Inhibition of mixed leukocyte culture responses in cadmium-treated mice

ENVIRONMENTAL

RESEARCH

inhibition

27, 421-432 (1982)

of Mixed Leukocyte Cadmium-Treated

Culture Mice

Responses

in

BRIAN E. BOZELKA Department

of Medicine, Medicine,

Clinical Imn~unology Section, T~dnnr 1700 Perdido. Nr,ls Or/ems. Louisiana

Uni\,ersity 70112

School

oj

PETERM. BURKHOLDER Kidney

Disease

Institute. State of Ne,t, York Department Albany. Nen, York 12201

of Health.

Received June 28. 1981 Previous studies have documented inhibition of antibody-mediated immunity by exposure of experimental animals to heavy metals. To date, very little is known of possible effects of heavy metal ions on cellular-mediated immunity. The mixed leukocyte culture (MLC) assay was used in this study to evaluate the effects on lymphocyte blastogenic response of chronic exposure of mice to cadmium (2 mg CdCl$kg body wtiday). Spleen cells from two strains of mice treated with CdClz were unable to respond as effectively as control normal spleen cells to stimulation with allogeneic leukocytes. Furthermore, X-irradiated splenocytes from the cadmium-stressed animals were less effective than splenocytes from control normal mice as stimulator cells in the MLC reaction. This comparative decrease in T-lymphocyte reactivity could not be attributed to loss of lymphocyte viability or to decrease in ability of lymphocytes from cadmium-treated animals to incorporate tritiated thymidine. Although spleen cells from cadmium-intoxicated mice responded poorly to stimulation with allogeneic leukocytes, they were capable of responding to a mitogenic stimulus. Possible explanations for these observations are discussed.

INTRODUCTION

Recent investigations concerning suppressive effects of heavy metals on the mammalian immune system have uncovered yet another biological target for these toxic elements. It is apparent that experimental animals exposed to certain metal ions are more susceptible to bacterial or viral challenge (Bozelka and Burkholder, 1979; Exon et al., 1975; Gainer, 1974). Furthermore, cadmium or lead intoxication cause a significant reduction in splenic IgG and IgM plaque-forming cells directed against selected antigens (Koller, 1974, 1975; Bozelka et al., 1978). Although the mechanisms responsible for these observations remain unclear, it is known that several heavy metals can alter various physiological functions, such as respiration, ATPase-dependent energy production, phagocytosis, motility, and surface receptor activity, not only in macrophages (Cross et al., 1970; Loose et al., 1977, 1978; Koller and Roan, 1977) but also in B cells (Shenker et al., 1977; Koller and Brauner, 1977), polymorphonuclear leukocytes (Ward, 1973, mast cells (Chvapil, 1976), and even fibroblasts (Ward, 1975). In addition to monitoring possible immunosuppressive effects of these metals, immunobiologists are discovering that these elements may be employed as biological probes. For instance, mercury and zinc both act as nonspecific mito421 0013-9351182/020421-12$02.00/O Copyright All rights

@ 1982 by Academic Press. Inc. of reproduction in any form reserved.

422

BOZELKAAND

BURKHOLDER

gens when supplemented to lymphocyte cultures in vitro (Chvapil, 1976; Berger and Skinner, 1974; Caron et al., 1970). However, Roa et al. (1979) have indicated recently that zinc also inhibits the blastogenic response of human lymphocytes cultured with phytohemagg!utinin (PHA). Surprisingly, this suppressive effect was noted only when the cultures comprised lymphocytes from aged donors. The work described in this study examines the mixed leukocyte culture response of splenic lymphocytes from mice chronically exposed to cadmium chloride. Exposure to this metallic ion limited the ability of mouse splenocytes to respond and/or stimulate in allogenic leukocyte culture reactions. MATERIALS

