4>EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600 1-78-071
December 1 978
Research and Development
Effects of
Environmental
Contaminants on
Cell Mediated
Immunity
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
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This document is available to the public through the National Technical Informa-
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EPA-600/1-78-071
December 1978
EFFECTS OF ENVIRONMENTAL
CONTAMINANTS ON CELL MEDIATED
IMMUNITY
by
Loren D. Koller
School of Veterinary Medicine
Oregon State University
Corvallis, Oregon 97331
Grant No. R 804200
Project Officer
Richard J. Bull
Toxicological Assessment Branch
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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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. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement of recommendations for use.
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FOREWORD
Man and his environment must be protected from the adverse effects of
pesticides, radiation, noise and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment require a
focus that recognizes the interplay between the components of our physical
environment—air, water, and land. In Cincinnati, the Environmental Research
Center possesses this multidisciplinary focus through programs engaged in
0 studies on the effects of environmental contaminants on man
and the biosphere, and
" a search for ways to prevent contamination and to recycle
valuable resources.
The Health Effects Research Laboratory conducts studies to identify
environmental contaminants singly or in combination, discern their relation-
ships, and to detect, define, and quantify their health and economic effects
utilizing appropriate clinical, epidemiological, toxicological, and socio-
economic assessment methodologies.
The immune system is one of the body's primary defense systems. If a
chemical interferes with the function of this system, an increased frequency
of infectious disease may be expected in a population exposed to such chemi-
cals. Newer scientific information also suggests that the immune system
plays an important role in preventing tumor growth.
Previous work has shown that both lead and cadmium acted synergistically
with certain infectious agents. The present work has established which com-
ponents of the immune system are compromised by exposure to these metals and
suggests an approach that may be useful ^fer^^detecting similar effects by
other environmental chemicals.
R.J". Garner
Director
Health Effects Research Laboratory
m
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ABSTRACT
The effect of lead and cadmium on mouse peritoneal macrophages was
investigated. Lead and cadmium given orally to mice for 10 weeks stimulated
phagocytosis and increased acid phosphatase levels in peritoneal macrophages.
It was concluded that these two environmental contaminants activated macro-
phages and, therefore, the macrophage does not contribute to the immunosup-
pressive activity produced by lead and cadmium. The effect of lead and cad-
mium on macrophage related functions is discussed.
The immune response in aged mice was studied. Mice were exposed to 0,
13, or 1,300 ppm lead in drinking water for 18 months. The immunological
assays performed were mitogen (lippolysaccharide E_. coli, concanavalin A, and
phytohemagglutinin-P) stimulation of lymphocytes; erythrocyte-antibody (EA),
erythrocyte-antibody-complement (EAC), phagocytosis of macrophages; and EAC
of splenic lymphocytes. As measured by the majority of these assays, the low
dosage (13 ppm) of lead tended to stimulate certain immune responses (lympho-
cyte mitosis, EA, and EAC) while the high dosage (1,300 ppm) did not provoke
any appreciable alteration. The results were interpreted by comparing data
on aged mice with data on young adult mice. It was apparent from this com-
parison that the aged mice are naturally immunosuppressed. Therefore, the
results obtained from lead-exposed aged mice were unpredictable.
B-lymphocyte response after exposure to lead and cadmium was studied.
CBA/J mice were exposed to lead acetate or cadmium, chloride in the drinking
water for 10 weeks. The ability of bone marrow-derived lymphocytes (B cells)
to form rosettes was measured using an erythrocyte-anti body-complement (EAC)
assay. Fewer EAC rosettes were formed by splenic B lymphocytes from mice
exposed to lead and cadmium than by B cells from control animals. The direct
effect of these compounds on B cells could account in part for suppression of
the humoral immune response reported in previous studies.
CBA/J mice were exposed to lead and cadmium, and the mitogen stimulation
of lymphocytes was studied. Mice were given lead or cadmium in their drink-
ing water for 10 weeks. In addition, some mice were injected with BCG. Con
A, IPS, or PPD was cultured with the splenic lymphocytes. The effect of PB,
Cd, and BCG on mitogen stimulation is discussed.
This report was submitted in fulfillment of Grant No. R 8042000 by
School of Veterinary Medicine, Oregon State University, under the sponsorship
of the U.S. Environmental Protection Agency. This report covers the period
February 1, 1976, to January 31, 1978, and work was completed as of February
28, 1978.
IV
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CONTENTS
Foreword iii
Abstract iv
Tables vi
1. Introduction 1
2. Conclusions 2
3. Recommendations 5
4. Materials and Methods 6
5. Results 10
References 23
Bibliography 25
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TABLES
Number Pa9e
1 Percent EAC Rosettes Formed by Spleen Cells from Mice
Exposed to Lead or Cadmium for 10 Weeks 13
2 Percent Phagocytosis by Peritoneal Macrophages from Mice . . 13
3 Percent Viability of Peritoneal Macrophages from Mice .... 14
4 Mean Concentration of Acid Phosphatase in Peritoneal
Macrophages from Mice 14
5 Comparison of Immunological Assay Values of Control Young
Adult Mice to Aged Mice 15
6 Mean Body Weight of Mice 15
7 Mean Renal Concentrations of Lead and Cadmium 16
8 Lymphocyte Proliferation Response of Mice Exposed to Lead
and Cadmium, Macrophages Removed 17
9 Lymphocyte Proliferation Response of Mice Inoculated with
BCG and Exposed to Lead and Cadmium, Macrophages Removed . 18
10 Lymphocyte Proliferation Response of Mice Inoculated with
BCG and Exposed to Lead and Cadmium, Macrophage Not
Removed 19
11 Summary of Lymphocyte Proliferation Response of Mice Exposed
to Lead and Cadmium 20
12 Lymphocyte Stimulation Induced by LPS, Con A, and PHA from
Mice Exposed to Lead for 18 Months 21
13 Percentage of Phagocytosis by Peritoneal Macrophages .... 21
14 Percentage of EA and EAC Rosettes Formed by Peritoneal
Macrophages and Splenic Lymphocytes from Mice 21
15 Kidney, Liver, and Brain Concentration of Lead from Mice . . 22
VI
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SECTION 1
INTRODUCTION
Cadmium (Cook et al., 1975), lead (Cook et al., 1975, Hemphill et al.,
1971), methylmercury (Keller, 1975), polychlorinated biphenyls (Friend &
Trainer, 1970) and sulfur dioxide (Fairchild et al., 1972) are a few of the
environmental contaminants that have been demonstrated to be synergistic to
infectious agents in animals.
