Report NO. 111*1-1,6061-8
(Final Report,)
INTERACTIONS OF VARIOUS AIR POLLUTANTS
ON CAUSATION OF PULMONARY DISEASE
Em/ ironmen tai Pro tection Agency
Research Triangle Park
North Carolina 27711
Attention: Dr. David Coffin
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Report No. IITRI-L6061-8
(Final Report)
INTERACTIONS OF VARIOUS AIR POLLUTANTS
ON CAUSATION OF PULMONARY DISEASE
Environmental Protection Agency
Research Triangle Park
North Carolina 27711
Attention: Dr. David Coffin
III RESEARCH INSTITUTE
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Report No. IITRI-L6061-8
(Final Report)
INTERACTIONS OF VARIOUS AIR POLLUTANTS
ON CAUSATION OF PULMONARY DISEASE
September 28, 1970 to September 27, 1972
Contract No. EHSD 71-37
IITRI Project L6061
Prepared by
Richard Ehrlich
and
James D. Fenters
of
IIT RESEARCH INSTITUTE
10 West 35th Street
Chicago, Illinois 60616
for
Environmental Protection Agency
Research Triangle Park
North Carolina, 27711
Attention: Dr. David Coffin
Copy No. October 30, 1972
IIT RESEARCH INSTITUTE
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FOREWORD
This is Report No. IITRI L6061-8 (Final Report), entitled
"Interactions of Various Air Pollutants on Causation of Pulmonary
Disease," Contract No. EHSD 71-37, IITRI Project No. L6061.
The studies were conducted by IIT Research Institute for the
Environmental Protection Agency during the period from
September 28, 1970 to September 27, 1972. The report summarizes
various studies on the effect of exposure to nitrogen dioxide,
chromium trioxide, and nickel oxide on the resistance of experi-
mental animals to bacterial and viral infection. A chronic
exposure study to determine the effects of nitrogen dioxide on
the immune system initiated during the program will continue
beyond September 27, 1972. However, all data available to
date are summarized in this report period.
In conducting the research reported, the investigators
adhered to the "Principles of Laboratory Animal Care" as
established by the National Society for Medical Research.
Dr. Richard Ehrlich served as the principal investigator
and Dr. James Fenters as the co-investigator. Other personnel
participating in the program were Mr. J. C. Findlay, serving
as the principal professional assistant, Mrs. Vivian Neary,
Mr. T. Sharp, Mr. W. Jeter, and Mrs. A. Garner. Dr. Curtis D. Port
was responsible for all histopathologic examinations, and in
conjunction with Miss Catherine Aranyi, performed the scanning
electron microscopic examinations.
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The experimental data are recorded in IITRI Logbooks
C20305, C20307, C20416, C20495, C20604, D1756, C20892, C20951,
and C21056.
Respectfully submitted,
IIT RESEARCH INSTITUTE
(2wg.
D. Fenters
Senior Microbiologist
Life Sciences Research
Approved by:
Richard Ehrlich •
Director
Life Sciences Research
JDF/kk
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ABSTRACT
Squirrel monkeys were challenged periodically with five
intratracheal injections of monkey adapted influenza A/PR/8 virus
while continuously exposed for 493 days to < 1 ppm of nitrogen
dioxide (NC^) or filtered air. Exposure to NC>2 appeared to
influence the serum neutralization (SN) antibody formation but
hemagglutination-inhibition (HI) antibody titers were similar
in control and experimental groups of monkeys. Monkeys exposed
to N(>2 produced SN antibody within 21 days after virus infection,
and after 294 days they consistently showed higher SN antibody
titers than the controls. After administration of an inactivated
A2/Taiwan vaccine, the control and experimental monkeys exhibited
similar immunogenic responses to both the A£/Taiwan and A/PR/8
influenza viruses. Throughout the exposure, no significant
differences were observed in body temperature, respiratory
function, body weight, and hematological values between control
and experimental monkeys. Histopathological examination of
lung tissue indicated slight emphysema and thickened bronchial
epithelium only in monkeys exposed to NC>2 and challenged with
influenza virus. Scanning electron microscopic examination also
suggested presence of emphysema in a monkey exposed to NC>2 and
influenza virus.
Enhanced mortality was observed among hamsters infected
with K. pneumoniae within 1 hr after intratracheal instillation
of 5 and 7.5 mg of nickel oxide (NiO). Similarily, increased
mortalities were observed in hamsters challenged with influenza
virus and exposed to NiO. However, the statistical significance
of this observation could be demonstrated only for hamsters
exposed to 5 mg of NiO 48 hr after the infectious challenge.
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Histopathologic examination of lung tissues indicated a more
severe pathologic response in hamsters challenged with the
virus and given NiO than in the various control hamsters.
Clearance studies of influenza virus from lungs indicated
little difference in virus titers between experimental and
control groups of hamsters.
Single or multiple exposures to chromium trioxide (C^O-j
aerosol did not enhance mortality in mice challenged with
K^ pneumoniae or A/PR/8 influenza virus. Furthermore, no
significant differences in mortalities were observed among
infected mice exposed to a combination of CroOo aerosol and
N02.
A study was initiated to determine the effects of
continuous, long-term exposure to NC^ on the immune response in
mice. Mice were exposed to either filtered air, 2 ppm of NOoj
or 0.5 ppm of NC^ with daily 1 hr peaks of 2 ppm NOo for 5 days
a week for 3 months prior to vaccination with an influenza
vaccine. After the vaccination, exposure to the various
environments was continued. The initial SN antibody response
was depressed in mice exposed continuously to 0.5 ppm NOo and
those held in filtered air prior to vaccination and exposed to
2 ppm or 0.5 ppm N02 after vaccination. Within 2 weeks after
vaccination the SN seroconversion rate was 100?0 in the filtered
air control mice, whereas no mice in the group exposed to
0.5 ppm of NC>2 seroconverted. Furthermore, the seroconversion
rate for the remaining experimental groups of mice ranged from
only 40 to 57%. After challenge with live A2/Taiwan influenza
virus, mortality rates and lung lesion scores indicated protec
tion in all vaccinated mice. However, more s.evere lung lesions
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were seen in vaccinated mice or control mice given saline and
held in NC>2 for 3 months prior to vaccination than in corres-
ponding groups of mice maintained in filtered air. Scanning
electron microscopic examination of lung tissues indicated
changes attributable to N02 in mice exposed to 2 ppm NC>2 for
16 weeks. Macrophage cells from mice exposed to either 2 or
0.5 ppm of NC>2 for 21 weeks showed distinct morphologic alter-
ations. In some instances, a complete deterioration of the
cells, with only some skeletal remnants remaining, was seen.
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TABLE OF CONTENTS
Page
Q
Foreword
Abstract
A. Long-Term Exposure of Squirrel Monkeys to NOp 10
B. Interaction of Nickel Oxide and Respiratory 12
Infection in Hamsters
I. Introduction 12
II. Materials and Methods 13
III. Results and Discussion 13
1. Klebsiella pneumoniae Infection and NiO 15
2. Influenza Infection and NiO 15
a. Mortality 15
bo Histopathology
c. Clearance of Virus from Lungs 27
IV. Summary 29
C. Interaction of Chromium Trioxide and 31
Respiratory Infection in Mice
I. Introduction 31
II. Materials and Methods 31
III. Results and Discussion " 32
1. Single Exposure to Ci^O-, 32
20 Combined Exposures to N0~ and Cr~0o 34
3. Multiple Cr20o Exposures 34
IV. Summary 36
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TABLE OF CONTENTS (cont.)
Page
D. Immune Response in Mice During Long-Term N©2 38
Exposure
I. Introduction 38
II. Materials and Methods 38
III. Results 44
IV. Summary 59
References 61
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A. LONG-TERM EXPOSURE OF SQUIRREL MONKEYS TO N02
The studies designed to determine the effects of long-
term exposures to nitrogen dioxide (N02) in squirrel monkeys
were described in detail in Report No. IITRI L6061-4 (Annual
Report) dated September 27, 1971. The following is a brief
summary of the results.
Squirrel monkeys were challenged periodically with five
intratracheal injections of monkey adapted influenza A/PR/8
virus while exposed 24 hr/day, 7 days/week, for 493 days to
<^ 1 ppm of N02 or filtered air. Exposure to N02 appeared to
influence the serum neutralization (SN) antibody formation.
