Effects of cadmium on natural killer and killer cell functions in Vivo

ENVIRONMENTAL RESEARCH 45, 71-77 (1988)

Effects of Cadmium on Natural Killer and Killer Cell Functions in Vivo N E I L L H . STACEY, G R E G C R A I G , AND L U D W I G M U L L E R

Occupational Health Division, National Occupational Health and Safety Commission, The University of Sydney, New South Wales 2006, Australia Received February 18, 1987

INTRODUCTION Cadmium is widely recognized as a metal of considerable toxicity in both an environmental and an occupational setting (Ryan et al., 1982; Bernard and Lauwerys, 1984). Recently, the effects of environmental and occupational agents on the immune system have received increasing attention. Vos (1977) has reviewed some of the earlier studies on the immunotoxicological effects of cadmium and other metals, while there have been several more recent studies reporting on interference of various aspects of immune function. The sometimes contradictory data of these reports have been mentioned by both of the recent review papers (Bozelka and Salvaggio, 1985; Luster and Rosenthal, 1986). Interestingly, there was virtually no mention of the effects of cadmium on NK or K cell functions in these reviews, even though they are considered important for immune surveillance (Herberman, 1983). Indeed, the paucity of information prompted a recent study which found that cadmium was inhibitory for NK and K cell functions when added in vitro (Stacey, 1986). In view of these data and the lack of similar studies in the whole animal, the current investigation has sought to determine the effects of cadmium administered in vivo on NK and K cell functions. Lymphocyte blastogenesis was also estimated in order to give an indication whether any observed responses may be due to an insult to lymphocytes in general. MATERIALS AND METHODS A n i m a l s . Male Sprague-Dawley rats (340-460 g) from the University of Sydney animal house were used in this study. They were allowed free access to food (Allied Stock Feeds, Sydney) and distilled water unless otherwise indicated. The cadmium content of food was found to be approximately 0.01 ppm, while that in distilled water was undetectable at 0.1-1 ng/ml, the limit of sensitivity of the atomic absorption spectrometer. T r e a t m e n t . Three different series of experiments were undertaken. Acute, high-dose administration was by intraperitoneal injection of 0, 0.25, 1, and 4 mg/kg cadmium chloride dissolved so that each rat received 1 ml/kg. Twenty-four hours later spleens were removed and weighed while the rats were under ether anesthesia. The second set of experiments involved gavage of cadmium acetate to rats five times a week for 2 or 6 weeks at individual doses of 2.5, 25, and 250 txg 71 0013-9351/88 $3.00 Copyright © 1988by AcademicPress, Inc. All rights of reproductionin any form reserve&

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STACEY, CRAIG, AND MULLER

cadmium/kg. Controls received sodium acetate such that the acetate dose was similar to that in the middle cadmium dose above. In the third experiment, as drinking water, rats received 0 or 8.2 mg/liter cadmium chloride (i.e., 5 ppm Cd) in distilled water for 6 weeks. Water consumption was monitored in order to estimate cadmium consumed. Control rats consumed an average of 32.7 and cadmium-treated 31.8 ml/day/rat. Thus the daily intake of cadmium per rat averaged 159 ~g/day which further approximates to 400 Ixg cadmium/kg/day using 400 g as the average rat weight. After 2 and 6 weeks in the gavage and 6 weeks in the drinking water experiments, rats were anesthetized with ether and their spleens were removed for processing. Splenocyte preparation. After spleens were removed and weighed they were placed in sterile saline. Each spleen was then teased apart and filtered through sterile cotton wool in Pasteur pipets. Splenocytes were then washed once with medium (RPMI 1640 + 10% heat-inactivated fetal calf serum) and resuspended in sterile water. After 7 sec 9 ml of medium was added and the cells were washed twice more with fresh medium. Cells were then counted and diluted to 1.33 x 106/ml for the T and B cell blastogenesis assay. The remaining cells were placed in tissue culture flasks and incubated overnight at 37°C. Effector cells. Splenocytes from the overnight incubation were poured into tubes leaving the monocytes attached to the flask, then centrifuged and washed twice with medium, before resuspension to concentrations of 2.5 x 106/ml for NK cell assay and 1.25 x 106/ml for K cell assay. Target cells. Yac-1 cells (Clinical Immunology Research Centre, University of Sydney), which provided the targets for the NK cell assay, were cultured continuously in suspension. The cell suspension was centrifuged at 350g for 7 min, and the cells were washed twice, followed by resuspension of the pellet to approximately 6 x 106/0.3 ml medium. This was incubated with 300 ~Ci SlCr (sodium chromate in 0.9% NaC1, Amersham) for 1 hr at 37°C, after which the suspension was centrifuged and washed three times with fresh medium. Yac-1 cells were then resuspended to t x 10S/ml. Chang liver cells (Commonwealth Serum Laboratories, Melbourne), which were used as the targets for the K cell assay, were cultivated continuously in medium and harvested using 0.05% Trypsin (Difco 1:250, Bacto Labs, Sydney)/ 0.02% EDTA (Ajax, Sydney). After washing, the cells were resuspended in fresh medium at 5 x 106/0.6 ml to which 300 txCi of 5~Cr was added. One 0.3-ml aliquot was incubated at 37°C for 1 hr with control rabbit serum and the other 0.3 ml with serum containing an antibody to human lung tissue (supplied by W. J. Halliday, University of Queensland). The cells were then washed twice with fresh medium and resuspended to a final concentration of 2.5 x 104/ml. Cytotoxicity assay. Aliquots (0.8 ml) of medium or splenocyte suspension were added to sterile polycarbonate centrifuge tubes (Disposable Products, South Australia), followed by target cells (0.4 ml) to give a final effector: target cell ratio of 50:1 for NK cell activity and 100:1 for K cell activity. Tubes were then centrifuged at 250g for 1 rain to establish cell-cell contact and incubated at 37°C. After 4 hr of incubation, tubes were centrifuged at 300g for 5 min. An aliquot of supernatant was then removed for the estimation of S~Cr using an LKB mini gamma counter.

