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
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.  Environmental Health  Effects Research
      2.  Environmental Protection Technology
      3.  Ecological Research
      4.  Environmental Monitoring
      5.  Socioeconomic Environmental Studies
      6.  Scientific and Technical Assessment Reports (STAR)
      7.  Interagency Energy-Environment Research and Development
      8.  "Special" Reports
      9.  Miscellaneous Reports
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
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical  instrumentation and  health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.

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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

-------
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.

-------
                                  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

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