Prolonged exposure to certain environmental contaminants may suppress
the immune system of a host (Keller, 1973; Keller and Thigpen, 1973; Keller
et al., 1977; Miller and Zarkower, 1974). Exposure to lead has resulted in
reduced circulating antibody titers to infectious disease (Keller, 1973),
decreased antibody synthesis of splenic lymphocytes (Keller and Kovacic,
1974), and inhibited formation of erythrocyte-antibody-complement (EAC) (B
cell) rosettes (Keller and Brauner, 1977).
Since the macrophage has an important role as an accessory cell by co-
operating with T cells in aiding the response of B cells to antigens, the
effect of lead and cadmium on peritoneal exudate cells of young adult mice
was investigated. The cells were examined for viability, phagocytic proper-
ties and acid phosphatase content. In addition, the response of B lympho-
cytes, T lymphocytes, and macrophages in aged mice chronically exposed to
lead was investigated.
Subpopulations of lymphoid cells have been differentiated on the basis
of characteristic cell surface antigens and receptors (Gmelig-Meyling et al.,
1976; Gormus and Shands, 1975). Splenic B cells were identified by detecting
their surface receptors for the activated third component of'complement (C'3
receptor) (Nussenzweig, 1974). The procedure used to detect B cells involved
the binding of sheep erythrocytes sensitized to antibody and complement (EAC
complexes) to the C'3 receptor on B cells to form rosettes (Bianco et al.,
1970). In this study, we were concerned with the effect of lead and cadmium
on the B cell population in murine spleens.
Mitogens can induce blast transformation in normal lymphocytes. Con-
canavalin A (Con A) and phytohemaglutinin (PHA) activate thymus derived (T)
lymphocytes while lipopolysaccharide £. coli (IPS) stimulates bone marrow
derived (B) lymphocytes. Interference of mitogen proliferation induced by
Con A and PHA suggests alteration of cell mediated immune responses while
IPS indicates humoral involvement. The purpose of this study was to deter-
mine if lymphocytes collected from CBA/J mice after exposure to lead or cad-
mium would respond abnormally to mitogen stimulation.
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SECTION 2
CONCLUSIONS
The support fron this grant enabled us to examine the effects that lead
and cad-iu-^ have on the immune system of mice. Several important features
were established which revealed that lead and cadmium are generally detri-
"-ental to the inr;une response in animals.
First, in previous studies, we demonstrated that both lead and cadmium
inhibited antibody production and thus, diminished circulating antibody
tlters to antigens. The lowest dosage examined (lead - 13 ppm, cadmium - 3
ppn) resulted in significant suppression.
We next wanted to determine if lead and cadmium directly affected the B
lyrphocyte which produces antibody. Swiss Webster mice were exposed to lead
or cadmi^- in the drinking water for 10 weeks. The ability of bone marrow-
derived lymphocytes (B cells) to form rosettes was measured using an erythro-
cyte-antibody-complement (EAC) assay. Fewer EAC rosettes were formed by
splenic B lymphocytes from mice exposed to lead (130 or 1,300 ppm) and cad-
-I'jr- (30 ppm) than B cells from control animals (Table 1). The direct effect
c* these compounds on B cells could account in part for suppression of the
humoral immune response reported in previous studies.
Macrophages indirectly influence the humoral immune system and were
investigated. Lead and cadmium given orally to mice for 10 weeks stimulated
phagocytoses (Table 2) and increased acid phosphatase levels (Table 4) in
peritoneal macrophages. Since lead and cadmium primarily suppress the secon-
dary innune response, the moderately reduced primary response may be due to
direct action of these compounds on B cells. The increased ability of macro-
phages to phagocytize and contribute to immunogenicity of the antigen may not
be great enough to compensate for this inhibition. Also, other factors such
as opsonins are involved in ij^ vivo studies. Lead and cadmium may inhibit
cpsonins or other soluble factors (alpha-2-globulin, etc.) that could perhaps
oppose the stimulatory effect observed in the in vitro experiments. Finally,
the results •r'rom the B cell and macrophage studies suggest that lead and cad-
mium may impair the complement receptor but not the Fc receptor of lympho-
cytes.
Concurrent with the above investigations was a study to determine the
immune response in aged nice exposed to lead. Mice were exposed to 0, 13, or
",300 ppm lead in drinking water for 18 months. The immunological assays
performed were mitogen (lipopolysaccharide £. coli, concanavalin A, and
chytohemagglutinin-P) stimulation of lymphocytes; erythrocyte-antibody (EA),
erythrocyte-antibody-coroplement (EAC), and phagocytosis of macrophages; an
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EAC of splenic lymphocytes. As measured by the majority of these assays, the
low dosage (13 ppm) of lead tended to stimulate certain immune responses
(lymphocyte mitosis, EA and EAC) while the high dosage (1,300 ppr.) did not
provoke any appreciable alteration. The results were interpreted by compar-
ing data on aged mice with data on young adult mice (Table 5). It was appar-
ent from this comparison that aged mice were naturally immunosuppressed.
Therefore, the results obtained from lead-exposed aged mice were unpredict-
able.
Mitogens can induce blast transformation in normal lymphocytes. Con-
canavalin A (Con A) and phytohemagglutinin (PHA) activate thymus derived (T)
lymphocytes while lipopolysaccharide £. coli (IPS) stimulates bone marrow
derived (B) lymphocytes. Interference of mitogen proliferation induced by
Con A and PHA suggests alteration of cell mediated immune responses while
IPS indicates humoral involvement. Few studies concerning lymphocyte blasto-
genesis after exposure of a host to environmental pollutants have been con-
ducted. The purpose of the following study was to determine if lymphocytes
collected from CBA mice exposed to lead or cadmium would respond abnormally
to mitogen stimulation.
Mice were exposed to 3, 30, or 300 ppm cadmium as cadmium chloride or
13, 130, 1,300 ppm lead as lead acetate orally in deionized water for 70
days. The controls were given deionized water.
Lead and cadmium had little effect on lymphocyte proliferation by Con A.