AND METHODS

Animals and Cd treatment. Twenty-five BlO.A(2R) and fifteen BlO.A(3R) mice, approximately 3 months old (Immunobiology Research Center Mouse Colony, University of Wisconsin, Madison, Wise.), were given 0.1 ml intraperitoneal injections of saline containing 2 ppm CdCl, (2 mg Cd/kg body wt) for a total of 40 days. A similar number of control mice received daily O.l-ml ip injections of saline. In addition, BlO.A(SR) and BlO.A(lR) animals (congenic, with respect to the LD locus of 2R and 3R mice) were available as a source of stimulator spleen cells in the MLC assay. All mice were maintained on Purina mouse chow and water ad libitum. Cellular assays: Mixed lymphocyte culture. The in vitro mixed lymphocyte culture (MLC) assay was performed as described by Peck and Bach (1973). Excised spleens from control and Cd-treated responder (2R) and (3R) mice and from the corresponding stimulator (5R) and (1R) animals were dissociated separately into 40 cc of sterile Hanks’ balanced salt solution (GIBCO, N.Y.). The cell suspensions were centrifuged at 2OOg for 10 min. and resuspended in 5 ml of proteinfree medium (EHAA) (Click et al., 1972). Next, the splenocytes were counted and viability was determined by trypan blue dye exclusion. The responding cells were then diluted to yield a final density of 5 x IO6 cells/ml, while stimulating cells were held at 7.5 x 10” cells/ml. Treatment of the stimulating lymphocytes with 2000 rad of X irradiation (Mark 1 Model 30 irradiation, 137C)ensured blastogenesis and thymidine incorporation only among the responding cell populations. Using a Hamilton syringe (Hamilton Co., Inc., Whittier, Calif.) 0. l-ml aliquots of the appropriate responding and stimulating cell suspensions were dispersed into each flat-bottom microtiter well (Linbro microtiter plates, IS-FB-96-TC). Plates were then incubated in a humidified 5% CO,-air atmosphere at 37°C for 120 hr. At this time 2 &i [3H]TdR/well (New England Nuclear; sp act 1.9 mCi/mmole) were added to 0.05 ml serum-free EHAA for an additional 12 hr. Our preliminary studies, as well as results reported by other investigators (Peck and Bach, 1973), have demonstrated that the peak spleen MLC response in these strains occurs at Day 5 (120 hr), which was also true for cadmium-treated BlO.A(2R) and (5R) animals. The cultures were next precipitated onto Whatman glass fiber filters using a multiple automated sample harvester (MASH, Otto Hiller, Madison, Wise.) with phosphate-buffered saline, and dried for 24 hr at 37°C. The uptake of

MLC

INHIBITION

BY

Cd-TREATMENT

423

isotope was determined by liquid scintillation counting (Packard Tricarb liquid scintillation spectrometer No. 3300) and expressed as counts per minute X 10m3. The following MLC combinations were tested (Control = Cont, Cadmium = Cd, X = irradiated): Cont (2R) Cd (2R) + Cont (2R) Cd (2R) + Cont (5R) Cont (5R)

+ Cont (2R)X Cd (2R)X + Cont (5R)X Cont (5R)X + Cont (2R)X + Cd (2R)X

(Control background stimulation) (Cd background stimulation) 12 microtiter wells per mouse 12 microtiter wells per mouse 12 microtiter wells per mouse 12 microtiter wells per mouse

An identical set of experiments was also implemented with lymphocytes from Cont and Cd (3R) animals stimulated with (1R) lymphocytes. MLC data were analyzed by Student’s t test at 95% confidence limits. The percentage standard deviation was less than 20% for all MLC combinations. Since background stimulation of 2R(2R)X and Cd2R(2R)X combinations were not significantly different, the MLC data are expressed as total counts per minute. Effect of exogenous cadmium upon the t?zouse MLC. Spleen cells from BlO.A(2R) responder and BlO.A(SR) stimulator mice were prepared for a one-way MLC (Cont 2R(5R)X) as described in the previous section. However, the stimulator cells were also supplemented with various concentrations of cadmium ions such that at least one triplicate series of wells contained the following metal ionic molarities: lop4 M Cd, 1O-5 M Cd, . . . , lo-lo M Cd. The metal was added to the stimulator (5R) cells as a 10 PLVrnl aliquot of a 200~ concentrated metallic solution. Upon the addition of responding cells, the desired molarity was achieved within the wells. Lymphocyte viability was also determined by trypan blue dye exclusion for each MLC combination during 120 hr of incubation. Effect of Cd upon [“H]TdR incorporation. Again, 2R(5R)X MLC combinations were constructed in triplicate sets. The reaction was allowed to proceed for 5 days, at which time the cultures were labeled with (a) metal-free [3H]TdR, (b) [3H]TdR with lo-lo M Cd, (c) [3H]TdR with lops M Cd, (d) p?H]TdR with 10m6 M Cd, and (e) [3H]TdR with lop4 M Cd. Following a 12-hr incubation period the cultured cells were washed, harvested, and processed for the scintillation counter. Assessment ofsuppressor cell activity. Dissociated spleen cells from BlO.A(2R) cadmium-treated mice were incubated with mitomycin C (25 p&ml, 20 min at 37°C). Next, on Day 0, 5 x lo4 of these cells were added to Cont 2R(SR)X/MLC combinations prepared as described above (final density = 1 Cd: 10 Cont responding (2R) cells.) The cultures were harvested on Day 5 and processed for scintillation counting. Mitogerz ussays: Spleen cells from control and cadmium-treated mice were prepared for culture as previously outlined. Lymphoid cells (5 x 105) and various concentrations of concanavalin A (Pharmacia) or phytohemagglutinin (Type M, GIBCO) were then incubated for 48 hr at 37”C, 95% sir/5% CO,. Cultures were then pulsed with 1 &i [3H]TdR for an additional 18 hr and blastogenesis was subsequently determined by scintillation counting.