All five monkeys exposed to N02 produced SN antibody within 21
days after virus infection, whereas only one control monkey
showed comparable response. After 294 days, the monkeys exposed
to N02 consistently showed higher SN antibody titers than the
controls. The hemagglutination-inhibition (HI) antibody titers
in the control and experimental groups of monkeys did not differ
significantly. After administration of an inactivated A2/Taiwan
influenza vaccine, the control and experimental monkeys exhibited
similar immunogenic responses to both the A2/Taiwan and A/PR/8
influenza viruses.
During the 493 day exposure no significant differences
were observed in body temperature,, respiratory function, body
weight, and hematological values between control and experimental
monkeys. Histopathological examination of lung tissue indicated
slight emphysema and thickened bronchial and bronchiolar epithel-
ium only in monkeys exposed to N0£ and challenged with the in-
fluenza virus. Scanning electron microscopic examination also
suggested the presence of emphysema in lungs of one monkey
exposed to N02 and challenged with the virus. Transmission
electron microscopic examination did not show any ultrastructural
changes which could be attributed to the experimental exposures.
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Increase in chromosome or chromatid breaks was not observed.
However, there was an unusually high number of tetraploid
metaphases in three of four monkeys exposed to NC>2 and in only
one of three controls.
The two publications resulting from work conducted during
this phase of the program were:
Antibody response in squirrel monkeys given influenza
virus and continually exposed to nitrogen dioxide.
J. D. Fenters, J. Findlay, and R. Ehrlich. Bacteriol.
Proceed., 1972.
Immunologic, physiologic, and pathologic effects of
chronic exposure to nitrogen dioxide in virus-challenged
squirrel monkeys. J. D. Fenters, J. Findlay, C. D. Port,
R. Ehrlich,, and D. L. Coffin. Submitted for publication
in Arch. Environ. Health.
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B. INTERACTION OF NICKEL OXIDE AND RESPIRATORY INFECTION
IN HAMSTERS
!_. Introduction
This study was conducted to determine the effects of
various concentrations of nickel oxide (NiO) on the resistance
of hamsters to influenza infection and bacterial pneumonia. The
parameters of interest were mortality rates, lung histopathology,
and clearance rates of virus from the lungs.
II. Materials and Methods
Experimental Animals. Male Syrian golden hamsters,
6 to 7 weeks old, were used. Throughout the experiments food
and water were provided .ad libitum..
Infectious Agents. Influenza virus A/PR/8 strain
was obtained from Dr. Max Rosenbaum, Naval Medical Research Unit
No. 4, North Chicago9 Illinois. This virus was passaged several
times in hamsters and a 2070 lung suspension was used for the
infectious challenge. Prior to use in the studies, the virus
was identified by use of specific A/PR/8 influenza virus anti-
serum obtained from the National Institutes of Health. Bacterial
challenge was made with mouse adapted Klebsiella pneumoniae
type A3 strain A-D.
Administration of NiO. Hamsters were anesthetized
with dry ice (C02) or sodium methohexital and injected intra-
tracheally with sterile 0.2 ml doses containing various concen-
trations of NiO. The NiO particles suspended in 0.5% gelatin-
saline were ^ 5p, in diameter.
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Histopathology. Upon autopsy lungs and hearts
of the hamsters were removed as a unit, and representative tissues
were fixed in a 10% phosphate buffered formalin solution. After
blocking in paraffins 4|u, thick sections were cut in a rotary
microtome and stained with hematoxylin and eosin.
Experimental Protocol. For challenge with K.
pneumoniae anesthetized hamsters were injected intratracheally
with sterile 0.2 ml of gelatin-saline containing 1, 5, 7.5, and
10 mg of NiO particles. At 1, 6, and 24 hr after the NiO in-
jections, the hamsters were challenged with the bacterial aerosol.
For influenza virus studies, anesthetized hamsters
were injected intratracheally with 0.2 ml doses of gelatin-saline
containing 1, 2.5, and 7.5 mg of NiO particles. In one group
of experiments the hamsters were challenged by intranasal in-
stillation with influenza virus at 1, 24, and 48 hr before the
NiO injection. In another group of experiments the effect of a
reverse sequence of exposures, namely intratracheal injection
of NiO followed by virus challenge, was investigated.
III. Results and Discussion
1. Klebsiella pneumoniae Infection and NiO
Results of two replicate experiments, summarized in Table
1, indicate that enhanced mortality was observed among hamsters
infected with K. pneumoniae within 1 hr after the intratracheal
instillation of 5 or 7.5 mg of NiO. However, increase in death
rates was not seen in hamsters given 1 or 10 mg of NiO nor in
those challenged with the infectious agent 6 or 24 hr after the
intratracheal instillation of NiO.
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Table 1
MORTALITY OF HAMSTERS AFTER EXPOSURE TO NiO
AND CHALLENGE WITH K. PNEUMONIAE
Interval between NiO
and K. pneumoniae
challenge, hr
1
6
24
NiO Control
NiO, mg
0* 1
D/T**
0/12
4/12
0/12
7,, D/T
0 0/12
33 0/12
0 0/12
0/12
7o
0
0
0
0
5
D/T
5/12
3/12
0/12
0/12
7o
42
25
0
0
7.
D/T
2/6
0/6
0/6
0/3
,5
7o
33
0
0
0
10
D/T
0/6
1/6
1/6
0/6
7o
0
17
17
0
0.57o gelatin-saline.
•;'"'*
D/T = Dead/Total
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2:. Influenza Infection and NiO
a. Mortality
Replicate studies for the two virus-NiO sequences
are summarized in Table 2. The results suggest enhancement of
mortalities, especially upon exposure of infected hamsters to
NiO, Death did not occur among 143 hamsters challenged with
the influenza virus only, nor in 89 hamsters after intratracheal
injection of 0.570 gelatin-saline per se. Furthermore, only 3
out of 266 hamsters died after intratracheal injection of NiO
only. However, when infected hamsters were exposed to 2.5 and
5 mg of NiO within 24 and 48 hr after the infectious challenge,
increased mortalities were observed. Because of the high death
rates observed in control hamsters challenged with influenza
virus and exposed to gelatin-saline, the statistical significance
(P < 5%) of the increase could be confirmed only for the group
of hamsters exposed to 5 mg of NiOs 48 hr after the infectious
challenge.
Intratracheal injection of NiO before the in-
fectious challenge also appeared to enhance mortality rates;
however, the increases were relatively small. In general, the
death rates observed in this exposure sequence were markedly
lower than those seen in hamsters exposed to NiO after the
infectious challenge.
b. Histopathology
At 6, 243 and 48 hr after intranasal instillation
of 1/5 LE>5Q °f A/PR/8 influenza virus, groups of hamsters were
injected intratracheally with 1, 5, and 7.5 mg of NiO, or
gelatin-saline. All dead and moribund hamsters were autospied
and tissues were examined. The surviving hamsters were sacri-
ficed at 2, 4, and 10 weeks and the tissues were obtained for
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Table 2
MORTALITY OF HAMSTERS CHALLENGED WITH INFLUENZA VIRUS
AND EXPOSED TO NiO
Interval Between
NiO, mg
Virus and NiO
hr
0*
D/T
7
la
1.
D/T
2.5
%
D/T
7
10
5
D/T
7
/o
Virus -»• NiO
NiO
NiO
NiO
1
24
48
Control
— »• Virus
1
24
48
Control
0/30
30/48
4/25
0/45
0/30
1/29
0/30
0/44
0
63
16
0
0
3
0
0
1/30
26/47
2/25
2/42
0/29
2/30
1/26
0/44
3
55
8
5
0
7
4
0
0/30
24/30
4/25
1/45
0/29
3/30
3/30
0/45
0
80
16
2
0
10
10
0
0/29
31/47
12/25
0/45
1/29
7/28
2/29
0/45
0
66
48
0
3
25
7
0
0.5% gelatin-saline
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examination. Controls represented by hamsters given 1, 5, or
7.5 mg of NiO, gelatin-saline, saline, or the virus only were
sacrificed at 2, 4, and 10 weeks after the respective treatments.
All lung tissues were subjected to histopathological examination.
