Toxic. in Vitro Vol. 5, No. 5/6, pp. 525-528, 1991 088%2333/91 $3.00+0.00 Printedin Great Britain PergamonPressplc EFFECTS OF CADMIUM ON CYTOTOXIC FUNCTIONS OF HUMAN NATURAL KILLER CELLS M. G. CIFONE§,T. NAPOLITANO, C. FESTUCCIA, M. G. CANTALINI, G. DE NUNTIIS, G. SANTONI*,G. MARINELLI t and A. SANTONI~ Department of Experimental Medicine, University of L'Aquila, Collemaggio, *Institute of Legal Medicine, University of Macerata, "['Transfusion Center, Italian Red Cross, L'Aquila and :~Department of Experimental Medicine, University La Sapienza, Via Regina Elena, Rome, Italy Abstract--The effect of cadmium (Cd2+) was studied on natural killer (NK) cell-mediated cytotoxicity as well as on antibody-dependent cellular cytotoxicity (ADCC) of human peripheral blood lymphocytes. The results show that cadmium chloride inhibits human NK and ADCC activities against K562 and Ab-coated P815 target cells in a dose- and time-dependent manner. Maximal inhibition (80-90%) was observed in the presence of 100 #M-cadmiumchloride, and it does not appear to be due to toxic effects on effector cells. Purification of large granular lymphocytes by Percoll density gradient centrifugation suggests that Cd2+ acts directly on this cell population. No effects of Cd2+ could be found after pre-exposure of either effector or target cells separately. The inhibitory effect of Cd2+ was manifested only when effector and target cells were present simultaneously. The greatest inhibition of NK and ADCC activities occurred when cadmium chloride was added to the assay within the first 60 min, suggestingthat an early event was affected by Cd2+. However, Cd2+ did not block effector-target conjugate formation or affect leucocyte function associated Ag-I expression on effector cells. This indicates that an initial triggering of effector cells by target cells was required before Cd2+ could exert its effect. Cd2+-induced inhibition of cytotoxic functions of NK cells could be only partially prevented by increasing the external Ca2+ concentration or by adding Zn2+ to the culture medium. Introduction Cadmium is highly toxic and it is considered a potential hazard to the general population. There is frequent environmental pollution in food, water, air and cigarette smoke and it has a long half-life (20-30 years). Cadmium can influence several parameters of immune response both in vitro and /n vivo. Our previous studies have shown that Cd 2÷ interferes with lymphocyte activation induced in vitro by mitogens such as phytohaemagglutinin and phorbol myristate acetate (Cifone et al., 1989b). These effects seem to occur at different levels of cell metabolism, such as Ca 2÷ influx (Hinkle et al., 1987), protein kinase-C- dependent events (Cifone et al., 1989b), and calmod- ulin-mediated processes (Akerman et al., 1985). In vivo studies showed that cadmium produces either suppression or stimulation of humoral as well as cell-mediated immune responses depending on dose, route of exposure, duration of treatment and species (Blackley, 1985; Thomas et al., 1986). We have previously shown that natural killer (NK) activity of both peripheral blood lymphocytes (PBL) and splenocytes was altered in rats treated with cadmium chloride in drinking-water. The effects depended on the time of exposure. It was lower up to day 30 of treatment and higher in rats treated for longer §To whom correspondence should be addressed: Dip. to di Medicina Sperimentale, Catt. Patologia Generale, Uni- versit~i degli Studi di L'Aquila, Collemaggio, 67100 L'Aquila, Italy. Abbreviations: ADCC = antibody-dependent cellular cyto- toxicity; LFA-1 =leucocyte function associated Ag-l; LGL=large granular lymphocytes; moAb=mono- clonal antibody; NK = natural killer; PBL = peripheral blood lymphocytes; TC = total count. periods (Cifone et al., 1989a). In this study we examined the effects of cadmium chloride on in vitro cytotoxic functions of human NK cells, which are regarded as the first line of defence against both cancer and infectious diseases (Herberman, 1982). These cells could represent a good system to study the in vitro toxicology of cadmium and to identify the molecular and biochemical events underlying the cadmium-induced modulation of NK cell func- tions. Materials and Methods Effector cells. Heparinized peripheral blood from healthy subjects was fractionated by Lymphoprep gradient centrifugation (Nicamed, Oslo, Norway). Mononuclear cells were used as effector cells in the cytotoxicity assays. In a set of experiments, large granular lymphocytes (LGL) were purified on a discontinuous Percoll density gradient (Pharmacia Fine Chemicals, Uppsala, Sweden). LGL purity was more than 80% as judged by examining cyto- centrifuge preparations stained with Giemsa stain (Fisher Scientific Co., FairLawn, N J). Target cells. K562 cells, a line derived from the pleural effusion of a patient with chronic myelocytic leukaemia in blastic crisis, were used as the target cells for NK activity. P815 cells from an NK-resistant murine mastocytoma cell line were used in the anti- body-dependent cellular cytotoxicity (ADCC) assay after coating with a 1:100 dilution of a rabbit anti-P815 antiserum. Cytotoxicity assays. Target and effector cells were suspended in RPMI 1640 medium supplemented with 10% beat-inactivated foetal calf serum, 2mM-glu- tamine, 100 U penicilin/ml, 100/tg streptomycin/ml 525