424

BOZELKAANDBURKHOLDER

::: I

2R5Rx MLC FIG. 1. MLC responses between the spleen cells from eight groups of control and cadmium-treated BlO.A(ZR) mice (responding populations) and the spleen cells of BlO.A(SR), animals (X-irradiated stimulating population). Each bar represents the response of one animal whose spleen cells were assayed in at least twelve different MLC tests. Percentage standard deviation for each bar is 20% or less. P < 0.001 for all combinations except for one Cd mouse in group 3, whose response was not significantly different from that of the control. (The numbers directly above several of the bars represent the total spleen cell count.) Starred bars = Cont 2R. open bars = Cd2+ 2R, and dotted bars = background 2R2Rx.

RESULTS

MLC Reactivity in Cadmium-Treated and Control Mice MLC responses were tested in two different inbred strains of mice, BlO.A(2R) and BlO.A(3R), which had been treated with ip injections of CdCl, (2 ppm) for at least 40 days. Figure 1 reveals that the (2R) mouse spleen cells from metalintoxicated animals were incapable of mounting an antigen (SR&induced blastogenic response comparable to that of the controls. In most instances, incorporation of PH]TdR by responder Cd lymphocytes (Cd2R(5R)x) was below the background (2R2Rx) level. However, an occasional Cd-treated animal was able to respond in the MLC assay with radiolabel readings similar to nonmetal controls. Within this group of animals there appeared to be no direct correlation between spleen size and MLC reactivity. Cadmium-treated BlO.A(3R) mice also displayed a significant repression of the in vitro MLC response (Fig. 2), however, the degree of inhibition was not as great as that noted in the cadmium-treated (2R) animals. As observed above, spleen size

MLC INHIBITION

425

BY Cd-TREATMENT

ih l

: :

3RIRx MLC FIG. 2. MLC responses between the spleen cells from five groups of control and cadmium-treated BlO.A(3R) mice (responding populations) and the spleen cells of BlO.A( lR)x animals (stimulating populations). Each bar represents the response of one animal whose spleen cells were assayed in 12 different MLC tests. Percentage standard deviation for each bar is 20% or less. P < 0.01 for all combinations except for one Cd mouse in group 5, whose response was not significantly different from that of the control. (The numbers directly above several of the bars represent the total spleen cell count.) Starred bars = Cont 3R, open bars = Cd z+ 3R. and dotted bars = background 3R3Rx.

did not correlate with the reactivity of this T-cell response. Cd-treated animals with spleens smaller than the corresponding controls did not incorporate label to a greater degree than Cd-treated mice which had splenomegaly. The response among Cd-treated mice in this test group was always greater than the background, (3R(3R),), level of stimulation. Also, at Day 5 of the MLC, the viability of Cd (2R) and (3R) mouse spleen cells was comparable to that of spleen cells from control mice. Spleen cells from Cd-treated mice ((2R), (3R)) were also incapable of serving as efficient stimulator cells in the MLC assay (Fig. 3). X-Irradiated spleen cells from Cd-treated (2R) and (3R) mice in all instances induced significantly less [3H]TdR incorporation by responding normal BlO.A(SR) and BlO.A( 1R) splenocytes than that induced by control stimulator lymphocytes in respective control reactions. Effect of Exogenous Cadmium Ions upon the Mouse MLC Various concentrations (between 1O-4 and 10-l” M) of ionic cadmium chloride were added to the supporting medium on Day one in BlO.A(2R)(5R), MLC (Fig. 4). MLC reactivity was completely inhibited at Cd levels between 10m4and 10e5 M.