Control Hamsters. After administration of saline
and gelatin-saline the pathological findings consisted of mild
congestion with a mild interstitial response of a chronic nature
(Figures 1-4). Some alveolar hemorrhage and atelectasis were
observed, but these findings were attributed to the method of
sacrifice of the animals. Hamsters challenged with the virus
only, among which no deaths occurred, showed a mild chronic
interstitial response with some congestion. The response to
instillation of 1, 5, and 7.5 mg NiO was of an acute nature
and consisted of an infiltration of polymorphonuclear cells and
lung macrophages. The extent of the pathology appeared to be
related to the amount of NiO administered (Figures 5-7). In
lungs of hamsters receiving 7.5 mg of NiO, considerable con-
solidation, some alveolar hemorrhage, mild congestion, and
marginal emphysema were observed.
Experimental Hamsters. Most of the hamsters
challenged with the virus and given NiO, gelatin-saline, and
saline, respectively, were dead one week after the infectious
challenge. At necropsy, hamsters given 1, 5, and 7.5 mg of
NiO at 6, 24, and 48 hr after the infectious challenge, showed
a mild to severe acute interstitial response. The response
consisted of interstitial infiltration of heterophils and macro-
phages, with the more severe response observed in the 7.5 mg
NiO group. Lungs of hamsters challenged with the virus and
exposed to saline or gelatin-saline and sacrificed at the same
time periods (6, 24 and 48 hr post-challenge) did not show this
type of interstitial reaction. The pathology was comparable to
that found in hamsters given gelatin-saline or saline without
the infectious agent.
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Figure 1 VIRUS - GELATIN SALINE: Gelatin saline was
administered 48 hr after challenge with virus
and the hamster was examined 1 week post virus
infection. The lung showed an acute inter-
stitial reaction with mild congestion, and
alveolar hemorrhage. Note the "thickened"
bronchial epithelium.
95x
Figure 2 VIRUS -_GELATIN SALINE: The gelatin saline
was administered 48 hr after challenge with
the virus and the hamster was examined 1 week
after the virus infection. The pleural cells
are "rounded up". No evidence of an exudate
or fibrin deposition was present.
15 Ox
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Figure 1
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Figure 3 VIRUS -_GELATIN SALINE: The gelatin saline
was administered 24 hr after the challenge
with the virus and the hamster was examined
after 1 week. Beginning adenomatosis was
observed surrounding some bronchioles.
Note the "thickened'1 epithelium.
95x
Figure 4 VIRUS - SALINE; The saline was administered
24 hr after the challenge with the virus
and the hamster was examined 4 weeks later.
Extensive adenomatosis was present. Note
the "thickened" epithelium.
95x
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Figure 3
Figure 4
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Figure 5 1 mg NiO IN GELATIN SALINE: The hamster was
examined 2 weeks after NiO administration.
The NiO particles were sparse with an
accompanying mild interstitial reaction,
mild atelectasis and some alveolar hemorrhage.
Note the normal respiratory epithelium.
95x
Figure 6 5 mg NiO IN GELATIN SALINE: The hamster was
examined 2 weeks after NiO administration.
The NiO particles were distributed mainly in
the hilus and generally surrounded bronchioles.
Note the normal respiratory epithelium.
95x
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Figure 5
Figure 6
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Figure 7 7.5 mg NiO IN GELATIN SALINE: The hamster
was examined 2 weeks after NiO administration.
The NiO is heavily concentrated in the
parenchyma with an accompanying mild to
moderate interstitial reaction. Note the
normal bronchial epithelium.
95x
Figure 8 VIRUS - 1 mg NiO IN GELATIN SALINE; The NiO
was administered 24 hr after the virus and
the hamster was examined 2 weeks later. The
pleura has greatly "thickened" with no exudate
or other evidence of inflammation.
150x
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P?wBE^S»r!S5|5ftl
L t .tt>s. *V ». JV*» Sr*( A •'*-.^X">'> -'•j'*l
Figure 7
Figure 8
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In addition to the NiO response all three groups
of hamsters showed a markedly thickened bronchiolar and bronchial
epithelium, similar to those seen in Figures 1, 3, and 4. In
some instances, epithelial hyperplasia was present as evidenced
by the number of mitotic figures visible in the basilar cells
of the epithelium. In other instances, the epithelium appeared
hyperplastic9 without the presence of mitotic figures. Thus,
the term "thickened" was applied when mitosis could not be
demonstrated. Additionally, the pleural mesothelial cells in
these hamsters appeared to "round up", (Fig. 2) and an
occasional area of adenomatosis in the vicinity of a bronchiole
was observed.
Hamsters examined at the 2 week interval included
the various controls and those given 1, 5, 7.5 mg of NiO 6 hr
after infectious challenge. The pathological changes in the
lungs were similar to those seen in the 1 week group of hamsters,
and consisted of interstitial response to the NiO, bronchial
epithelial hyperplasia9 and a pleural reaction which appeared
as a thickened pleural lining (Fig. 8). Adenomatosis was more
prominent at this time period.
A chronic interstitial reaction, with many macro-
phages containing NiO particles, was observed in lungs of
hamsters examined 4 weeks after challenge with the virus. This
response varied with the amount of NiO given as well as with the
time period elapsed after the infectious challenge. The thickened
bronchial epithelium was present in all groups. The adenomatosis
(Fig. 4) was considerably more prominent in the 24 and 48 hr
groups than in the 6 hr group. The pleural thickening was
especially prominent in the 24 and 48 hr groups, in some cases
reaching a depth of almost 1 mm.
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Hamsters from all three time groups as well as
control animals, killed 10 weeks after administration of virus,
showed lung tissue changes similar to those found in those
hamsters sacrificed 4 weeks after virus infection.
In general, an acute reaction related to the dose
of NiO was observed upon histopathological examination of lung
tissues. Furthermore, there appeared to be an additive effect
of virus and NiO, virus and gelatin-saline or virus and saline.
The effect appeared to be related to the time interval between
infectious challenge and administration of NiO or gelatin-
saline, as well as to the time elapsed after the challenge
with the virus. Thickened or hyperplastic epithelium, adeno-
matosis, and thickened pleura were seen only in hamsters
challenged with the virus in conjunction with NiO, gelatin-
saline, or saline. In addition, there was a difference between
hamsters exposed to the virus and NiO and those exposed to the
virus and gelatin-saline or virus and saline, in that the NiO
appeared to elicit an additional response above that produced
by the gelatin-saline or saline.
c. Clearance of Virus from Lungs
To determine the effects of an intratracheal in-
jection of NiO on the clearance rate of virus from lungs, groups
of hamsters were challenged with influenza virus and 24 and 48
hr later were given 5 mg NiO, gelatin-saline, or saline. Ham-
sters, challenged with influenza virus only served as controls.
Three hamsters from each group were sacrificed at 1 hr and at
1, 2, 3, 4, 5, and 6 days after the infectious challenge and
the lungs were removed. The virus in the lungs was assayed from
a 20% lung suspension titered in eggs and measured by hemagglu-
tination (HA) titers. The data, including the number of hamsters
that died during the experiment, are shown in Table 3.
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Table 3
CONCENTRATION OF INFLUENZA VIRUS IN LUNGS OF HAMSTERS
(Log10/0.l ml)
Time After Interval Between Virus Challenge, and Exposure
00
Virus Challenge3 Virus
Day Control
1 hr
1
2
3
4
5
6
2
8
6
6
6
4
4
.5
.0
.5
.5
,33
.66(1)
.33
NiO
8,0
7.45(3)*
6.75(7)
6.39(1)
=**
2
4 hr
GeljiSal
6
7
5
5
3
.5
.33
.55(5)
.75
.5
48 hr
Sal NiO Gel-Sal Sal
8
7
6
4
3
oO
.0 7.0 7o5 7.39
.0(3) 5.5(1) <4.0 6.66(1)
.5(2) 5.17(3) 5,5 5.33
.5 3.75 3.39 4.5
Number of dead hamsters in parenthesis
-.
No surviving hamsters.
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Among the hamsters challenged with the virus only,
1/23 died while in the groups given intratracheal injection of
NiO 24 and 48 hrs after the infectious challenge, 11/22 and 4/16
died, respectively. In groups of hamsters given gelatin-saline
24 and 48 hr after the infectious challenge, 5/24 and 0/16 died
respectively, and among those given saline 24 and 48 hrs after
the challenge 5/21 and 1/16 died. Thus, in this experiment the
mortality rate of hamsters challenged with virus and exposed
to NiO was approximately twice that of the controls. In general,
there was little difference in virus titers between the various
experimental and control groups. However, it appeared that the
concentration of virus was somewhat higher and the virus per-
sisted for a longer time in lungs of hamsters exposed to NiO
within 24 hr after the infectious challenge.