Counts per minute (CPM) and relative proliferation indices (RPI) were similar
for lead, cadmium and control animals from non-BCG treated mice (Table 8) and
from BCG treated mice without removing macrophages (Table 10). However, when
lymphocytes from BCG treated animals were used after removal of macrophages,
13 ppm lead and 30 ppm cadmium resulted in a slight increase in CPM without a
noticable change in RPI (Table 9). Conversely, the low cadmium dose (3 ppm)
impaired lymphocyte stimulation by Con A as seen by a decrease in CPM and
RPI.
Lead reduced lymphocyte CPM and RPI by LPS from non-BCG and BCG treated
mice at all levels except 13 ppm in BCG mice (Tables 8 and 9). However, when
lymphocytes from BCG treated mice with macrophages were stimulated by LPS,
1,300 ppm lead enhanced CPM and RPI (Table 10). Cadmium, on the other hand,
inhibited LPS stimulation of lymphocytes in most treatments at 3 ppm but
resulted in significantly elevated CPM and RPI at 30 and 300 ppm (Tables 8,
9 and 10).
Response to PPD was similar to LPS. Lead in most cases decreased CPM
and RPI and in some instances was as much as one-half that of the controls
(Tables 1, 2 and 4). Cadmium at 3 ppm tended to inhibit CPM and RPI stimu-
lation while a slight increase in CPM and RPI often occurred at 30 and 300
ppm cadmium dosages. In the BCG treated animals, the RPI were especially
high (Table 9).
Table 11 is a summary of the mitogen studies and indicates changes for
each compound and mitogen. The interpretation of proliferative responses to
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mitogens has been difficult primarily due to assay variations since these
measures are not stable. Furthermore, mitogens may dysfunction and vary
between lots in absolute CPM which makes it difficult to combine data from
identical experiments for purposes of longitudinal monitoring and analysis.
Since assay variation and data analysis is a common problem encountered to
measure lymphocyte function with mitogens or alloantigens by tritiated
thymidine, alterations in CPM were considered significant when they were
10-15 percent larger or smaller than the control and RPI when they were
greater than 1.15 or less than .85.
A summary of the work completed in the two year study revealed that
lead (130 and 1,300 ppm) and cadmium (30 ppm) directly affect B lymphocytes
by reducing numbers of rosettes in the EAC assay. The complement receptor
site on the surface of the B cell was apparently altered by these two com-
pounds. Lead and cadmium, on the other hand, stimulated phagocytosis and
digestive functions of mouse peritoneal macrophages in vitro. However,
macrophages release several soluble factors which are regulated by T cells.
These factors which were not examined in this study often influence the
immune response.
Finally, lead, even at low dosages, tended to inhibit mitosis of B
lymphocytes while cadmium stimulated proliferation. Therefore, lead should
inhibit antibody synthesis which has been demonstrated by other studies while
cadmium may amplify the response. However, B lymphocytes are regulated by
T cells in amount of antibody produced. T cell proliferation in this study
was not altered by lead or cadmium but this technique does not directly
measure helper or suppressor activity that is responsible for B cell perfor-
mances.
An observation discovered during these studies is that strains of mice
responded differently to lead in particular. CBA mice seem to be very resis-
tant to lead since 1,300 ppm given orally for 18 months did not result in
death. Also, antibody synthesis is not affected in these animals by lead as
it is in Swiss Webster mice. We have examined other inbred strains of mice
which also have not exhibited an appreciable affect to lead. Furthermore,
inclusion bodies in the kidneys cannot be demonstrated by special strains as
they are in other animals even though they appear to be present in H & E
sections. We are currently investigating other inbred strains of mice to
determine if they respond accordingly.
The smallest dose of lead used in these studies was 13.75 ppm lead in
the drinking water, so the mice were ingesting about 0.069 mg of lead per
day. As approximately 10 percent of ingested lead is absorbed, each mouse
was actually receiving about 0.0069 mg of lead into its system each day. A
significant decrease in antibody forming cells, particularly 7A, occurred at
this dose. The adult human normally ingests 0.3 mg of lead per day and 2 mg
per day can produce toxicity.
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SECTION 3
RECOMMENDATIONS
I feel these studies have been valuable by providing data which helps to
better understand the mechanism by which two environmental contaminants (lead
and cadmium) affect the immune system of a host. These studies not only lead
us closer to describing the complete mechanism of action but also provide
dose response for each compound and assay. It is apparent that subclinical
dosages of lead and cadmium alter the immune system of a host. Furthermore,
these assays and techniques that were developed will greatly benefit future
environmental investigations.
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SECTION 4
MATERIAL AND METHODS
ANIMALS AND DIET
Swiss Webster and CBA/J mice 28 days of age were given 3, 30, or 300 ppm
cadmium as cadmium chloride or 13, 130, or 1,300 ppm lead as lead acetate
orally in deionized water for 70 days. CBA/J mice were also given 13, 130,
or 1,300 lead orally as lead acetate in deionized water for 18 months. The
controls were given deionized water. Oregon State University Rodent Chow fed
to all mice contained less than 1.12 ppm lead by analysis. There were 300
mice in each group. Mice were housed in polycarbonate cages (five per cage)
with stainless steel lids and cedar shavings for bedding. These mice were
free of apparent infections and ectoparasites during the experiments.
PHAGOCYTOSIS
Five days before termination, mice were inoculated intraperitoneally
(ip) with three ml mineral oil. Mice were killed by cervical dislocation and
peritoneal exudate cells (PEC) were obtained by injecting seven ml cold
Hank's balanced salt solution (HBSS, Flow Laboratories) containing heparin
(10 U/ml) ip using a 20 gauge 1.5 in. disposable needle. Leaving the needle
in place, the cavity was gently massaged and the fluid containing PEC was
withdrawn. The collected PEC were washed three times on cold HBSS and cen-
trifuged at 200 x g for 10 min. to remove excess oil. The washed pellet was
transferred to a clean tube and contaminating red blood cells were lysed in
0.832 NH.CL. The pellet was then diluted to six ml in culture medium com-
posed ofWdium 199, 15% fetal calf serum (FCS), 100 U/ml penicillin, 100
ug/ml streptomycin, and N-2-hydroxyethyl-piperazine-N'-2~ethanesulfonic acid
(HEPES) buffer, pH 7.2. Two-milliliter aliquots of the culture media were
transferred to Leighton tubes (Bellco) containing a cover slip (Wheaton no.
1, 9x50mm). The cells were incubated for two hrs. at 37° C and washed once
in warm phosphate-buffer saline (PBS) followed by the addition of two ml of
10% opsonized sheep red blood cells (SRBC). The preparation was incubated
one hr. at 37° C.
