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Effect ofin vitrocadmium exposure on natural killer (NK) cells of catfish,Ictalurus melas
Fish & Shellfish Immunology (1996) 6, 167–172
Effect of in vitro cadmium exposure on natural killer (NK) cells of catfish, Ictalurus melas A. VIOLA, G. PREGNOLATO
AND
V. ALBERGONI
Department of Biology, University of Padova, Via Trieste 75, 35121 Padova, Italy (Received 2 February 1995, accepted in revised form 24 September 1995) The e#ect of in vitro cadmium exposure on catfish natural killer (NK) cells was investigated. Purification of NK cells by nylon-wool columns and density gradient centrifugation indicated that they were a population of nylon-wool adherent cells. Subsequently NK cells were separated only by density gradient centrifugation, where these cells band at the 43/45·5% Percoll interface. In some cytotoxicity assays, catfish NK cells were exposed to 5, 10, 30 and 50 ìM cadmium (CdCl2); 10 ìl of Cd solutions were added to NK cells simultaneously with the target cells. In vitro, 5 ìM Cd inhibited the ability of catfish NK cells to kill the human erythroleukemic cell line K-562. In the presence of 10 ìM Cd, the antibody-independent cytotoxic activity was totally suppressed. ? 1996 Academic Press Limited
Key words:
cadmium, natural killer cells, catfish.
I. Introduction Fish are often exposed to sublethal toxic doses of cadmium, which is an industrial waste product in the aquatic ecosystem. Several studies have documented the toxic e#ects of Cd on the immune system in fish: in vivo Cd causes both suppressive and stimulatory e#ects on humoral and cellular immune responses (Robohm, 1986; Thuvander, 1989); haematological studies report Cd-induced alteration of the percentage distribution of various kinds of white blood cells (Gill & Pant, 1985) and increased numbers of circulating eosinophils (Gardner & Yevich, 1970) and blood lymphocytes (Johansson-Sjöbeck & Larsson, 1978; Sjöbeck et al., 1984). In earlier investigations, it was reported that catfish exposed to 20 ìg l "1 Cd, in vivo, showed a modified immune response against sheep red blood cells (Albergoni & Viola, 1995a) and inhibition of B- and T-cell proliferation (Albergoni & Viola, 1995b). A series of investigations on the e#ects of in vitro exposure to Cd on various functional parameters of the immune response of I. melas were thus initiated. In vitro exposure to 2–40 ìM Cd inhibited catfish lymphocyte proliferation and enhanced superoxide anion production by activated macrophages (Albergoni & Viola, 1995b). 167 1050–4648/96/030167+06 $18.00/0
? 1996 Academic Press Limited
168
A. VIOLA ET AL.
These investigations were extended in the present study, to ascertain whether cadmium a#ects the antibody-independent cytotoxic activity of catfish natural killer (NK) cells in vitro.
II. Methods FISH
Outbred catfish (Ictalurus melas) of both sexes, weighing 100–150 g, were obtained from a local commercial farm. Fish were kept in 150-litre glass aquaria and temperature was maintained at 17) C throughout the experiments. Acclimatisation lasted for two months.
EFFECTOR CELLS
Fish were killed by anaesthetic overdose (MS222). The head kidney was aseptically dissected from each fish by ventral incision and transferred to RPMI 1640 medium supplemented with 50 ng ml "1 gentamycin sulphate and 2 g l "1 NaHCO3. Cell suspensions, prepared by macerating the kidneys, were layered on Histopaque-Ficoll and centrifuged at 500 g for 30 min at 17) C. Cells were removed manually, washed twice in RPMI-HEPES for 10 min, then resuspended in RPMI 1640 medium and counted; their viability (95%) was determined by trypan blue exclusion. Collection of non-NK cells Columns (Polyscience Europe) containing 0·5 g of nylon wool fibre were pre-equilibrated with RPMI 1640 plus 10% foetal bovine serum (FBS) for 1 h at 26) C. The column was then charged with 1–2#108 anterior kidney cells, in 2 ml of medium, and incubated for 1 h at 26) C. Nonadherent cells were collected using two washes in RPMI 1640 supplemented with 0·05 ìg ml "1 gentamycin sulphate and 2 g l "1 NaHCO3. Collection of NK cells See Evans et al., 1984. RPMI 1640 plus 10% FBS was adjusted to 250 mOsm by diluting with distilled water. Percoll (Sigma) was adjusted to 250 mOsm using PBS. The osmolarity adjusted Percoll and the osmolarity adjusted RPMI were mixed in appropriate ratios to obtain 41, 43, 45·5, 47·5 and 53% Percoll. Each solution was layered in 2 ml volumes and 1–2#108 cells (obtained after Ficoll centrifugation) were layered on top, in a 1 ml volume. Gradients were centrifuged at 700 g for 30 min, and cells at each interface were collected, washed twice in RPMI 1640 plus 10% FBS and used for the cytotoxicity assay.
TARGET CELLS
K-562 cells, a human erythroleukemic cell line, were used as targets for catfish NK activity.