IV. Summary
Enhanced mortality was observed among hamsters infected
with K. pneumoniae within 1 hr after intratracheal instillation
of 5 and 7.5 mg of NiO. Similarily, increased mortalities were
observed among hamsters challenged with influenza virus and
exposed to NiO» However, because of the high death rate in
control hamsters challenged with the virus and exposed to
gelatin-saline, the statistical significance of the mortality
increase could be demonstrated only for hamsters exposed to
5 mg of NiO within 48 hr after the infectious challenge.
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Upon histopathologic examination of lung tissues,
hyperplastic epithelium, adenomatosis, and thickened pleura
were seen in hamsters challenged with the virus in conjunction
with NiO, gelatin saline, or saline. In hamsters infected with
the virus and exposed to NiO, the NiO appeared to elicit an
additional response above that produced by gelatin-saline or
saline.
Clearance studies of virus from lungs indicated little
difference in virus titers between experimental and control
groups of hamsters. Only in the group of hamsters exposed to
NiO within 24 hr after virus challenge was there a somewhat
higher concentration of virus which persisted for a longer
period of time.
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C. INTERACTION OF CHROMIUM TRIOXIDE AND RESPIRATORY
INFECTION IN MICE
!_. Introduction
This phase of the studies was conducted to determine the
effects of single and multiple exposures to chromium trioxide
(C^Oo) aerosol on the resistance of mice to influenza infection
and bacterial pneumonia. In addition, limited studies were con-
ducted to determine the effects of combined exposures to C^Oo
aerosol and nitrogen dioxide (NCO on resistance to respiratory
infections.
II. ' Materials and Methods
Experimental Animals. Specific pathogen free male
Swiss-Webster mice, 5 to 6 weeks old, were used. Food and water
were provided ad libitum.
Respiratory Agents. Influenza virus A/PR/8 was ob-
tained from Dr. Max Rosenbaum, Naval Medical Research Unit No. 4,
North Chicago, Illinois. This virus was passaged several times
in mice and a 2070 lung suspension was used for the infectious
challenge. Prior to use in the studies, the virus was identified
by use of specific A/PR/8 influenza virus antiserum obtained
from the National Institutes of Health. Bacterial challenge was
made with mouse adapted Klebsiella pneumoniae type A, strain A-D.
Administration of CroOo. Mice were exposed for
2 hr inside a plexiglass chamber to C^O- aerosol produced by
the Wright Dust Feed Mechanism. The estimated concentration of
C^Oo aerosol in the chamber ranged from 3 to 12 |j,g/liter of air.
Prior to dissemination, the C^Oo was passaged through a 100
mesh sieve to remove all particles larger than 20|i.
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NOp Exposure. Mice were held in wire cages in a
plexiglass chamber for the 2 hr exposure to 5 ppm of NC^. The
NC>2 concentration in the chamber was monitored at 30 min intervals
using the Saltzman method (Anal. Chem. 26: 1949, 1954).
Histopathology. Upon autopsy, the mice were anes-
thetized with carbon dioxide (002), the lungs and heart removed
as a unit, and representative tissues were fixed in a 107o phos-
phate buffered formalin solution. After blocking in paraffin,
4|a thick sections were cut in a rotary microtome, stained with
hematoxylin and eosin, and examined.
III. Results and Discussion
!_. Single Exposure to Cr^Oo
In a preliminary study, mice were exposed to C^Oo aero-
sol for 1, 2, and 2-1/2 hr. Microscopic examination of lungs
of the mice showed clumps of Cr^Oo particles distributed over
the bronchial tree. Some particles were seen in the lower
bronchioles with few particles found in alveolar macrophages.
A slight acute interstitial response with some congestion was
present.
In subsequent experiments mice were challenged with an
estimated one I^r of airborne A/PR/8 influenza virus and at
1, 24, and 48 hr after the infectious challenge were exposed for
2 hr to C^O-j aerosol. The results of four replicate experiments
summarized in Table 4 indicate that exposure to Cr^Oo aerosol
had no effect on the susceptibility of mice to influenza in-
fection. A second series of experiments were completed using
the reverse sequence, i.e., groups of 20 mice were exposed to
CO aerosol for 1, 24, and 48 hr before the viral challenge.
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The results of two replicate studies shown in Table 4 again
indicate that inhalation of C^O., did not enhance the suscepti-
bility of mice to the influenza infection.
Table 4
MORTALITY OF MICE CHALLENGED WITH INFLUENZA VIRUS
AND EXPOSED TO Cr203 AEROSOL
Interval Between
Mortality
Virus
Virus
Virus
Cr2°3
Cr20,
Virus
Cr203
and Cr000 , Hr
-* Cr000
Control
1
24
48
Control
— »• Virus
Control
1
24
48
Control
D/T
9/42
8/40
10/40
10/40
1/32
30/80
10/40
9/40
17/40
0/8
7o
21
20
25
25
3
38
25
22
43
0
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2. Combined Exposures to NOp and CrpOg
Two replicate experiments were carried out to determine
the effects of combined exposures to NO^ and CroO^ aerosol on
the resistance of mice to influenza infection. The infectious
challenge with the A/PR/8 influenza virus was followed 1 or 24
hr later by a 2 hr exposure to 5 ppm of NC^ which was followed
immediately by a 2 hr exposure to Cr90~ aerosol. At 7 and 14
^- J
days after the infectious challenge the surviving mice were
sacrificed and lung tissues were obtained for histopathological
examination.
The results summarized in Table 5 suggest slight enhance-
ment in mortality when mice were exposed to Cr^Oo aerosol or a
combination of NC>2 and C^O- within 24 hr after the infectious
challenge. However, the differences in mortalities between the
virus control and experimental groups of mice were not signi-
ficant at P <0.05. Mortalities did not occur among non-infected
mice exposed to N09 or C^O-, aerosol or to the combination of
the two pollutants.
3. Multiple CrpOp Exposures
To determine the effects of multiple exposures to C^O-o,
mice were exposed for 2 hr daily for 5 consecutive days to
Cr^O- aerosol. At the end of 1, 3, and 5 exposures, two mice
were sacrificed and their lungs were subjected to histopatho-
logical examination.
After one exposure, large Cr90o particles were sparsely
distributed in the alveoli and alveolar ducts and occasional
particles were seen in the macrophages. Large numbers of
particles were observed in the esophagus.
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Table 5
MORTALITY OF MICE CHALLENGED WITH INFLUENZA VIRUS
AND EXPOSED FOR 2 HR TO N02 AND 2 HR TO C^O-j AEROSOL
Treatment
Controls
Virus
N02
Cr2°3
N02 -. Cr203
1 hr*
Virus -* N02
Virus -* Cr20o
Virus — >• N02 — »• Cr20,
24 hr*
Virus -» N02
Virus -+ Cr20o
Virus -»• N02 -*• Cr20^
D/T
6/16
0/8
0/8
0/12
6/12
4/12
; 5/20
3/9
5/10
1 9/16
7o
38
0
0
0
50
33
25
33
50
56
jr.
Interval between infectious
challenge and initiation of ex-
posure to the pollutants.
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After three daily exposures, a greater number of clumped
large particles was seen in the main bronchioles. The number of
smaller individual particles lining the alveoli and respiratory
bronchioles as well as the number of phagocytized particles
within the macrophages were greater than after the single
exposure.
After five daily exposures to C^O-, aerosol even more
clumped particles were present in the alveoli and respiratory
bronchioles. Clumps of macrophages containing C^Oo particles
were more prominent even at low magnification. A mild inter-
stitial reaction to the particles was also observed.
Replicate studies were also conducted to determine the
effects of repeated exposures to C^O., aerosol on the mortality
rate of mice challenged with K. pneumoniae and A/PR/8 influenza
virus. Both experimental sequences, namely the infectious
challenge followed by exposure to Cr^O-i aerosol and vice-versa,
were included. Results of the experiments summarized in Table
6 indicate that repeated exposures to C^Oo aerosol did not
enhance the mortality rates.