426

BOZELKA

AND BURKHOLDER

0: :: L:

5R2Rx MLC IR3Rx MLC 3. MLC responses between the spleen cells from BlO.A(SR) and BlO.A( IR) mice (responding populations) and the spleen cells from control and cadmium-treated BIO.A(ZR), and BlO(3R)x animals (X-irradiated stimulating populations.) Starred bars = 5R or 1R responses induced by 2Rx or 3Rx Cont, open bars = 5R or 1R responses induced by 2Rx or 3Rx Cd”+, and dotted bars = 5R5Rx or IRlRx background. Percentage standard deviation for each bar is 20% or less. P < 0.05 except for one 5R animal in each of groups 1 and 4. FIG.

At 10e6 M, thymidine incorporation began to approach control values, while 10d7 M Cd had a negligible effect. Seventy-five percent of the cells in higher concentrations (10e4) were not viable after 24 hr, and at 5 days, very few of the cells (<3%; 10p4- 10-j M Cd) remained viable. Cd at 10m6 M reduced viability to 70% after 120 hr of culture conditions. Lower levels of cadmium in the culture medium produced no detectable effects on lymphocyte viability as determined by trypan blue dye exclusion. Effect of Cadmium upon the Incorporation of [3H]TdR Cadmium ions were added to the [3H]TdR cocktail in one of four different molarities (10p4, lO-‘j, 1O-8, lo-lo) immediately prior to the 12-hr labeling period (Fig. 5). Only the highest metal concentration, 10e4 M CdCl,, affected the uptake of the radioisotope by blastogenic spleen cells. At this level, virtually no [3H]TdR was incorporated by the cadmium-supplemented spleen cells. The inclusion of lO-‘j M Cd or less produced no adverse effect on PH]TdR incorporation. Mitogenic Responses of Cadmium-Treated Mice Spleen cells from control and Cd-treated mice were stimulated in vitro with both Con A and PHA. The results (Table 1) indicate that splenocytes from many of the

MLC INHIBITION

BY Cd-TREATMENT

427

450-

350.

N g )<

250.

3 v

150.

so-

FIG. 4. Effect of various concentrations of cadmium ions upon the MLC response resulting from 2R5Rx spleen cell combinations. Cadmium ions were added to the stimulator 5R, cells as 200x concentrated solutions immediately prior to the addition of the 2R responding cells. Percentage standard deviation of each bar is 15% or less.

metal-stressed animals responded to mitogenic stimulation comparably to those from controls. However, blastogenesis was inhibited among certain Cd-treated mice, particularly to suboptimal levels of mitogen, i.e., Con A 2 pLg/well, 0.25 &well, PHA l/400. Assessment of Suppressor

Cell Activity

in Cadmium-Treated

Mice

The possible presence of suppressor cell activity in the spleens of Cd-treated mice was evaluated by a cell mixing experiment. Control (BlO.A(2R) lymphocytes (5 x 105), supplemented with up to 10% (5 x 104) mitomycin-inactivated Cd splenocytes, responded to allostimulation without significant inhibition of the MLC response as compared to controls (Table 2). DISCUSSION

The MLC reaction is an assessment of cell-mediated immunity and detects genetic I region disparities between allogeneic lymphocyte populations. It requires the recognition of foreign surface antigens on stimulator cells by responding T lymphocytes which subsequently undergo blastogenic transformation (Bach et al., 1977). Viable macrophages, syngeneic to the responding T lymphocytes, are also required for the MLC (Mann and Abelson, 1980). These data demonstrate that in vivo cadmium-treated spleen cells from BlO.A(2R) and

428

BOZELKAANDBURKHOLDER

350.

250. (Y P

i v

150.

so-

0

1

FIG. 5. Effect of various concentrations of cadmium ions upon the incorporation of rH]TdR into MLC-generated blastogenic spleen cells. Cadmium ions were added to the rH]TdR-enriched medium immediately before the spleen cells were labeled (Day 5 of the MLC response). Percentage standard deviation is 15% or less.