CATFISH NATURAL KILLER CELLS
169
CYTOTOXICITY ASSAY
Target and e#ector cells were suspended in RPMI 1640 medium supplemented with 10% FBS, 0·05 ìg ml "1 gentamycin sulphate and 2 g l "1 NaHCO3 (Sigma). One million target cells were labelled with 200 ìl sodium 51chromate (Na251CrO4, 1 mCi ml "1, Amersham) for 1 h at 37) C. After washing, 5#103 target cells in 100 ìl of medium were distributed in round-bottomed 96-well plastic microtitre plates. E#ector cells in 100 ìl of medium were added to triplicate wells to give E:T ratios of 400:1, 200:1, 100:1, 50:1, 25:1, 12:1 and 6:1. Each plate also comprised three wells of spontaneous release (SR) (100 ìl target cells+100 ìl medium) and three wells of total release (TR) (100 ìl target cells+10 ìl HCl 2N). After incubation in a 5% CO2 atmosphere for 3 h at 26) C, the plates were centrifuged at 300 g for 10 min at 4) C. Then, 100 ìl medium from each well were carefully removed and counted in a gamma counter. Percent cytotoxicity was determined as follows: % cytotoxicity =
cpm test release"cpm SR cpm TR"cpm SR
# 100.
IN VITRO CD TREATMENT
Cd solutions were prepared by dissolving CdCl2 in double distilled water and sterilising by filtration. Cd concentrations were measured on an atomic absorption spectrophotometer (Perkin Elmer 4000). In some cytotoxicity assays, catfish NK cells were exposed to 5 and 10 ìM cadmium, by addition of 10 ìl of Cd solutions added to the NK cells simultaneously with the target cells. STATISTICS
Data were compared for statistical significance by Student’s t-test (P values obtained on the sum of the treatment results).
III. Results Ictalurus melas NK cells were nylon-wool adherent, in fact, the cytotoxic activity of head kidney cells (separated using Percoll or unfractionated) was absent in nonadherent cells collected by washing the columns. Nevertheless, catfish NK cells could be separated from the other head kidney cells by density gradient centrifugation where they banded at the 45·5% Percoll interface. Gradient-purified NK cells showed a five-fold increase in cytotoxic activity compared with unfractionated cells (Fig. 1). Figure 2 shows the e#ect of exposure to 5 or 10 ìM Cd on the cytotoxic activity of I. melas NK cells. In vitro, 5 ìM Cd significantly inhibited the antibody-independent cytotoxic activity of both unfractionated head kidney cells (not shown) and purified natural killer cells (P<0·01). Higher doses caused loss of the response.
A. VIOLA ET AL.
170
60
% Cytotoxicity
50
40
30
20
10
400:1 200:1 100:1
50:1 E:T
25:1
12:1
6:1
Fig. 1. Antibody-independent cytotoxic activity of catfish lymphocytes. Cells purified by density gradient centrifugation (-) show a five-fold increased cytotoxicity compared with unfractionated cells (4). (The results are expressed as means (& S.D.) of 10 fish determinations.)
To control for direct toxicity of cadmium to cells, the survival of e#ector cells after Cd treatment was measured. The viability of e#ector cells was determined by trypan blue exclusion. Unexposed e#ector cells showed a mortality of 5%, but this mortality rose to 13% after 5 ìM Cd treatment and to 17% after 10 ìM Cd treatment. IV. Discussion Nylon-wool columns have been extensively used for the fractionation of mammalian natural killer cells (Herberman & Holden, 1978). It is now accepted that nylon-wool filtration does not a#ect the cytotoxic activity of lymphoid cells and that NK cells are generally nonadherent to this fibre (Stutman et al., 1980). However, some NK activity has also usually been found in the nylon adherent population, although to a lesser extent (Kiessling et al., 1976; Wolfe et al., 1977). In fish, natural killer cells are known to be involved in the rejection of tumours and transplanted tissues (Anderson, 1990) and they share properties with mammalian NK cells (Evans et al., 1992). In the present experiments, Ictalurus melas natural killer cells were found to be nylon-wool adherent and no sign of cytotoxic activity was evident in the
CATFISH NATURAL KILLER CELLS
171
60
% Cytotoxicity
50 40 30 20 10
0
5 [Cd] µ M
10
Fig. 2. In vitro CdCl2 inhibition of catfish natural killer cell activity. E:T ratios: 400:1 (/); 200:1 (.). (The results are expressed as means (& S.D.) of 10 fish determinations.)
nonadherent cell fractions. I. melas NK cells were able to kill the human erythroleukemic cell line K562, and a five-fold increase in cytotoxicity of target cells was produced by purified cytotoxic cells compared with unfractionated ones. The ability of I. melas NK cells to kill transformed target cells was partially inhibited by in vitro exposure to 5 ìM Cd and totally suppressed by exposure to 10 ìM Cd. In vitro, Cd has been shown to inhibit human NK activity at a postbinding (e#ector-target) level (Cifone et al., 1990). Several studies report that Cd can block calcium channels in several cell types (Nelson, 1986; Hinkle et al., 1987; Verbost et al., 1987): the e#ect of Cd on NK activity may be due to the impaired ability of NK cells to accumulate optimal concentrations of Ca2+ . Considering that NK cells seem to play a leading role in host defence against tumours and infectious diseases (Herberman, 1982), the demonstration that heavy metals alter NK cell activity provides insight into the enhanced susceptibility to bacterial, fungal or viral infections observed in fish exposed to sublethal levels of toxicants (Ziskowski & Murchelano, 1975; Mearns & Sherwood, 1977; Welling et al., 1977). Supported by a grant from the ‘Murst-Sistema Lagunare Veneziano’.