IV. Summary
Single or multiple exposures to Cr90o aerosol did not
£~ -J
enhance mortality in mice challenged with K. pneumoniae or
A/PR/8 influenza virus. Similarly, no significant increases
in mortalities were observed among infected mice exposed to a
combination of Cr^Oo aerosol and NO^. Histopathological exam-
ination of lung tissues indicated distribution of Cr^O., particles
in the alveoli and alveolar ducts and presence of phagocytized
particles within macrophages. Thus, it would appear of interest
to further determine the effects of exposure to C^O- aerosol
on phagocytic activity of macrophages and on the retention of
infectious microorganisms in lungs.
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Table 6
MORTALITY OF MICE CHALLENGED WITH INFECTIOUS AGENTS
AND REPEATEDLY EXPOSED TO CO- AEROSOL
Mortality
Expt. Treatment
A. Agent
Agent
Cr2°3
Agent
Bf1 V C\
. \_>I7oUo
Agent
Cr203
L« 3T r>vj o
-»• Cr000*
Control
Control
-* Cr2°3
-»• Agent**
Control
Control
-»• Agent
Influenza Virus
D/T
12/20
0/10
13/20
12/20
0/10
7/20
70
60
0
65
60
0
35
K. pneumoniae
D/T
31/60
0/10
28/60
13/60
0/20
12/60
7o
52
0
47
22
0
20
""Influenza virus challenge was 24 hr prior to first
exposure. K. pneumoniae challenge was 1 hr prior to first
C^O^ exposure.
**A11 infectious challenges were 24 hr after final Cr,,0o
exposure.
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D. IMMUNE RESPONSE IN MICE DURING LONG-TERM NOp EXPOSURE
I. Introduction
Previous studies in our laboratories indicated that ex-
posure of squirrel monkeys to low concentrations (1 to 5 ppm)
of NC>2 affected the production of serum neutralization antibodies
(1, 2). Therefore, a more extensive study was initiated to
determine the immunological response in mice vaccinated with
a highly purified influenza virus vaccine and exposed to various
low concentrations of NC^. Parameters of interest were
hemagglutination-inhibition (HI) and serum neutralization (SN)
antibody formation, mortality rate in mice challenged with live
virus, lung histopathology, extent of lung edema, and serum
immunoglobulin levels. In addition, scanning electron micro-
scopic examination of lung tissue and macroph.age were performed.
This long-term exposure study is continuing and only data
accumulated to date are presented.
II. Materials and Methods
Experimental Animals. Specific pathogen free male
Swiss-Webster mice 4 weeks old were obtained from Charles River
Laboratories. After a two week quarantine period, the mice were
placed in the respective environmental chambers and held for
2 days before initiation of the exposures. During the study mice
were removed from the chambers for maintenance 3 times a week
for 1 hr and clean cages were provided once a week. Throughout
the experiments food and water were provided ad libitum.
Exposure to NOp. To maintain control and experi-
mental mice under similar conditions, three identical walk-in
aluminum-lined chambers (4 x 6 x 6.5 ft) were used. Ambient air
was passed through conventional fiber-glass filters before
entering the exposure chambers. Air flow through the chambers
was identical, and was at a rate of 20 changes/hour. The mean
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temperature in the chambers was 75° + 2°F.
Randomly distributed mice were housed in suspended
wire cages, which were moved periodically to various positions
on the cage racks. This permitted a thorough and unbiased ex-
posure to the experimental environments.
For NC>2 exposures, a minute amount of NOo was con-
tinuously passed from a cylinder through a stainless steel tube
into a glass mixing vessel where it was diluted and mixed with
filtered air. The mixture was then passed into the NOo exposure
chambers. To verify the homogeneity of NOo, air samples were
taken from different sections of the chamber using a 100 ml flask
as the sampler. The NOo concentration was determined and cal-
culated by the Saltzman method (3) and in addition, a Mast gas
analyzer was used for continuous monitoring of NOo.
£• ^
In one chamber used for continuous exposure to NOo,
the mean NOo concentration was 2.0 + 0.3 ppm and ranged from
1.3 to 2.8 ppm over a 7 month period. In the second chamber
in which mice were exposed to 0.5 ppm of NOo with daily 1 hr
peaks of 2.0 ppm of NOo, the actual mean concentration was 0.56
+ 0.07 of NOo with a range of 0.4 to 0.9 ppm. The mean con-
centration for the 1 hr peaks was 1.9 + 0.2 of NOo and ranged
from 1.4 to 2.4 ppm over the 7 month exposure period.
Influenza Virus Aerosol Challenge. Mouse adapted
influenza Ao/Taiwan/I/64 was obtained from Mr. Robert Bower,
Abbott Laboratories, North Chicago, Illinois. This virus was
passaged several times in mice and a 207<> lung suspension was
used in all challenge studies. Prior to use, the virus was
identified by use of specific A?/Taiwan/I/64 virus antiserum
obtained from the National Institutes of Health.
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Infectious aerosol challenge was conducted in a 350
liter plastic chamber (60 x 60 x 95 cm) installed within a micro-
biological safety cabinet. A University of Chicago Toxicity
Laboratory type atomizer was used to produce airborne particles
of 1 to 5|a mass median diameter. The virus suspension was fed
to the atomizer by a 50 ml syringe activated by a motor-driven
piston delivering 0.4 ml/min. Filtered air was supplied to
primary and secondary inlets of the atomizer at a flow rate of
approximately 33 1/min. The humidity in the chamber was main-
tained at 78 + 6% RH.
The aerosol was sampled with an all-glass impinger
(AGI-30) employing PBS with 0.2% bovine serum albumin (BSA) as
a collecting fluid. The inhaled dose of the virus was a function
of the concentration of the virus in the air, respiratory minute
volume of the mice, and the duration of exposure to the viral
aerosol (4).
For infectious challenge, groups of vaccinated mice
were placed in the aerosol chamber and exposed for 6 min to the
mouse adapted influenza virus. After challenge, the mice were
air washed for 10 min, removed from the aerosol chamber and held
for 14 days in an isolated animal room in filter-capped cages.
Non-vaccinated mice challenged with airborne influenza virus
or 0.27o BSA served as controls.
Vaccine. Chicken embryo A2/Taiwan/l/64 vaccine
(Zonomune), lot No. BP0549, was supplied by Eli Lilly and Co.,
Indianapolis. Mice were given one subcutaneous injection of
279 CCA units in 0.1 ml vaccine.
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HI and SN Titrations. Tests were performed in
duplicatate by the microtiter method (5) in disposable "V"
plates (Cooke Engineering Co., Alexandria, Va.) as described
by Davenport and Minuse (6). In all tests, 17o chicken red
blood cells were used.
For the hemagglutination-inhibition (HI) test, four
hemagglutinating units of antigen were used, all antisera were
heated at 56°C for 30 min and were treated with trypsin-periodate
to remove nonspecific inhibitors of hemagglutination.
The protocol used for serum-virus neutralization
(SN) test was similar to that described in the USPHS Require-
ments (7). Sera were heat inactivated at 56°C for 30 min,
serially diluted, and incubated for 1 hr at 4°C with an equal
volume of influenza virus. The serum-virus mixture was then
tested in 10 day old embryonated chicken eggs. The embryonated
eggs were inoculated by the allantoic route with 0.1 ml of the
virus-serum mixture, incubated at 37°C and harvested when an
EIDcQ dose of 32 to 320 was attained as indicated by hemagglu-
tinat ion of the virus control. The EIDcQ was determined by
parallel infectivity tests in eggs by using a 0.1 ml virus-
saline mixture. As controls, phosphate-buffered saline (PBS),
PBS plus normal mouse serum, and normal mouse serum plus virus
were inoculated into the eggs.
Scoring of Pulmonary Lesions. The extent of pulmonary
lesions was expressed as a percentage of the total lung con-
solidated (8). A score of 1 represented 25% lung consolidation,
2 = 50%, 3 = 75%, 4 = 100%, and a score of 5 represented death.
Lung Edema. To detect the extent of lung edema, three
pools each consisting of 3 to 5 lungs for each experimental
group were weighed immediately after removal from the mice. The
lungs were then held in a vacuum desiccator and reweighed at
24 hr intervals until no additional weight loss was apparent.
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The ratio of the wet-dry weights was used to express the extent
of edema.
Lung Tissue - SEM. Lungs were fixed by airway per-
fusion with a paraformaldehyde-glutaraldehyde fixative (9). The
mice were anesthetized and exsanguinated, and the lungs and
trachea were removed and attached to a perfusion apparatus.