The cover slips were then carefully removed, rinsed in two solutions of
PBS, fixed in methanol, stained with Giemsa, mounted onto slides, and the
cells counted. A macrophatje was considered positive for phagocytosis if two
or more SRBC were seen engulfed. A separate count was taken counting cells
with three or more SRBC around the macrophage as in the form of a rosette.
Two slides of macrophages were prepared from each mouse and 200 cells were
counted per slide.
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OPSONIM
To obtain optimal phagocytosis, a 10% solution of washed SRBC's was
incubated for one hour with an equal amount of diluted opsonin (rabbit anti-
SRBC, Cappell Laboratories, Inc.). Preliminary testing showed the optimal
opsonin dilution to be 1:1600 hemagglutination units in microtiter plates.
The opsonized SRBC's were washed three times in PBS and diluted to 10% in
culture medium before addition to PEC monolayers.
ACID PHOSPHATASE
Acid phosphatase concentrations in macrophages were determined using a fi
Coleman U.V. Digital Spectrophotometer. A 0.2 ml solution containing 1 x 10
macrophages was added to 1.0 ml of a buffered substrate (Boehringer Manneheim
Corporation, Acid Phosphatase Test Kit, Catalog No. 15988) which contained
50 mM citrate buffer (pH 4.8), 550 u moles p-nitrophenylphosphate and 12.8 M
sodium chloride. This solution was kept at 37° C for 30 min. at which time
the reaction was terminated by adding 10 ml of 0.02 M sodium hydroxide.
Color change was determined with the spectrophotometer at a wavelength of 405
nm by comparing treatment samples to a reagent blank.
SPLENIC LYMPHOCYTES
At termination, mice were etherized, their spleens sterilly removed,
scissor minced, and smashed lightly through a stainless steel screen. The
spleen cells were washed three times in cold HBSS at 200 x g for ten minutes.
The spleen cell suspension was then layered on top of a Ficoll-Hypaque den-
sity gradient (Ficoll, 400,000 molecular weight, Sigma; Hypaque, 50% Win-
throp) with a specific gravity of 1.09 and spun at 400 x g for thirty
minutes. The interface band containing mononuclear cells was removed, washed
three times in cold HBSS at 200 x g for ten minutes, counted, and checked for
viabilityfiusing the trypan dye exclusion test. The cells were then diluted
to 1 x 10 /ml in culture medium (medium 199, Flow; 10% Fetal Calf Serum,
Flow; Penicillin, 100 U/ml, Squibb; Streptomycin, 100 ug/ml, Squibb; Genta-
micin, 50 ug/ml; L-Glutamine 2 mM/ml, Microbiological Associates; 2 mM HEPES
buffer, pH 7.2, Sigma; 2-Mercaptoethanol 5 x 10" M, Baker Co.).-, Additional
mononuclear cells were plated at a cell concentration of 1 x 10 in 75 cm
tissue culture flask (Corning, £25110). The cells were incubated in culture
medium at 37° C 5%CO? for two hours after which time the flasks were gently
shaken to remove the non-adherent mononuclear cells. The non-adherent cells
were spun, diluted to a concentration of 1 x 10 /ml and pipetted 0.2 ml per
microtiter well (Flow). Percent macrophages before and after removal by
adherence was determined by using the non-specific esterase stain procedure
of Koski (IN VITRO METHODS IN CELL-MEDIATED AND TUMOR IMMUNITY, ed. Bloom and
David).
MITOGEN STIMULATION
Stock mitogens were prepared in aliquots with sterile HBSS and stored at
-20° C until used. These solutions were then diluted in culture medium to
the concentration required. Optimal concentrations of mitogens were
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determined to be: lipopolysaccharide E.. coli (IPS), 055:65 (Difco), 160 ug/
ml; IPS 0111:84 (Difco), 40 ug/ml; phytohemagglutinin-P (PHA-P) (Difco), 4
ug/ml; and concanavalin A (Con A) (Sigma, IV), 2 ug/ml, and purified protein
derivative (PPD) (Connought Laboratories, LTD.), 40 ug/ml. All cells, mi to-
gen-stimulated and unstimulated, were cultured in quadruplicate for 72 hr. at
37° C in an atmosphere of 5% C0?. At 48 hr. 0.5 uCi tritiated thymidine (6.7
Ci/mmol, New England Nuclear) w5s added to each well; 24 hr. later the cells
were harvested using the Analog Digital Automatic Program Systems (Dedham,
Massachusetts) automated system and collected on glass fiber filter paper
strips (Reeve-Angel). Each filter disk was placed in a minivial (New England
Nuclear) with three ml Aquasol (New England Nuclear) scintillation fluid and
counted for 10 min. in a Packard Tri-Carb liquid scintillation counter (model
3375). The final values in counts per min. (CPM) were reported as the means
of quadruplicate samples. The stimulation index (SI) was calculated by
dividing the CPM of spleen cells with mitogen by that of spleen cells with
culture medium only. A relative proliferative index (RPI) was calculated
by dividing the net CPM of the test groups (mitogen stimulated CPM minus
background CPM) by the net CPM of the control group (mitogen stimulated CPM
minus background CPM). An RPI value greater than 1.15 was arbitrarily picked
to represent an increase from the control, and a value of less than 0.85 to
represent a decrease from the control. Values between 0.85 and 1.15 were
considered to be of no change from the control.
MACROPHAGE EAC
The PEC's were pooled according to their respective groups and charac-
terized by the EAC rosette assay. Sheep red blood cells were stored at 4° C
in Alsever's solution for no longer than seven days. Before use, the cells
were washed three times in PBS and resuspended to a final concentration of 5%
Rabbit anti-sheep red blood serum (Cappel Laboratories, Inc.) was heat inac-
tivated at 56° C for thrity min. and stored at -20° C. The hemmagglutination
titer was 1:400. Autologous mouse complement serum was obtained from blood
collected by cardiac puncture and stored at -70° C.
Sheep erythrocytes were sensitized with an equal volume of a 1:800 dilu-
tion of rabbit anti-sheep red blood cell serum for thirty min. at 37° C. The
EA complex was an equal volume of a 1:10 dilution of mouse complement serum
for thirty min. at 37° C. After two washes in VBS, the EAC complex was
resuspended in HBSS, eight times the initial volume of SRBC's. A suspension
of EA was prepared in a similar manner.