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EFFECTS OF CADMIUM ON NK AND K CELLS

Tubes without lymphocytes were run concurrently for determination of spontaneous 51Cr release. Cytotoxicity was calculated using the formula C = csup - csp° × 100, Ctot

where csup, cspo, and Ctot are the supernatant, spontaneous release, and total counts, respectively. T and B cell blastogenesis. Splenocytes (2 × 105 cells per well) were placed in a 96-well plate (Linbro, U-shaped well, Flow Laboratories). Mitogens were conconavalin A (Pharmacia) at 2.5 p~g/ml, 50 ~I per well, for estimation of T cell blastogenesis while the B cell response used lipopolysaccharide (Salmonella typhimurium, TCA extract, Sigma) at l0 ~g/ml, 50 txl per well. Control wells received saline, 50 txl per well. Plates were incubated at 37°C, 5% CO2, and 95% humidity for 48 hr, followed by the addition of [3H]thymidine (Amersham) at 0.5 txCi/well in 50 t~1, and incubated for a further 18 hr. Plates were harvested with a Titertek Cell Harvester 550, (Flow Laboratories). Radioactivity was quantitated in ACSII with a Minaxi Tri-Carb 4000 scintillation counter. Statistics. Data were analyzed by analysis of variance and Duncan's test or by unpaired t test. A level of probability of 0.05 was used and analyses were performed on log-transformed values. RESULTS

The majority of rats receiving the highest acute dose (4 mg/kg) showed general signs of toxicity such as ruffled coat and lethargy, while the lower-dose groups generally appeared similar to controls. Rats administered the highest dose of cadmium showed a decreased spleen weight and yield of splenocytes (Table 1). Both N K and K cell activity were found to be inhibited by the highest dose of cadmium administered (Table 2). On the other hand, neither Con A- nor LPS-induced blastogenesis was different in cadmium-treated as opposed to control rats (Table 3). Splenocytes from rats treated with the highest dose of cadmium showed a statistically significant elevation in [3H]thymidine incorporation. TABLE 1 THE EFFECTS OF ACUTE CADMIUM ON SPLEEN WEIGHT AND CELLULARITY CdC12 (mg/kg, ip)

N

0 0.25 1.0 4.0

10 4 4 8

Spleen/body weight (g/kg) 2.40 2.07 2.38 1.43

_+ 0.08 b (A) ¢ -+ 0.08 (A) + 0.11 (A) _+ 0.11 (B)

Cellularity ~ 28.1 31.8 19.5 9.7

+- 3.4 +_ 4.8 _+ 3.6 - 1.1

(A,B) (A) (B,C) (C)

Cells × 107/g wet wt spleen. Values are m e a n s +_ SE. c T h o s e values with the s a m e capital letter in p a r e n t h e s e s are not statistically different within that set. Analysis of variance and D u n c a n ' s test, P < 0.05.