BlO.A(3R) mice displayed severely diminished MLC reactivity. Not only were the splenic lymphocytes from Cd-treated mice unable to function as responder cells in MLC reactions, but they also were inefficient stimulators in MLC (i.e., Cont 5R (Cd2RJ) (Fig. 3). Similarly, both cadmium-treated BlO.A(2R,) and (3R.J lymphocytes exhibited this functional defect. Our previous study, which examined antibody-mediated immune responses in cadmium-treated mice (Bozelka and Burkholder, 1979), revealed several interesting alterations in the splenic morphology of these animals, which may be pertinent to the above results. First, many of the mice exhibited impressive splenomegaly, and histological examination confirmed a “loss of normal splenic architecture.” Most obvious was the appearance within the red and white pulp compartments of increased numbers of large blastoid cells. Second, immune adherence (with C4-coated sheep erythrocytes) and rosetting (with IgG-coated SRBC) assays demonstrated positive adherence of the indicator cells throughout the spleen tissue. The location of these Fc and complement receptor positive cells therefore paralleled that of the large blastoid cells. Finally, immunofluorescence preparations suggested that many of these cells also harbored surface immunoglobulins (IgG and IgM). Thus it is conceivable that dilution of responding T

MLC

INHIBITION

TABLE MITOGEN

RESPONSES

429

BY Cd-TREATMENT 1

OF CONTROL

AND

Cd-TREATED

Stimulation” Mitogen

Dilution

Con A (&well)

PHA”

(cpm)

Cont

Cd

2.0 1.0 0.5

63,320 162,239 169,938 129,609

t t -c ”

0.25

138,659 151,060

t 3,696 + 7,867

11200

119,411 107,045 115,840 120,695

t + + ?

l/400

U Each value represents D PHA = Type M, final

MICE

the mean cpm of four microtiter dilution 11200 or 11400 per well.

9,440 12,385 1,107 3,207

15,530 2,223 6,527 5,986

wells

per animal

27,261 153.630 123.994 120,701 30,171 82.474 151.925

+ 2 k + 2 k ”

4,603 20,860 4,168 8,954 6,259 2.735 7,789

177,794 60.718 122,623 24,313 83.000

i + ‘I k k

3,726 11,456 20,220 2,989 6,786

? SEM

lymphocytes by the non-T cells responsible for the apparent splenomegaly among Cd-treated mice might explain the altered MLC responses. However, equivalent inhibition of MLC was noted in Cd-treated animals whose spleen size was comparable to controls (Figs. 1 and 2). Another theoretical consequence of cadmium exposure may be the selective elimination of T lymphocytes responsive to allostimulation. Indeed, a very recent report has suggested that mercury can alter T-cell mitogenic activity and suppressor cell function via a direct effect on lymphocytes (Weening et al., 1981). Indirect evidence, obtained through preliminary mitogenic assays (Table l), confirmed the presence and blastogenic capability of T lymphocytes in Cd-treated mice. From these initial data it appears though, that a defect in mitogenesis, particularly at suboptimal mitogen levels, may also exist, and the possibility is currently being investigated. In addition, future studies will compare the proportions and functional integrity of T and B lymphocytes, as well

ASSESSMENT

-

OF SUPPRESSOR

MLC

combination

Cont

2R5Rx

2R5Rx 2R5Rx

2

CELL

ACTIVITY

IN Cd-TREATED

MICE

Stimulation

(cpm)”

22,747

Cd 2R5Rx Cd 2R5Rx Cont Cont

TABLE

+ 892

12.922 + 446 14,059 ” 312 + Cdb f Cd

22,699 20,850

r 543 k 672

” Each value represents the mean stimulation of six microtiter wells per MLC combination 6 5 x IO4 mitomycin-treated Cd spleen cells were added to each control MLC.

+ SEM.