The PEC's (1.5 x 106 in 15 ml HBSS) were incubated with 0.5 ml EAC at
37° C for five min. After centrifugation at 200 x five min., the cells were
incubated in ice for one hr. without removing the supernatant (Jondal et al.,
1972). After gentle resuspension, an aliquot of cells was mixed with an
equal volume of crystal violet solution (1 mg/ml in minimal essential medium
with 10% PCS). Two hundred cells were examined microscopically in a hemacy-
tometer. Only stained cells with three or more bound erythrocytes were con-
sidered as EAC rosettes. The PEC's were similarly assayed with EA.
8
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LYMPHOCYTE EAC
Spleen lymphocyte cells derived from a Ficoll-Hypaque gradient were
similarly characterized by the EAC rosette assay and the direct B-cell
fluorescent antibody technique. Fluorescein-conjugated rabbit anti-mouse IgG
(FITC-Rxmlg) (Cappel Laboratories, Inc.) was filtered through a 0.45 urn
Millipore filteg stored at -70° C. An equal volume of spleen lymphocyte
cells (1.5 x 10° in 0.05 ml HBSS with 0.1% NaNJ was incubated with the FITC-
Rxmlg for thirty min. on ice. The cells were washed twice by centrifugation
at 200 x g at 4° C for six min. on a gradient of 2.5 ml HBSS with 30% FCS
and 0.1% NaN~. After resuspension in 0.05 ml HBSS with 0.1% NaNv the cells
were stored on ice until they were examined by fluorescent microscopy.
BCG
Six weeks before termination 75 of the lead exposed, 75 of the cadmium
exposed and 25 control mice were injected subcutaneously with BCG (Bacillus-
Calmette Guerin; University of Illinois) diluted to 1 mg with equal amounts
of CFA (Complete Freund's Adjuvant; Difco). Backs of mice were shaved and a
total of four injection sites, 0.125 ml/site, or a total of 0.5 ml was
injected. One week before termination the BCG treated mice received an addi-
tional injection of 0.25 mg BCG intraperitoneally.
TISSUE RESIDUES
The brain, liver, and one kidney from each mouse was collected at
necropsy and stored at -70° C for lead and cadmium analysis. These tissues
were analyzed for content by atomic absorption spectrophotometry using a
microcarbon furnace.
PATHOLOGY
Sections of liver, kidney, brain, lungs, heart, urinary bladder, muscle,
stomach, intestine, and spleen were corected at necropsy, fixed in 10%
buffered formalin, and stained with Harris1 hematoxylin and eosin (H&E). In
addition, kidney sections were stained by the periodic acid-Schiff (PAS),
Ziehl-Neelsen acid-fast, and Masson Trichrome methods.
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SECTION 5
RESULTS
The percent of EAC rosette formation by spleen cells from mice exposed
to lead and cadmium for 10 weeks was generally less than rosette formation by
control animals (Table 1). The number of EAC rosettes was significantly less
in animals which received 130 or 1,300 lead or 30 ppm cadmium. The impaired
resetting was not due to toxicity of lead and cadmium since the percent
viability was similar for treated and control groups.
The inhibition of EAC rosettes produced by cadmium at 30 ppm but not 300
ppm demonstrates the variable response of animals to different dosages of
cadmium which we so often observe in our laboratory. A similar response
occurred with acid phosphatase concentrations in macrophages that were col-
lected from cadmium-exposed mice (Koller and Roan, 1977).
Antibody responses to many antigens require cooperation between at least
two types of lymphocytes for optimal expression. One cell type is thymus
derived (T cells). T cells may amplify, help, or suppress B cells as well as
function as cytoxic cells. T cells do not produce antibody. The other cell
type is bone marrow derived (B cell) that mature independently of thymic
influence. B cells differentiate into antibody-producing cells and are often
influenced by T cells. A third cell type is the macrophage, which is impor-
tant as an accessory cell in cooperating with T cells and aiding B cells in
response to antigens.
Humoral antibody response to sheep red blood cells (SRBC) requires coop-
eration between B and T cells (Elliott and Haskill, 1974). Recent studies
demonstrated suppressed antibody synthesis to SRBC in mice exposed to lead or
cadmium (Koller and Kovacic, 1974; Koller et al., 1974). It was postulated
that the T helper cell may be the primary target for these two environmental
contaminants since the secondary immune response was most severely inhibited.
However, in this study, we demonstrated that these two compounds also
affected the B cells. B cells from animals exposed to lead and cadmium were
inhibited in rosette formation. This could account for the diminished anti-
body response that was reported to occur during primary immunity after expo-
sure to lead and cadmium (Keller and Kovacic, 1974; Koller et al., 1975).
Recently it was reported that lead and cadmium fed to mice stimulated both
ingestion and digestive functions of peritoneal macrophages (Koller and Roan,
1977). Studies of the effects of these contaminants on T cells are currently
in progress.
Only B cells formed rosettes by the EAC technique. This was confirmed
by examining nylon well-separated T and B cells with fluorescein-conjugated
10
-------
antiglobulin. All resetted cells fluoresced with antiglobulin, indicating
that they were B cells. T cells do not react with antiglobulin, nor do they
form rosettes by the EAC technique. The complement receptor site on the sur-
face of the B cell was apparently altered by lead and cadmium since lymphocyte
viability and percentage T and B cells were similar in all groups.
Macrophages from CBA and Swiss Webster mice exposed to lead and cadmium
were stimulated to phagocytize SRBC. Phagocytosis increased as the dose
increased in the Swiss Webster mice (Table 2) while the greatest response in
the CBA mice was at the high dose of both lead and cadmium and the low dose
of lead. When macrophages were examined for SRBC rosettes, the response was
similar for cells prepared from both treated and control animals.
Viability for macrophages was comparable for treated and control groups
(Table 3). Cell viability was considered excellent for most groups and four
of the six exposure groups had 96 to 99% viability.
Acid phosphatase levels were increased in macrophages of each cadmium
exposure and the two highest lead exposures (Table 4). The greatest concen-
tration of the enzyme occurred in macrophages from the medium dosages of both
lead and cadmium.
The body weight of mice that received 300 ppm cadmium were significantly
lower than those of animals in the other treated and control groups (Table 6).
Renal concentrations of lead and cadmium increased significantly as the metal
dosage increased (Table 7).