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STACEY, CRAIG, AND MULLER TABLE 2 THE EFFECTS OF ACUTE CADMIUM ON N K AND K CELL ACTIVITIES CdC12 (mg/kg, ip)

N

0 0.25 1.0 4.0

10 4 4 8

N K cell (%) 24.6 28.4 23.6 13.7

K cell (%)

_+ 2.4 a (A) b _+ 5.7 (A) _+ 1.0 (A) ± 2.3 (B)

11.0 13.4 10.3 4.3

_+ 2.0 _+ 3.1 ± 3.0 ± 0.9

(A) (A) (A) (B)

a Values are means _+ SE. b Those values with the same capital letter in parentheses are not statistically different within that set. Analysis of variance and Duncan's test, P < 0.05.

Administration of cadmium to rats by gavage did not result in any changes in body weight, spleen weight, or yield of splenocytes (data not shown). Neither NK nor K cell activity at 2 or 6 weeks was significantly altered by this protocol of cadmium administration (Table 4). Splenocyte blastogenesis induced by Con A and LPS was found to be significantly elevated after 6 weeks of treatment with the highest dose (250 fxg/kg) of cadmium by gavage (Table 5). Dosage of rats with cadmium in the drinking water for 6 weeks did not provide any indication of changes in body weight, spleen weight, or spleen cellularity (data not shown). As shown in Table 6 no differences in NK or K cell activities were noted but Con A-induced blastogenesis was significantly increased in spleen cells from those rats which had received cadmium. DISCUSSION

The current study has demonstrated that acute administration of a high dose of cadmium causes an inhibition of NK and K cell function. Taken in conjunction with the decreased spleen weight and cellularity, this indicates a substantial decrease in splenocyte NK and K cell function. The lack of effect on mitogen-induced blastogenesis indicates that the observed decrease in activity is more specific than a general inhibition of splenocyte function. These data would appear to be consistent with the inhibitory effects of cadmium on NK and K cell activity when added in vitro (Stacey, 1986) although the mechanism of interference need TABLE 3 THE EFFECTS OF ACUTE CADMIUM ON SPLENOCYTE BLASTOGENESIS

Mitogen CdClz (mg/kg ip) 0 0.25 1.0 4.0

None 2980 3277 3763 6543

± ± _+ +_

524 a (B) b 206 (A,B) 716 (A,B) 1660 (A)

Con A 35718 53823 48760 53222

_+ 9368 (A) _+ 4965 (A) ± 8180 (A) _+ 12577 (A)

LPS 9374 9083 10056 9725

+± -+ ±

2722 1888 4086 1906

(A) (A) (A) (A)

Values are means -+ SE, N = 8. b Those values with the same capital letter in parentheses are not statistically different within that set. Analysis of variance and Duncan's test, P < 0.05.

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E F F E C T S OF C A D M I U M ON N K A N D K C E L L S TABLE 4 EFFECTS OF CADMIUM BY GAVAGE ON N K AND K CELL ACTIVITIES N K cell (%)

Cadmium acetate (Ixg/kg) 0 2.5 25 250

2 weeks 27.3 36.0 40.3 30.4

K cell (%) 6 weeks

± 8.6 a ± 12.2 ± 12.5 _+ 9.9

23.5 27.7 33.4 39.7

± ± ± ±

6 weeks

2 weeks

4.0 8.1 6.3 3.0

12.2 14.9 11.3 11.9

± ± ± ±

1.6 1.8 2.7 1.9

10.8 10.3 13.3 13.2

± ± ± ±

2.1 2.1 4.0 3.2

a Values are means ± SE, N = 4.

not necessarily be similar. It should be noted that the interference with NK and K cell functions is occurring at very high doses, which are associated with general signs of toxicity and changes in other organs as well (Ryan et al., 1982). This acute high-dosage regime is somewhat unrealistic both in quantity and in route of administration. An oral administration of much lower levels is more appropriate to the exposure situation of the general population. Thus the experiments where gavage and drinking water were the sources of cadmium were carried out. Under these conditions no effect on NK or K cell activity was observed, although there was an elevation in Con A-induced blastogenesis after 6 weeks of administration both by gavage and in drinking water. The dose of 25 txg/kg was chosen to represent that closest to the WHO level for maximum tolerable intake (70 jxg/day). This, assuming 5% absorption, results in an average intake of 0.05 txg/kg/day while 25 ixg/kg/day to rats by gavage has been shown to result in a daily intake of about 0.075 ixg/kg/day (Muller et al., 1986). The decrease in spleen weight and cellularity at high acute cadmium exposure was quite marked. Another study has reported a decrease in cellularity at lower TABLE 5 EFFECTS OF CADMIUM BY GAVAGE ON SPLENOCYTE BLASTOGENESIS