430

BOZELKA

AND

BURKHOLDER

as other cell types, within the lymphoid organs of cadmium-treated and control mice. Furthermore, the apparent lack of T-lymphocyte MLC reactivity was not due to disproportionately high cell death among cultured spleen cells from Cd-treated mice. After 5 days of continuous culture, viability tests revealed no difference in trypan blue dye exclusion between cells from metal-treated and control animals. However, mouse spleen cells did prove to be sensitive to rather high in vitro concentrations of cadmium ions. At a concentration of lo-” M Cd, lymphocyte viability was reduced to less than 25% after 24 hr of culture. After 5 days, mortality had increased to 98% at 1Oe1 M and 10e5 M Cd, while inclusion of 1O-6 M Cd proved to be much less cytotoxic (70% viability at 120 hr of culture). Certainly, these results explain the inhibitory effect of supplemented cadmium ions on control MLCs noted in Figs. 4 and 5. Flame atomic absorption spectrophotometric analysis of tissue culture media obtained after 72 hr of continuous incubation of splenocytes from metal-treated mice revealed ionic cadmium levels below 1OV M Cd. This level of residual concentration of metal did not interfere with [3H]TdR incorporation or cell viability in the MLC reaction and therefore Cd was not acting as an in vitro toxin in Cd(Cont), combinations. Previous studies documenting splenomegaly, as well as deficient antibody responses in metal-stressed mice (Bozelka et nl, 1978), could raise the question of suppressor cell activation in the spleens of these animals (Oehler et al, 1977; Baird and Kaplan, 1977). Indeed, the dramatic reduction in MLC reactivity among Cd-treated mice might be explained on this basis. The addition of up to 5 x lo4 mitomycin C-treated Cd spleen cells to normal control 2RSRx MLCs (Table I), however, did not result in any significant inhibition of this response. The inability of Cd-treated spleen cells to stimulate in MLC as effectively as controls suggests that the metal could be altering or masking cell surface histocompatibility glycoproteins. Such interactions might prevent significant access of responding lymphocytes or macrophages to these proteins, thereby inhibiting allostimulation. Alternatively, surface-bound metal ions on stimulating cells could depress MLC responses by a direct effect on the responding population. Biochemical differences in these glycoproteins may also affect Cd-lymphocyte interactions, thereby rendering certain allotypes more susceptible to the metal ions. Interestingly, this study has shown that spleen cells from BlO.A(3R) Cd-stressed mice were more responsive in the MLC than congeneic Cd(2R) metal-treated animals when compared to controls. In a recent study, Cd was shown to be actively absorbed by pulmonary alveolar macrophages (Hart, 1978). Preliminary work in our laboratory employing ultrastructural cytochemistry has indicated that splenic macrophages and lymphocytes also take up the metal. However, at this time the specific cellular components participating in MLC reactivity which are affected by Cd remain unidentified. Proposed future investigations will employ cell substitution experiments to determine which immunocompetent cell is deficient in Cd-treated animals, and which cell component(s) is altered by the metal. Finally, our results are in contrast to those recently reported by Koller and Roan (1980). These authors found that neither lead, methyl mercury, nor cadmium significantly altered MLC responses in mice (all metals at certain concentrations

MLC INHIBITION

BY Cd-TREATMENT

431

stimulated MLC). However, their animals were fed the heavy metals and route of administration may certainly affect the interaction between metals and the immune system, In addition, our work suggests that Cd-induced inhibition of MLC may vary between inbred strains, which may further explain the discrepancy between these data (Koller and Roan exposed DBA/U mice). This hypothesis is further supported by Lawrence (1981), who demonstrated significant suppression of MLC in CBA/J mice which were fed lead acetate. Thus, cadmium, in addition to being a potent inhibitor of antibody-mediated immunity, may also deleteriously affect the cellular-mediated immune system. Such an interaction could alter host responses to fungal and viral infections as well as to carcinogenesis. ACKNOWLEDGMENTS The authors wish to thank Drs. John E. Salvaggio and Christopher F. Bryan for their helpful suggestions in preparing this manuscript. The expert secretarial assistance of Mrs. Gail White is greatly appreciated.