Lymphocytes from control and lead-exposed mice cultured in the presence
of IPS 055:B5, IPS 0111:B4, Con A and PHA-P were classified according to the
SI values (Table 12). The SI values for IPS 055:B5, IPS 0111:64, and Con A
were markedly elevated in mice exposed to 13 ppm lead compared to controls or
to mice given 1,300 ppm lead. However, the SI of PHA-P was elevated in the
mice exposed to 1,300 ppm lead compared to controls or to mice exposed to 13
ppm lead.
The ability of macrophages to phagocytize SRBC's was inhibited in mice
exposed to 13 ppm lead (Table 13). However, the EA and EAC values for macro-
phages (Table 14) and lymphocytes were slightly elevated in the same 13 ppm
lead group where as they were suppressed in the 1,300 ppm group compared to
controls. Lymphocyte viability before culturing for mitogen stimulation and
the EAC test was generally 85-95%.
Tissue residues of lead increased with an increase in dosage (Table 15).
The kidneys contained the largest quantities of lead in both exposures; larger
quantities of lead were found in the brain than in the liver in the 13 ppm
group, while the converse was found in the 1,300 ppm group.
Histopathology disclosed a wide zone of necrosis at the cortical medul-
lary junction in the kidneys of mice exposed to 1,300 ppm lead. The principle
lesion was necrosis of tubular epithelial cells. Within these necrotic areas
there were frequent granulomas composed of large mononuclear cells, occasional
11
-------
multinucleated giant cells, and fibrous connective tissue. Many intranuclear
inclusion-like bodies were identified in H&E sections, but they did not stain
well by the acidfast method.
The lesions in mice exposed to 13 ppm lead were much less severe and
were characterized by necrosis of the tubular epithelial cells at the corti-
cal medullary junctions. Granulomas were infrequent in these lesions. The
PAS stain demonstrated thickening of the glomerular basement membranes and
Bowman's capsule in the lead-exposed animals. Masson's trichrome stain
revealed a slight increase of fibrous connective tissue in the necrotic areas
of the kidneys of many of the lead-exposed mice. Spontaneous tumors were
found in four lead-exposed mice and in one control animal.
Lead and cadmium had little effect on lymphocyte proliferation by Con A.
Counts per minute (CPM) and relative proliferation indexes (RPI) were similar
for lead, cadmium and control animals when lymphocytes were examined from
non-BCG treated mice (Table 8) and from BCG treated mice without removing
macrophages (Table 10) in the lymphocyte suspensions. However, when lympho-
cytes from BCG treated animals were used after removal of macrophages, 13 ppm
lead and 30 ppm cadmium resulted in a slight increase in CPM without a notice-
able change in RPI (Table 9). Conversely the low cadmium dose (3 ppm)
impaired lymphocyte stimulation by Con A reflected by both decreased CPM and
RPI.
Lead reduced lymphocyte CPM and RPI by LPS from non-BCG and BCG treated
mice at all levels except 13 ppm in BCG mice (Tables 8 and 9). However, when
lymphocyte from BCG treated mice with macrophages were stimulated by LPS,
1,300 ppm lead enhanced CPM and RPI (Table 10). Cadmium, on the other hand,
inhibited LPS stimulation of lymphocytes in most treatments at 3 ppm but
resulted in significantly elevated CPM and RPI at 30 and 300 ppm (Tables 8,
9, and 10).
Response to PPD was similar to LPS. Lead in most cases decreased CPM
and RPI and in some instances were as much as one half that of the controls
(Tables 8, 9, and 10). Cadmium at 3 ppm tended to inhibit CPM and RPI of PPD
stimulation while a slight increase in CPM and RPI often occurred at 30 and
300 ppm cadmium dosages. In the BCG treated animals, the RPI were especially
high (Table 9).
Cell viability averaged 87 percent from non-BCG mice and slightly less
(84%) for the BCG treated animals. Most of the macrophages were removed by
the Ficoll-Hypaque density gradient but for the non-BCG and BCG without macro-
phate groups, the cells were plated in plastic flasks for two hours. Two to
three percent were present after plating. However, in the 300 ppm cadmium
group, eight percent macrophages were present before plating and three percent
after.
12
-------
TABLE 1. PERCENT EAC ROSETTES FORMED BY
SPLEEN CELLS FROM MICE EXPOSED
TO LEAD OR CADMIUM FOR 10 WEEKS +
Dose (ppm)
EAC (%)
Lead Cadmium
0 0
13 3
130 30
1,300 300
Lead Cadmium
53.5 45.1
46.5 42.9
41. 5*1 30. 9*2
41. 3*1 42.7
+ = 25 mice per group
* = Significant P < 0.05 (one way analysis of variance)
1
= LSD - 9.64
= LSD - 13.63
TABLE 2. PERCENT PHAGOCYTOSIS BY PERITONEAL
MACROPHAGES FROM MICE EXPOSED TO
LEAD OR CADMIUM FOR 10 WEEKS
Phagocytosis (%)
Dose (ppm)
Lead
0
13
130
1,300
Cadmi urn
0
3
30
300
CBA
Lead
56
67
