrane
ruffling, and cytoplasmic projections.
All of the ultrastructural and morpho-
logical differences among control cells
were also seen among cells exposed to
fly ash, for all exposure periods.
The tissues of mice exposed to fly ash
for 6 weeks (short-term) and 31 weeks
(long-term) were histologically exam-
ined. Pigmented alveolar macrophages
were observed in the lungs after both
short- and long-term exposures. The
black non-birefringent pigment resem-
bled anthracotic pigment; particles that
were birefrmgent resembled silica.
Pigment was more randomly dispersed
in the lung after short-term than after
long-term exposure. In the long-term-
exposed lungs, pigment was more
prominent and tended to localize in
alveoli immediately adjacent to terminal
bronchioles. Uptake of pigment by
mediastinal lymph nodes was more
intense in long-term-exposed mice.
Other reticuloendothelial tissues (i.e.,
Peyer's patches, mesenteric lymph
nodes, spleen, and liver) did not show
evidence of fly-ash-pigment uptake.
Lymphoid accumulation associated
with fly-ash-pigment deposition was
more prominent in the long-term-exposed
mice. For both exposure times, lymphoid
accumulation is interpreted as a reaction
to the presence of fly ash pigment.
Conclusions
Fly ash in relatively high concentra-
tions had little effect on antimicrobial
and antitumor activities of lymphocytes
and macrophages. Inflammatory changes
suggestive of early pneumoconiosis
were seen in the lungs after 217 days of
exposure to fly ash.
A. Zarkower, J. Davis, F. Ferguson, andD. Stricklerare with the Pennsy/via State
University, University Park, PA 16802.
Judith A. Graham is the EPA Project Officer (see below).
The complete report, entitled "Determining Effect of Pollutants on the Immune
System," (Order No. PB 81-171 829; Cost: $9.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Ftoyal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U. S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use S300
t O
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