REFERENCES Bach, F. H., Kuperman, 0. J., Sollinger, H. W., Zarling, J. M., Sondel, P. M., Alter. B. J., and Bach, M. L. (1977). Cellular immunogenetics and LD-CD collaboration. Transplant Proc. 9, 859. Baird, L. G., and Kaplan, A. M. (1977). Macrophage regulation of mitogen-induced blastogenesis. Cell. Immltnol. 28, 22. Berger, N. A., and Skinner, A. M. (1974). Characterization of lymphocyte transformation induced by zinc ions. J. Cell. Bio[. 61, 45. Bozelka, B. E., and Burkholder. P. M. (1979). Increased mortality of cadmium-intoxicated mice infested with BCG strain of Mycobacterium bovis. J. Reticll/oendothrl. SW. 26, 229. Bozelka, B. E., Burkholder, P. M., and Chang, L. W. (1978). Cadmium. a metallic inhibitor of antibody-mediated immunity in mice. Environ. Rrs. 17, 390. Caron, G. A., Poutala. S.. and Provost, T. T. (1970). Lymphocyte transformation induced by inorganic and organic mercury. ht. Arch. Allergy 37, 76. Chvapil, M. (1976). Effect of zinc on cells and biomembranes. Med. Clit~. North Amer. 60, 799. Click, R. E., Benck, L., and Alter, B. J. (1972). Immune responses in vitro. I. Culture conditions for antibody synthesis. Cell. Imm~nol. 3, 264. Cross, C. E., Ibrahim, A. M., and Mustafa, M. G. (1970). Effect of cadmium on respiration and ATPase activity of the pulmonary alveolar macrophage: A model for the study of environmental interference with pulmonary cell function. Bzriron. Rrs. 3, 512. Exon. J., Patton, N.. and Keller. L. (1975). Hemamitiasis in cadmium-exposed mice. Arch. En\irorl. Health

30, 463.

Gainer, J. (1974). Lead aggravates viral disease and represses the antiviral activity of interferon inducers. O~viron. Heulfh Perupect. 7, 113. Hart, B. A. (1978). Transport of cadmium by the alveolar macrophage. J. R~ticlr/o~ndotl~~/. SW. 24, 363. Keller, L. D. (1974). Decreased antibody formation in mice exposed to lead. Nature (London) 250, 148. Keller, L. D. (1975). Antibody suppression by cadmium. Arch. Environ. Health 30, 598. Keller, L. D., and Brauner, J. A. (1977). Decreased B-lymphocyte response after exposure to lead and cadmium. Toricol. App/. Pharmacol. 42, 62 I. Keller, L. D., and Roan, J. G. (1977). Effects of lead and cadmium in mouse peritoneal macrophages. .I. Reticuloendothel. Sot. 12, 1. Koller. L. D., and Roan, J. G. (1980). Response of lymphocytes from lead, cadmium, and methyl mercury exposed mice in the mixed lymphocytic culture. J. Environ. Pathd. Toxicol. 4, 393.

432

BOZELKA

AND

BURKHOLDER

Lawrence, D. A. (1981). In viva and in vifro effects of lead on humoral and cell mediated immunity. Infect. Immun. 31, 136. Loose, L. D., Silkworth, J. B., and Warrington, D. (1977). Cadmium-induced depression of the respiratory burst in mouse pulmonary alveolar macrophages, peritoneal macrophages and polymorphonuclear neutrophils. Biochem. Biophys. Res. Commun. 79, 326. Loose, L. D., Silkworth, J. B., and Simpson, D. W. (1978). Influence of cadmium on the phagocytic and microbicidal activity of murine peritoneal macrophages, pulmonary alveolar macrophages and polymorphonuclear neutrophils. Infect. Immun. 22, 378. Mann, D. L., and Abelson, L. (1980). Monocyte function in mixed lymphocyte reactions. Cell. Immunol.

56, 357.

Oehler, R. J., Herberman, R. B., Campbell, D. A.. and Djeu, J. A. (1977). Inhibition of rat mixed lymphocyte cultures by suppressor macrophages. Cell. Immunol. 29, 238. Peck, A. B., and Bach, F. H. (1973). A miniaturized mouse mixed lymphocyte culture in serum free and mouse serum supplemented media. J. Immutd. Methods 3, 147. Roa, K. M. K., Schwartz, A, and Good, R. A. (1979). Age dependent effects of zinc on the transformation response of human lymphocytes to mitogens. Cell. Immunol. 42, 270. Shenker. B. J., Mataruzzo, W. J., Hirsch, R. J. and Gray, I. (1977). Trace metal modification of immunocompetence. Cell. Immunol. 34, 19. Ward, P. A. (1975). Suppressive effects of metal salts on leukocyte and fibroblast function. J. Reticuloendothel.

Sot.

18, 5.

Weening, J. J., Hoedemacker, P. H. J., and Bakker, W. W. (1981). Immunoregulation and antinuclear antibodies in mercury-induced glomerulopathy in the rat. Clin. Exp. Immwol. 45, 64.