54
72*
Cadmi urn
54
56
56
63*
SW
Lead
43
49
57*
62**
Cadmi urn
43
38
45
59*
* = Significant at P < 0.05 (t test)
** = Significant at P < 0.01 (t test)
13
-------
TABLE 3. PERCENT VIABILITY OF PERITONEAL MACROPHAGES
FROM MICE EXPOSED TO LEAD OR CADMIUM FOR
10 WEEKS
Dose (ppm)
Lead Cadmium
Macrophage viability (%)
Lead Cadmi urn
0
13
130
1,300
0
3
30
300
94
96
96
89
93
89
99
96
= Ten mice in each exposure group; 2 samples per mouse
TABLE 4. MEAN CONCENTRATION OF ACID PHOSPHATASE IN
PERITONEAL MACROPHAGES FROM MICE EXPOSED
TO LEAD OR CADMIUM FOR 10 WEEKS
Dose (ppm)
Lead Cadmium
Concentration acid phosphatase0
Lead Cadmium
0
13
130
1,300
0
3
30
300
18.37
12.81
57.63**
24.50
21.72
28.12
34.80*
28.40
0 = SI units
* = Significant at P < 0.05 (t test)
** = Significant at P < 0.05 (t test)
14
-------
TABLE 5. COMPARISON OF IMMUNOLOGICAL ASSAY VALUES
OF CONTROL YOUNG ADULT MICE TO AGED MICEC
Immunological assay
Young adult mice
Erythrocyte-antibody-complement (EAC)
Lipopolysaccharide £. coli (LPS)
Concanavalin A (Con A)
Phytohemagglutinin-P (PHA-P)
Young adult mice were 14 weeks old
TABLE 6. MEAN BODY WEIGHTS OF MICE EXPOSED TO
LEAD OR CADMIUM FOR 10 WEEKS
Aged mice
Phagocytosis by macrophages (%)
EAC lymphocyte rosettes (%)
Lymphocyte stimulation index
LPS 055:B5
LPS 0111 :B4
Con A
PHA-P
56
54
27.5
17.9
32.6
3.2
40
43
6.9
4.6
9.3
1.8
Dose (ppm)
Lead Cadmium
Body weight (g)
Lead Cadmium
0
13
130
1,300
0
3
30
300
26
27
27
24
27
25
26
18**
a = Ten mice each exposure group
** = Significant at P < 0.01
15
-------
TABLE 7. MEAN RENAL CONCENTRATIONS OF LEAD AND
CADMIUM FROM MICE EXPOSED FOR 10 WEEKS
Dose (ppm) Renal concentration (ppm)3'
Lead Cadmium Lead Cadmium
0
13
130
1,300
0
3
30
300
0.07
0.35
1.29
8.10
0.31
1.07
8.35
44.80
a Wet weight
Ten mice in each exposure group
16
-------
TABLE 8. LYMPHOCYTE PROLIFERATION RESPONSE OF MICE EXPOSED TO LEAD AND CADMIUM,
MACROPHAGES WERE REMOVED FROM THE LYMPHOCYTE SUSPENSIONS BY PLATING
Dosage
Control
Lead
13
130
1300
Cadmi urn
3
30
300
CPM1
(CV)
108,000,
(.36)4
100,000
(.23)
105,000
(.17)
119,000
(.25)
102,000
(.23)
109,000
(.12)
105,000
(.07)
(.20)
Con A
SI2
(CV)
47
(.58)
79
(.66)
64
(.39)
52
(.35)
52
(.18)
29
(.35)
24
(.19)
(.39)
RPI3
(CV)
1.01
(.40)
1.08
(.42)
1.18*
(.38)
1.04
(.45)
1.10
(.37)
1.05
(.38)
(.40)
CPM1
(CV)
38,000
(.49)
25,000
(.08)
28,000
(.22)
31 ,000
(.65)
26,000
(.12)
55,000
(.28)
*64,000
(.14)
(.28)
LPS
SI2
(CV)
15
(.27)
19
(.53)
17
(.47)
11
(.23)
13
(.04)
14
(.19)
15
(.17)
(.27)
RPI3
(CV)
0.80
(.56)
0.88
(.52)
0.95
(.64)
0.84
(.61)
1.61
(.34)
2.17
(.71)
(.56)
CPM1
(CV)
27,500
(.08)
13,000
(.24)
19,500
(.33)
18,000
(.86)
*14,500
(.05)
32,500
(.11)
32,500
(.11)
(.25)
PPD
SI2
(CV)
(.20)
12
(.71)
13
(.62)
6
(.13)
7
(.20)
6.5
(.33)
.9
(.04)
(.32)
RPI3
(CV)
0.52
(.41)
0.77
(.49)
0.66
(.93)
*0.51
(.14)
1.15
(.06)
1.22
(.22)
(.38)
= mean
= mean
3
= mean
" — ~**v*s4?^
counts per minute
stimulation index
relative proliferative
F -i r» -i *N »•» 4" f\4- v/^v^-iairt^rt
index
* = Significantly different from control population at P < 0.05
-------
TABLE 9. LYMPHOCYTE PROLIFERATION RESPONSE OF MICE INOCULATED WITH BCG AND
EXPOSED TO LEAD AND CADMIUM. MACROPHAGES WERE REMOVED FROM THE
00
LYMPHOCYTE
SUSPENSIONS BY PLATING
Dosage
(ppm)
Control
Lead
13
130
1300
Cadmium
3
30
300
CPM1
(CV)
Con A
SI2
(CV)
RPI3
(CV)
125,000, 43
(.18)4 (.53)
139,000
(.18)
133,000
(.26)
126,000
(.15)
108,000
(.28)
139,000
(.16)
128,000
(.19)
(.20)
38
(.33)
52
(.27)
56
(.30)
40
(.23)
33
(.67)
39
(.50)
(.40)
1.13
(.22)
1.09
(.29)
1.03
(.17)
0.86
(.17)
1.07
(.08)
1.03
(.16)
(.19)
CPM1
(CV)
52,500
(.48)
54,000
(.36)
41 ,000
(.26)
37,000
(.34)
49,000
(.29)
67,000
(.44)
61 ,000
(.30)
(.35)
LPS
SI2
(CV)
15
(.14)
14
(.12)
16
(.21)
15
(.12)
19
(.36)
15
(.15)
17
(.24)
(.19)
RPI3
(CV)
1.12
(.38)
0.98
(.59)
0.77
(.33)
1.16
(.56)
1.31
(.21)
1.44
(.52)
(.43)
CPM1
(CV)
9,000
(.56)
11,000
(.92)
5,000
(.74)
4,000
(.30)
7,000
(.68)
12,000
(.50)
9,000
(.29)
(.57)
PPD
SI2
(CV)
2.5
(.40)
2.4
(.44)
1.8
(.54)
1.8
(.12)
2.7
(.55)
2.5
(.42)
2.4
(.35)
(.40)
RPI3
(CV)
1.09
(.52)
0.54
(1.42)
0.40
(.36)
0.93
(.97)
1.34
(.08)
1.52
(.96)
(.72)
= mean
2
= mean
= mean
counts per minute
stimulation index
relative proliferative
index
4
= coefficient of variance
-------
TABLE 10. LYMPHOCYTE PROLIFERATION RESPONSE OF MICE INOCULATED WITH BC6 AND EXPOSED
TO LEAD AND CADMIUM. MACROPHAGES WERE NOT REMOVED FROM THE LYMPHOCYTE
SUSPENSION
BY PLATING
Dosage
(ppm)
Control
Lead
13
130
1300
Cadmium
3
30
300
CPM1
(CV)
Con A
(CV)
RPI3
(CV)
169,000. 40
(-07)4 (.29)
170,000
(.11)
181,000
(.16)
166,000
(.07)
172,000
(.10)
178,000
(.12)
177,000
(.10)
(.10)
41
(.07)
47
(.12)
36
(.21)
38
(-32)
30
(.99)
25
(.14)
(.31)
1.01
(.12)
1.07
(.13)
0.98
(.13)
1.02
(.11)
1.03
(.06)
1.03
(.10)
(.11)
CPM1
(CV)
58,000
(.35)
53,000
(.28)