Mitogen Cadmium acetate (Ixg/kg)

None

Con A

0 2.5 25 250

3668 3590 3782 4470

± ± ± ±

225a(A) b 528 (A) 428 (A) 103! (A)

119489 117489 132511 136281

2 Weeks ± 19661 (A) _+ 18923 (A) ± 34922 (A) ± 15419 (A)

0 2.5 25 250

2950 2970 3499 5221

± ± ± ±

264 598 883 974

48106 69929 93023 122320

6 Weeks _+ 18681 (B) _+ 19438 (A,B) ± 28765 (A,B) _+ 24335 (A)

(A) (A) (A) (A)

LPS

6675 8155 6357 6212

-± -±

645 (A) 1505 (A) 2049 (A) 392 (A)

4990 8364 7940 13700

± + ± ±

855 (B) 1806 (A,B) 1504 (A,B) 2542 (A)

a Values are means _+ SE, N = 4. b T h o s e values with the same capital letter in parentheses are not statistically different within that set. Analysis of variance and Duncan's test, P < 0.05. The 2-week and 6-week data were analyzed separately.

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STACEY, CRAIG, AND MULLER TABLE 6 EFFECTS OF CADMIUM IN DRINKING WATER (5 PPM) ON N K AND K CELL ACTIVITIES AND SPLENOCYTE BLASTOGENESIS Parameter N K cell (%) K cell (%) Blastogenesis (no mitogen) Blastogenesis (Con A) Blastogenesis (LPS)

Control (N) 36.6 9.1 5499 97695 23586

_+ _+ +_ _+

6.5 a (5) 2.6 (5) 335 (5) 5044 (5) 2928 (5)

Cd-treated (N) 33.2 12.8 6195 142601 28753

_+ 1.6 (5) _+ 1.1 (5) _+ 1200 (3) +_ 25289 b (3) _+ 3010 (3)

a Values are m e a n s _+ SE. b Significantly different to control. Unpaired t test, P < 0.05.

and longer-term exposure (Malave and de Ruffino, 1984). In our 6-week exposure experiments no change in either parameter was observed. However, our oral exposure levels were 10-fold lower than the above study. Absence of change in spleen weight under a similar exposure range has been noted by Thomas et al. (1985). The observation that NK cell function is not affected by oral exposure is consistent with the work of Thomas et al. (1985). These authors reported a nonsignificant augmentation of N K cell activity. This did not show dose dependency. It is interesting that our data show a trend to augmentation with increasing cadmium exposure after 6 weeks (Table 4). However, lack of a similar effect at 2 weeks and in the drinking water data suggests that this is a spurious trend. With regard to K cell function, which is responsible for an antibody-dependent cell-mediated cytotoxicity (ADCC), Chopra et al. (1984c) reported that this process was increased after administration of cadmium. Exposure was higher than that in our oral experiments which may account for the differences. More importantly, as discussed by these authors, their effector cell population also contained macrophages, which confounds interpretation of their data with regard to K lymphocytes. A human study also assessed ADCC in patients with chronic cadmium disease and found no difference to the control group (Williams et aI., 1983). The increases in Con A- and LPS-induced blastogenesis that we observed were of interest because of the literature in this area. As reviewed by Koller (1979) there are studies that show increases, decreases, and no change in T cell blastogenesis. Other studies since this review continue to report varying responses. For example, three studies report an increase in Con A-stimulated blastogenesis (Muller et al., 1979; Chopra et al., 1984b; Malave and de Ruffino, 1984), two a decrease (Chopra et al., 1984c; Thomas et al., 1985), and four no change (Bozelka and Burkholder, 1982; Wesenberg and Wesenberg, 1983; Chopra et al., 1984a; Blakley, 1985). A recent study has addressed this problem with the observation that different effects on mitogen-induced blastogenesis are strain dependent (Ohsawa et al., 1986). Thus it is difficult to make meaningful interpretation of data on mitogen-induced blastogenesis, especially on consideration of the large variability that seems inherent in the assay. This problem was also noted and discussed by Koller (1979). The important point regarding the current study is that similar values in control and cadmium rats in the acute high-dose experiment indicate