Perfusion was continued for at least 2 hr with the lungs com-
pletely immersed in the fixative. Upon completion of airway
perfusion, the trachea was ligated and the lungs were floated
in fixative. The fixed tissue was washed in distilled water and
dehydrated with increasing concentrations of ethyl alcohol.
Thereafter, emyl acetate was substituted for the alcohol and
the tissue was dried by the critical point method in carbon
dioxide. The samples were mounted with an adhesive on copper
specimen holders and to render their surface conductive they
were covered by high vacuum evaporation with a thin layer of
carbon followed by gold. The specimens were examined in a JEOL
scanning electron microscope.
Alveolar Macrophages - SEM. Macrophages were ob-
tained from mice through tracheobronchial lavage by the method
developed for rabbits by Myrvik et al (10) and modified by
Coffin et al (11).
Alveolar macrophages from combined lavage fluids of
three mice were isolated from the surfactant by centrifuging at
300 xg for 10 min and subsequent repeated washing and centri-
fugation in saline. Total macrophage counts were made utilizing
a white blood cell diluting pipette and counting the cells in a
hemocytometer (11). Viability was determined by the dye ex-
clusion technique (12).
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The isolated macrophages were resuspended in PBS and
placed in plastic Petri dishes containing a glass cover slip
used as a substrate for the macrophages to attach. After 1 hr
incubation in a constant temperature bath at 30°C the glass
cover-slips were removed, rinsed with warm PBS and fixed in 170
glutaraldehyde solution. The fixed macrophages were washed
with distilled water, immediately frozen in Freon-22 and freeze-
dried in vacuum. The non-adherent macrophages remaining in the
Petri dishes were centrifuged, washed in PBS and resuspended in
I7o glutaraldehyde. After fixation the macrophages were washed
in distilled water, smeared on coverslips, immediately frozen
in liquid Freon, and freeze-dried.
For examination the coverslips were attached to metal
specimen holders and the macrophages were covered with a thin
layer of carbon followed by gold to render them conductive.
Scanning electron micrographs were taken at 5 and 25 KV, re-
spectively on a JEOL scanning electron microscope.
Experimental Protocol
Mice were exposed continuously to the following three
environmental conditions:
A. 2 ppm of N02
B. 0.5 ppm of N02 with daily 1 hr peaks of 2 ppm of
NC>2 for 5 days/week
C. filtered air
After a 2 month exposure, the mice were vaccinated by a
single subcutaneous injection of the influenza vaccine and were
held in either NC^ environment or filtered air. Groups of 14
to 20 mice were sacrificed at 2, 4, 8, and 12 weeks and the sera
were pooled in groups of two. The sera were assayed for HI and
SN antibodies, and portions of the sera were saved for detection
of immunoglobulin levels. Additional bleedings are planned at
16, 20, 24, and 28 weeks of exposure.
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To determine the protective effect of the vaccine as
measured by mortality rates and lung lesion scores, groups of
20 mice were challenged with approximately one LDcQ of A2/Taiwan
influenza virus 4 weeks after the vaccination. Additional
challenges will be made at 16 and 28 weeks post vaccination.
Table 7 outlines in detail the protocol for the entire study.
III. Results
Body Weight. Mice from two randomly selected cages
in each of the three chambers were weighed weekly. The initial
mean body weights of 22 mice in 2.0 ppm NC^ chamber, 21 in 0.5
ppm NC>2 chamber, and 30 mice in the control chamber were 30.0,
30.0, and 32.5, respectively. At the time of vaccination, 3
months after entering into the chambers, the mice exposed to
NC>2 had gained 13 g while the control mice gained 9.5 g. At 9
weeks after vaccination, all three groups of mice weighed 44.5 g
and at 16 weeks after the vaccination the mean weights were
44.2, 45.9 and 45.1 g, respectively. Thus, all groups of mice
showed a consistent weight gain throughout the study, and all
mice appeared healthy.
Serology. An HI antibody response was noted in all
groups of mice 2 weeks after vaccination (Table 8). When the
HI titers of mice exposed to filtered air were considered as
control baseline value, no significant (4-fold) differences
in HI titers were observed throughout the 12 week period.
The initial 2 week response as measured by SN anti-
body titers appeared to be depressed in mice exposed to NC^
(Table 8). The mean SN antibody titer for the control group of
mice was 1:34, whereas for mice exposed continuously to 0.5 ppm
of NC>2 or those held prior to vaccination in filtered air and
after vaccination at 2 or 0.5 ppm of NC^, the titers ranged
from <1:8 to 1:8. At the 4 and 8 weeks periods, however, the
SN titers were comparable for all groups.
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Table 7
PROTOCOL FOR IMMUNOLOGICAL STUDY IN MICE EXPOSED TO NITROGEN DIOXIDE
Ul
Exposure
Group
2.0
(n -
Pre-vaccinatton
Treatment
for 3 Months
1. Initial Ab
(n + 20)
Vaccinate
(a) A2 Vaccine
(n = 220)
2.
NO? exposure
(n = 280)
Initial Ab
(n - 20)
(b) Saline
(n = 60)
(a) A2 Vaccine
(n - 220)
Vaccination followed by
Continuous NOo Exposure
and AB Titers at 2, 4, 8,
12, 16, 20. 24. 28 Weeks
(a) Ab titer on 20 mice
given vaccine at
each of above times.
(n = 160)
(b) Do not bleed saline
mice.
(ab) Same as #1 above.
Infectious Challenge for Mortality
Study at 4, 16. 28 Weeks
(a) Challenge 20 vaccinated mice
at each of the above times.
(n = 60)
(b) Challenge the saline mice as
above.
(n = 60)
(ab) Same as #1 above.
Filtered Air
(n « 280)
(b) Saline
(n + 60)
B.
0.5 ppm
with daily
1 hr peaks
of 2.0 ppm
(n - 600)
Same groups and exposures as above but with intermittent NO 2 exposures
-------
Table 7 (Continued)
PROTOCOL FOR IMMUNOLOGICAL STUDY IN MICE EXPOSED TO NITROGEN DIOXIDE
Exposure
Group
C. Controls
(n - 900)
Pre-vaccinatton
Treatment
for 3 Months
1. Initial Ab
(n - 20)
Filtered Air
(n = 280)
2.
Initial Ab
(n = 20)
Vaccinate
(a) A2 Vaccine
(n - 220)
(b) Saline
(n = 60)
(a) A2 Vaccine
(n = 220)
Vaccination follwed by
Filtered Air Exposure
and AB Titers at 2, 4, 8,
12, 16. 20. 2A, 28 Weeks
(a) Ab titer on 20 mice
given vaccine at
each of above times.
(n = 160)
(b) Do not bleed saline
mice.
(ab) Same as #1 above.
Infectious Challenge for Mortality
Study at 4. 16, 28 Weeks
(a) Challenge 20 vaccinated mice
at each of the above times.
(n - 60)
(b) Challenge the saline mice as
above.
(n - 60)
(ab) Same as #1 above.
3.
2 ppm NOo
(n - 280)
Initial Ab
(n - 20)
(b) Saline
(n = 60)
(a) A2 Vaccine
(n = 220)
(ab) Same as #1 above.
(ab) Same as #1 above.
0.5 ppm NO?
with inter-
mittent ex-
posure
(n - 280)
(b) Saline
(n - 60)
Ab Studies: HAI and SN on pooled serum samples (pool consists of 7-10 groups of 2 mice)
Immunoglobulin assays on pooled serum samples (pool consists of 7-10 groups of 2 mice)
Total number of mice needed: 2100
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Table 8
MEAN HI AND SN RECIPROCAL TITERS IN VACCINATED
MICE EXPOSED TO N02
Reciprocal Titer at Weeks after
Vaccination
AU2,
Pre-
Vacc .
0
0.5
2
0
0
0.5
2
ppm
Post-
Vacc.
0
0.5
2
0.5
2
0
0
HIa
2
18
13
17
33
12
9
6
4
16
16
11
16
8
19
23
8
14
10
7
11
8
16
12
12
14
12
7
30
14
14
15
i_
2
34
<8
12
8
8
10
20
SN
2
23
31
46
26
26
15
40
8
23
29
26
19
36
21
21
3.
Four antigen units of egg grown A9/Taiwan virus
were used in the HI test.
b Tested against 258-320 EID5Q mice.
c Tested against 21-32 EID,-n virus.