54,000
(.04)
67,000
(.29)
53,000
(.33)
76,000
(.45)
78,000
(.13)
(.27)
LPS
(CV)
13
(.12)
13
(.16)
15
(.25)
14
(.12)
11
(.19)
10
(.25)
11
(.14)
(.18)
RPI3
(CV)
0.93
(.17)
1.00
(.31)
1.17
(.06)
0.90
(.15)
1.29
(.45)
1.42
(.33)
(.25)
CPM1
(CV)
11,000
(.30)
11,000
(.49)
5,500
(.63)
13,000
(.44)
10,000
(.45)
12,000
(.54)
17,000
(.50)
(.48)
PPD
SI2
(CV)
2.6
(.07)
2.5
(.36)
2.1
(.03)
2.8
(.31)
2.1
(.24)
2.3
(.27)
2.3
(.50)
(.25)
RPI3
(CV)
0.85
(.40)
0.68
(.47)
1.16
(.22)
0.77
(.42)
1.12
(.12)
1.49
(1.02)
(.44)
= mean
= mean
= mean
counts per minute
stimulation index
relative proliferative
index
= coefficient of variance
-------
TABLE 11. SUMMARY OF LYMPHOCYTE PROLIFERATION RESPONSE
OF MICE EXPOSED TO LEAD AND CADMIUM. GROUP 2
AND 3 IN ADDITION WERE INOCULATED WITH BCG WHILE
GROUP 2 ALSO HAD MACROPHAGES REMOVED FROM THE
LYMPHOCYTE SUSPENSION BY PLATING
Concanavalin A
Lead
13
130
1300
Cadmium CPM
_
-
-
3
30
300
Non-BCG
RPI
_
-
I
-
-
-
BCG
CPM
-
-
-
-
-
-
RPI
-
-
-
-
-
-
BCG w/Macs
CPM RPI
-
-
-
-
-
-
Li popolysacchari de
13
130
1300
13
130
1300
D
D
D
3 D
30 I
300 I
D
D
D
3 D
30 I
300 I
D
-
-
D
I
I
Purified Protein
D
D
D
D
-
I
~
D
D
-
I
I
-
-
D
I
I
I
-
-
I I
-
I I
I I
Derivative
-
D
D
D
I
-
-
D
D
-
I
I
D
D D
I I
D
-
I I
I = increase
D = decrease
- = no significant change
20
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TABLE 12. LYMPHOCYTE STIMULATION INDUCED BY LPS, CON A,
AND PHA FROM MICE EXPOSED TO LEAD FOR 18 MONTHS3
Lead
(ppm)
0
13
1,300
LPS 055:B5
6.9
11.1
7.9
Stimulation
LPS 0111:84
4.6
9.0b
5.7
index
Con A
9.3
12.2
7.1
PHA-P
1.8
2.0
2.7
Thirty mice
per group; LPS, 1
ipopolysacchride E.
coli; Con A,
concanavalin
A; PHA-P, phytohemagglutinin-P
Significant at P < 0.05 (one-way analysis of variance; LSD = 4.27)
TABLE 13. PERCENTAGE OF PHAGOCYTOSIS BY PERITONEAL MACROPHAGES
FROM MICE EXPOSED TO LEAD FOR 18 MONTHS
Lead Phagocytosis
(ppm) (%)
0 40
13 32b
1,300 42
a Thirty mice per group
Significant at P < 0.05 (one-way analysis of variance; LSD = 5.86)
21
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TABLE 14. PERCENTAGE OF EA AND EAC ROSETTES FORMED BY
PERITONEAL MACROPHAGES AND SPLENIC LYMPHOCYTES
FROM MICE EXPOSED TO LEAD FOR 18 MONTHS
Macrophages
Lead
(ppm)
0
13
1300
EA
(X)
53
55
50
EAC
/ Ql \
\ fO j
57
59
52
Lymphocytes
EAC
(X)
44
45
39
EA, erythrocyte-antibody; EAC, erythrocyte-antibody-complement
TABLE 15. KIDNEY, LIVER, AND BRAIN CONCENTRATIONS OF
LEAD FROM MICE EXPOSED FOR 18 MONTHS
Lead
(ppm)
Brain
Mean lead
(ppm)
Liver
Ki dney
0
13
1300
0.10
0.51
5.94
0.10
0.25
7.32
0.14
0.97
17.79
Wet weight
22
-------
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27
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-78-071
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Effects of Environmental Contaminants on Cell
Mediated Immunity
5. REPORT DATE
December 1978
issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Loren D. Koller
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
School of Veterinary Medicine
Oregon State University
Corvallis, Oregon 97331
10. PROGRAM ELEMENT NO.
ICC 614
11. CONTRACT/GRANT NO.
R804200
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory - Cinn, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT ~~
The effect of lead and cadmium on cell-mediated immunity was studied in peritoneal
macrophages, B-, and T-lymphocytes of mice. Lead and cadmium were administered in
drinking water for 10 weeks in short-term experiments and up to 18 months to deal with
immune responses in aged mice.
Lead and cadmium both tended to stimulate phagocytosis in peritoneal macrophages.
Consequently, depressed humoral immune response could not be explained on the basis of
an effect on the macrophage. The splenic B-lymphocyte response was depressed by both
lead and cadmium treatment. The direct effect of these metals on B cells could account
at least in part, for the suppression of the humoral immune response reported in
previous studies.
In long-term studies in aged mice low doses of lead (13 mg/1) tended to stimulate
certain immune responses. Results obtained with higher doses (up to 1300 mg/1) were
complicated by a natural immunosuppression in aged mice. As a consequence, no signifi-
cant alterations were observed with high doses and the impact of Pb on the immune
system in the long term cannot be predicted on the basis of this limited experimen-
tation.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Lead (metal)
Cadmium
Lymphocytes
Toxicity
Mice
Immune response
Peritoneal macrophage
13B
B. DISTRIBUTION STATEMENT
Release to public.
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
34
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE 28
«U.S GOVtRMUEKTPSmiHCOFFICE 1979 -657-060/1555
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