EFFECTS OF CADMIUM ON NK AND K CELLS

77

that the inhibition of NK and K cell functions is not a simple nonspecific lymphocyte response. In conclusion, it has been shown that NK and K cell functions are inhibited by in vivo cadmium administration. This is in addition to a decrease in spleen weight and cellularity. However, examination of lower-dose, longer-term oral administration of cadmium did not result in any indication of inhibition of these cell-mediated cytotoxicities. This suggests that under conditions relevant to human exposure, NK and K cell functions are not compromised by exposure to cadmium. REFERENCES Bernard, A., and Lauwerys, R. (1984). Cadmium in human population. Experientia 40, 143-152. Blakley, B. R. (1985). The effect of cadmium chloride on the immune response in mice. Canad. J. Comp. Med. 49, 104-108, Bozelka, B. E., and Burkholder, E M. (1982). Inhibition of mixed leukocyte culture responses in cadmium-treated mice. Environ. Res. 27, 421-432. Bozelka, B. E., and Salvaggio, J. E. (1985). Immunomodulation by environmental contaminants: Asbestos, cadmium, and halogenated biphenyls: A review. Environ. Carcinog. Rev. 3, 1-62. Chopra, R. K., Kohl, K. K., and Nath, R. (1984a). Effect of dietary chronic cadmium exposure on cell-mediated immune response in rhesus monkey (Macaca Mulatta). Toxicol. Lett. 23, 99-107. Chopra, R. K., Prasad, R., Sharma, N., Paliwal, V. K., and Nath, R. (1984b). Effect of dietary chronic cadmium exposure on cell-mediated immune response in rhesus monkeys (Macaca mulatta): Role of calcium deficiency. Arch. Toxicol. 56, 128-131. Chopra, R. K., Sehgal, S., and Nath, R. (1984c). Cadmium an inhibitor of lymphocyte transformation and stimulator of antibody-dependent cell-mediated cytotoxicity (ADCC) in rats: The role of zinc. Toxicology 33, 303-310. Herberman, R. B. (1983). Possible role of natural killer cells in host resistance against tumors and other diseases. Clin. Immunol. Allergy 3, 479-495. Koller, L. D. (1979). Some immunological effects of lead, cadmium, and methylmercury. Drug Chem. Toxi¢ol. 2, 99-110. Luster, M. I., and Rosenthal, G. J. (1986). The immunosuppressive influence of industrial and environmental xenobiotics. Trends Pharmacol. Sci. 7, 408-412. Malave, I., and de Ruffino, D. T. (1984). Altered immune response during cadmium administration in mice. Toxicol. Appl. Pharmacol. 74, 46-56. Muller, L., and Abel, J., and Ohnesorge, E K. (1986). Absorption and distribution of cadmium (Cd), copper and zinc following oral subchronic low level administration to rats of different binding forms of cadmium (Cd-acetate, Cd-metallothionein, Cd-glutathione). Toxicology 39, 187-195. Muller, S., Gillert, K. E., Krause, C., Jautzke, G., Gross, U., and Diamantstein, T. (1979). Effects of cadmium on the immune system of mice. Experientia 35,909-910. Ohsawa, M., Masuko-Sato, K., Takahashi, K., and Otsuka, F, (1986). Strain differences in cadmiummediated suppression of lymphocytes proliferation in mice. Toxicol. Appl. Pharmacol. 84, 379-388. Ryan, J. A., Pahren, H. R., and Lucas, J. B. (1982). Controlling cadmium in the human food chain: A review and rationale based on health effects. Environ. Res. 28, 251-302. Stacey, N. H. (1986). Effects of cadmium and zinc on spontaneous and antibody-dependent cell-mediated cytotoxicity. J. Toxicol. Environ. Health 18, 293-300. Thomas, E T,, Ratajczak, H. V., Aranyi, C., Gibbons, R., and Fenters, J. D. (1985). Evaluation of host resistance and immune function in cadmium-exposed mice. Toxicol. Appl. Pharmacol. 80, 446-456. Vos, J. G. (1977). Immune suppression as related to toxicology. CRC Crit. Rev. Toxicol. 5, 67-101. Wesenberg, G. B. R., and Wesenberg, E (1983). Effects of cadmium on the immune response in rats. Environ. Res. 31,413-419, Williams, W. R., Kagaminori, S., Watanabe, M., Shinmura, T., and Hagino, N. (1983). An immunological study on patients with chronic cadmium disease, Clin. Exp. Immunol. 53, 651-658.