47
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The rate of HI and SN seroconversion in vaccinated
mice held in the various environments in shown in Table 9. The
initial HI seroconversion rate was lowest in mice exposed to
2 and 0.5 ppm of NO^ before vaccination and held in filtered air
after the vaccination. The seroconversion rates were 29 and 20%
respectively. Thereafter, only mice exposed to 2 ppm of NC^
before and after vaccination, and those exposed to filtered air
before and to 2 ppm of NOj after the vaccination showed con-
sistently lower HI seroconversion rates.
The SN seroconversion rate presented an entirely
different pattern. All mice in the control group had SN anti-
body within 2 weeks of vaccination, whereas none seroconverted
among those continuously exposed to 0.5 ppm of NC^ and the sero-
conversion rate for the remaining experimental groups ranged
from only 40 to 57%. However, 4 weeks after vaccination, the
seroconversion rate ranged from 80 to 1007<> in the various experi-
mental groups. These data are in agreement with those previously
reported from our laboratories, whereby squirrel monkeys in-
jected with mouse-adapted influenza virus showed an initial
delay in SN, but not in HI antibody response.
Infectious Challenge. Vaccinated mice and control
mice injected with saline were challenged with airborne A2/Taiwan
influenza virus 4 weeks after vaccination. Mortality data
shown in Table 10 indicate that the vaccine afforded satisfactory
protection against challenge with the infectious virus in all
mice. Because of the low challenge dose, which resulted in
only 15% mortality in the control mice, the significance (p <
0.05) of the observed differences in protection between saline
and vaccinated mice could be ascertained only for the following
groups of mice:
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Table 9
RATE OF SEROCON'VERSION IN VACCINATED MICE"
f.
Pre-
Vacc.
0
0.5
2
0
0
0.5
2
Post-
Vacc.
0
0.5
2
0.5
2
0
0
HI Response \ 8, 7o
2
71
60
70
100
50
20
29
4
75
67
25
71
30
60
86
8
71
29
29
67
33
67
44
12
57
57
14
83
43
56
44
SN Response > 8, %
2
100
0
50
43
40
56
57
4
100
100
88
86
80
80
86
8
100
71
86
67
67
63
78
JU
Based on 14 to 20 mice per group.
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Table 10
MORTALITY RATE AND LUNG LESIONS IN MICE CHALLENGED
WITH INFLUENZA VIRUS 4 WEEKS AFTER VACCINATION -
N02,
Pre-
Vacc.
0
0.5
2
0
0
0.5
2
ppm
Post-
Vacc.
0
0.5
2
0.5
2
0
0
Mortality
Saline
D/T
3/20
9/20
1/19
3/20
7/20
9/20
7/20
70
15
45<**>
5
15
35
45<**>
35<**>
Vaccine
D/T
0/20
0/20
0/20
0/19
0/18
2/20
3/19
Lung Lesion Score
7o Saline
o<*>
0<*>
0
0
0(*)
10<*>
16
1.
3.
2.
2.
3.
3.
2.
95
20<**>
37
05
00<**>
40<**>
90
0
0
0
0
0
1
1
Vaccine
.30
.35
.35
.42
.44
.15
.21
(*)
(*)
(*)
(*)
(*)
(*)
(*)
(**)
(**)
' ' Significant differences (P < 0.05) between vaccinated and
corresponding saline-injected mice.
' JSignificant increase (P < 0.05) within vaccinated or saline control
groups when compared to mice held in filtered air only.
-------
a. mice exposed to filtered air,
b. mice continuously exposed to 0.5 ppm of N0?,
c. mice exposed to 0.5 ppm of N©2 before
vaccination and held in filtered air after
vaccination,
d. mice held in filtered air before vaccination
and exposed to 2 ppm of N©2 after vaccination.
When lung lesion scores were used as the indicator
of influenza infection (Table 10) all vaccinated mice showed
significantly lower scores than the corresponding control mice..
While the differences in mortality rate within the
various vaccinated groups were not significant, lung lesions
were found to be significantly more severe in mice exposed to•
0.5 ppm or 2 ppm of NC^ before vaccination and held in filtered
air after the vaccination than in the other vaccinated groups.
Thus, a 3 month exposure to NOp before vaccination
exposure to filtered air appeared to reduce the resistance to
infection of vaccinated mice as measured by lung lesions.
Several significant differences were observed when
the mortality rates were compared within the various groups of
mice given saline and exposed to the experimental conditions.
The death rates among the mice exposed to 0.5 or 2 ppm of N02
before injection of saline and then maintained in filtered
air were significantly higher (p < 0.05) than among those ex-
posed continuously to 2 ppm of N02 or to filtered air. In
addition, the mortality rates were significantly higher in
groups of mice exposed continuously to 0.5 ppm of NOj after
injection of saline than among those exposed continuously to
2 ppm of N02.
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Using lung lesion scores as another indicator of
influenza infection, it was again noted that mice exposed to
0.5 ppm of N(>2 for 3 months before saline injection and then
held in filtered air exhibited significantly (p < 0.05) higher
lung lesion socres than control mice, mice exposed continuously
to 2 ppm of NO^j and those held in filtered air prior to
saline injection and then exposed to 0.5 ppm of NO^. Inter-
estingly, mice exposed continuously to 0.5 ppm of NC^ exhibited
more severe lung lesions than control mice or mice exposed to
filtered air prior to saline injection and then held at 0.5
ppm of NO^. Therefore, it appeared that 3 months exposure to
N02 before injection of vaccine or saline resulted in the
development of more severe influenza lung lesions. In the
saline groups a higher mortality rate was found in the same
pre-exposure groups than in the control group.
Lung Edema. The wet:dry lung weight ratios are
shown in Table 11. At 2, 4, and 8 weeks after vaccination,
no significant differences were found between the filtered
air control and the experimental values. However, 12 weeks after
vaccination, i.e., approximately 6 months after initial NO^
exposure the ratios were significantly higher in mice con-
tinuously exposed to 0.5 ppm of NO^ than in the control mice.
All other values were not significantly different from the
controls.
SEM of Lung Tissue. Lung tissues from mice ex-
posed to 2 ppm of NO^ for 3 months appeared normal when
examined by the SEM. The alveoli were cup shaped and not
distended. Interalveolar septa were not broken and possessed
small interalveolar pores (Fig. 9A and 9B). Tracheobronchial
epithelium and lung tissues of mice exposed to 2 ppm of NO^
for 4 months showed changes attributable to NO^. The non-
ciliated epithelial cells of the tracheobronchial tree showed
loss of microvilli and the presence of small holes or pits
(Fig. 9C). These cells were considered to be degenerating
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Table 11
WET/DRY LUNG WEIGHT RATIO
N02,
Pre-
Vacc .
0
0.5
2.0
0
0
0.5
2.0
ppm
Post-
Vacc.
0
0.5
2.0
0.5
2.0
0 0
0
Post-Vaccination Means
2 wk
4.53
4.54
4.70
4.78
4.66
4.58
4.46
4 wk
4.68
4.50
4.54
4.52
4.39
4.58
4.87
8 wk
4.64
4.55
4.65
4.48
4.64
4.50
4.54
12 wk
4.11
4.79*
3.88
4.72
4.08
4.44
4.60
Significant difference (P < 0.05) from
other values at a given time period.
cells and were not seen in control animals exposed to filtered
air (Fig. 9D). Furthermore, the lung tissues from mice exposed
to NC>2 showed emphysematous changes which, although not numerous
could be readily recognized. The alveoli were enlarged and
coalesced to form enlarged airspaces. The alveolar septa were
lost and strands of tissue or trabeculae remained. Inter-
alveolar pores, visible in some septa, were enlarged and
irregularly shaped (Fig. 9E and 9F).
SEM of Macrophages. Macrophages isolated from
vaccinated mice after 21 weeks of continuous exposure to 2 ppm
of NC>2, 0.5 ppm of NC^ with daily peaks of 2 ppm of NC^, and
to filtered air, were examined in the SEM. The macrophages were
pooled from lung lavages obtained from 3 or 4 mice in each ex-
posure group and the total counts and viability are shown in
Table 12.
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Figure 9 SCANNING ELECTRON MICROGRAPHS OF MOUSE LUNG TISSUE:
(A & B) Lung tissue from mice exposed to 2 ppm
N02 for 3 months. (C & D) Non-ciliated epithelial
cells of the tracheobronchial tree exposed to
2 ppm N02 for 4 months and the air control,
respectively. (E & F) Lung tissue from mice ex-
posed to 2 ppm N02 for 4 months.
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.*
•*/- >* ; .
f ** •^^. ~
•fe 4ft
Figure 9
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Table 12
TOTAL COUNTS AND VIABILITY OF
ALVEOLAR MACROPHAGES -OBTAINED FROM MICE
N02, ppm
0
2
Q. 5
J.wi,aj- \_iyuiii_
x 10°
5.55
9.15
4.05
v j-au JL j. J-1- y
96.0
93.5
94.5
Scanning electron micrographs are shown in Fig. 10.
Macrophages grown and fixed directly on cover slips are shown
in Fig. 10A-10C. The intricate surface structure of a normal
alveolar macrophage with the surface membrane extentions
attaching to the glass substrate is seen in Fig. 10A. Macro-
phages obtained from mice after continuous exposure to 2 ppm
of N02 for 21 weeks showed distinct alteration in the surface
structure as seen in Fig. 10B. The changes became* more extreme
after 21 weeks of continuous exposure to 0.5 ppm of N©2 with
daily peaks of 2 ppm of N0£ (Fig. IOC), whereby a complete
deterioration of the macrophages with only some skeletal
remnants still attached to the glass was seen.
Macrophages not attached to the glass substrate but
remaining in suspension were fixed and smeared onto 'cover slips,
Normal macrophages shown in Fig. 10D showed the intricate
structure with numerous surface projections. However, the
attachment to the glass cannot be seen and the macrophage
appeared to be somewhat more rounded than the one shown in
Fig. 10A. Unattached macrophages obtained from the 2 ppm of
N02 exposure group (Fig. 10E), were more severely damaged than
those grown on the cover glass (Fig. 10B). An almost complete
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Figure 10 SCANNING ELECTRON MICROGRAPHS OF MOUSE PULMONARY
ALVEOLAR MACROPHAGE CELLS;(A, B & C) grown
onto glass substrate and fixed in situ; (D, E &
F) recovered from the cell suspension after re-
moval of the glass substrate and fixed in sus-
pension. (A & D) Normal controls, (B & E)
Macrophages obtained from animals after 21 weeks
of continuous exposure to 2 ppm N02, and (C & F)
after 21 weeks of continuous exposure to 0.5 ppm
NC>2 with a superimposed 1 hr long daily peak of
2 ppm NOo.
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absence of surface processes and the presence of numerous
fenestrae characterized the morphological condition of these
cells. The distinct difference between the appearance of these
cells and those spontaneously grown on cover slips may reflect
the ability of the macrophages to attach to glass and their
state of morphological deterioration. It is interesting to make
the same comparison for macrophages from the 0.5 ppm of NQ2
exposure group. The unattached macrophage shown in Fig. 10F,
while quite similar to the unattached cell from the 2 ppm of
NOp exposure group, is different from the macrophages isolated
from mice exposed to 0.5 ppm of NO^ that did attach to the glass
substrate (Fig. IOC). The macrophages attached to the glass
appeared in a more advanced state of deterioration than those
found in the maintenance medium.
IV. Summary
A study was initiated to determine the effects of con-
tinuous long-term exposure to NC^ on the immune response in mice,
After 3 months exposure to NO^ or to filtered air, mice were
injected with &.*/Taiwan influenza virus vaccine or saline and
placed in various experimental environments. The NO^ exposure
was either to 2 ppm or to 0.5 ppm of NC^ with daily 1 hr p^aks
of 2 ppm for 5 days a week. The initial SN antibody response
was depressed in mice exposed continuously to 0.5 ppm of NO 9
or those held in filtered air prior to vaccination and exposed
to 2 ppm or 0.5 ppm of NC^ after vaccination. The SN sero-
conversion rate was 100?0 in the control mice 2 weeks after
vaccination, whereas no mice exposed to 0.5 ppm of N0« sero-
converted. In addition, the rate for the remaining experimental
groups ranged from only 40 to 57%. No significant differences
in HI titers were noted throughout the study.
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After challenge with live Ap/Taiwan influenza virus,
mortality rates and lung lesions scores indicated protection in
all vaccinated mice. In both vaccinated mice as well as those
given saline, more severe influenza lung lesions were found in
those animals held in NC^ for 3 months prior to vaccination
than in the filtered air controls.
The wet: dry weight lung ratios were examined at 2, 4, 8,
and 12 weeks after vaccination. Lung edema was found only in
mice held for 6 months (12 weeks post vaccination. Lung edema
was found only in mice held for 6 months (12 weeks post vaccin-
ation) in 0.5 ppm of
Scanning electron microscopic examination of lung tissues
indicated that mice exposed to 2 ppm of NC^ for 4 months showed
changes attributable to NC^. The non-ciliated epithelial cells
of the tracheobronchial tree showed loss of microvilli and
emphysematous changes appeared in the lung tissues.
Scanning electron microscopic examination was made of
alveolar macrophages from vaccinated mice exposed to either
2 ppm or 0.5 ppm of N0« for 21 weeks. Macrophages from both
groups showed distinct morphologic alterations when compared
to the filtered air controls . In some cases there was complete
deterioration of the cells with only some skeletal remnants.
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REFERENCES
1. Fenters, J. D., R. Ehrlich, J. Findlay, J. Spangler,
and V. Tolkacz, 1971. Serologic response in squirrel
monkeys exposed to nitrogen dioxide. Am. Rev. Resp.
Dis. 104: 448.
2. Fenters, J. D., J. Findlay, and R. Ehrlich, 1972.
Antibody response in squirrel monkeys given influenza
virus and continually exposed to nitrogen dioxide.
Bacteriol. Proceedings, E7.
3. Saltzman, B. E. 1954. Tentative method for analysis
for nitrogen dioxide and nitric oxide in the atmos-
phere. Anal. Chem. 26; 1949.
4. Rosebury, T., 1947. Experimental airborne infection,
p. 62, The Williams & Wilkins Company, Baltimore.
5. Sever, J. L., 1962. Application of a microtechnique
to viral serological investigations. J. Immunol. 88;
320.
6. Davenport, F. M., and E. Minuse, 1964. Influenza
viruses, p. 455 In E. H. Lennette and N. J. Schmidt
(ed), Diagnostic procedures for viral and rickettsial
diseases, 3rd ed. American Public Health Association,
New York.
7. Division of Biologies Standards. National Institutes
of Health. 1947. Minimum requirements: Influenza
virus vaccines, types A and B, 6th rev.
8. Horsfall, F. L., Jr., 1939. Neutralization of epidemic
influenza virus. J. Exp. Med. 70; 209.
9. Karnovsky, M. J., 1965. A formaldehyde-gluteraldehyde
fixative of high osmolality for use in electron micro-
scopy. J. Cell. Biol. 27: 137A.
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REFERENCES (Cont.)
10. Myrvik, Q. N., E. S. Leake, and B. Fariss. 1961.
Studies of pulmonary alveolar macrophages from the
normal rabbit. J. Immunol. 86; 128.
11. Coffin, D., D. E. Gardner, R. S. Holzman, and F. J.
Wolock, 1968. Influence of ozone on pulmonary cells.
Arch. Environ. Health 16; 633.
12. Weissbecker, L., R. D. Carpenter, P. C. Luchsinger, and
T. S. Osdene. 1969. In vitro alveolar macrophage
viability. Arch. Environ. Health 18; 756,
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DISTRIBUTION LIST
Copies of this report are being distributed as follows
Copy No. Recipient
1 Environmental Protection Agency
Durham Contract Operations
Research Triangle Park
North Carolina 27711
Attn: D. K. Richmond
Contracting Officer
Mail Stop DCO-6
2-8 Environmental Protection Agency
Office of Technical Information &
Publications
Research Triangle Park
North Carolina 27711
9-25 Environmental Protection Agency
Research Triangle Park
North Carolina 27711
Attn: Dr. David Coffin
Project Officer
26 IIT Research Institute
T. R. Plonis
27 IIT Research Institute
G. Burkholder/Main Files
28 IIT Research Institute
J. D. Fenters
29 IIT Research Institute
Division L Files
30 IIT Research Institute
R. Ehrlich
31 IIT Research Institute
J. Findlay
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