References Albergoni, V. & Viola, A. (1995a). E#ects of cadmium on catfish, Ictalurus melas, humoral immune response. Fish & Shellfish Immunology 5, 89–95. Albergoni, V. & Viola, A. (1995b). E#ects of cadmium on lymphocyte proliferation and macrophage activation in catfish, Ictalurus melas. Fish & Shellfish Immunology 5, 301–311. Anderson, D. P. (1990). Immunological indicators: e#ects of environmental stress on immune protection and disease outbreaks. In Biological Indicators of Stress in Fish (S. M. Adams, ed.) pp. 38–50. American Fisheries Society, Symposium 8.
172
A. VIOLA ET AL.
Cifone, M. G., Procopio, A., Napolitano, T., Alesse, E., Santoni, G. & Santoni, A. (1990). Cadmium inhibits spontaneous (NK), antibody-mediated (ADCC) and IL-2stimulated cytotoxic functions of natural killer cells. Immunopharmacology 20, 73–80. Evans, D. L. & Jaso-Friedman, L. (1992). Non specific cytotoxic cells as e#ectors of immunity in fish. Annual Review of Fish Diseases 2, 109–121. Gardner, G. R. & Yevich, P. P. (1970). Histological and hematological responses of an estuarine teleost to cadmium. Journal of Fisheries Research Board of Canada 27, 2185–2196. Gill, T. S. & Pant, J. C. (1985). Erythrocytic and leukocytic response to cadmium poisoning in a freshwater fish, Puntius conchonius Ham. Environmental Research 36, 327–337. Herbermann, R. B. & Holden, H. T. (1978). Natural cell-mediated immunity. Advances in Cancer Research 27, 305–377. Hinkle, P. M., Kinsella, P. A. & Osterhoudt, K. C. (1987). Cadmium uptake and toxicity via voltage-sensitive calcium channels. Journal of Biological Chemistry 262, 16333–16337. Johansson-Sjöbeck, M. L. & Larsson, Å. (1978). The e#ect of cadmium on the hematology and the activity of delta-aminolevulinic acid dehydratase (ALA-D) in blood and hematopoietic tissue of the flounder, Pleuronectes flesus L. Environmental Research 17, 191–204. Kiessling, R., Petranyi, G., Ka¨rre, K., Jondal, M., Tracey, M. & Wigzell, H. (1976). Killer cells: a functional comparison between natural, immune T-cell and antibody-dependent in vitro systems. The Journal of Experimental Medicine 143, 772–780. Mearns, A. J. & Sherwood, M. J. (1977). Distribution of neoplasms and other diseases in marine fishes relative to the discharge of waste water. In Aquatic Pollutants and Biological Effects with Emphasis on Neoplasia (H. F. Kraybill, C. J. Dawe, J. C. Harshbarger & R. G. Tardi#, eds). Annales of the New York Academy of Sciences 298, 210–224. Nelson, M. T. (1986). Interactions of divalent cations with single calcium channels from rat brain synaptosomes. Journal of General Physiology 87, 201–222. Robohm, R. A. (1986). Paradoxical e#ects of cadmium exposure on antibacterial antibody responses in two fish species: inhibition in cunners (Tautogolabrus adspersus) and enhancement in striped bass (Morone saxatilis). Veterinary Immunology and Immunopathology 12, 251–262. Sjöbeck, M. L., Haux, C., Larsson, Å. & Lithner, G. (1984). Biochemical and hematological studies on perch, Perca fluviatilis, from the cadmium-contaminated river Eman. Ecotoxicology and Environmental Safety 8, 303–312. Stutman, O., Figarella, E. F., Paige, C. J. & Lattime, E. C. (1980). Natural cytotoxic (NC) cells against solid tumors in mice: general characteristics and comparison to natural killer (NK) cells. In Natural Cell-Mediated Immunity Against Tumors (R. B. Herbermann, ed.) pp. 187–229. New York: Academic Press. Thuvander, A. (1989). Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: e#ects on immune functions. Journal of Fish Biology 35, 521–529. Verbost, P. M., Senden, M. H. M. N. & van Os, C. H. (1987). Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium. Biochimica et Biophysica Acta 902, 247–252. Welling, S. R., Alpers, C. E., McCain, B. B. & Myers, M. S. (1977). Fish disease in the Bering Sea. In Aquatic Pollutants and Biological Effects with Emphasis on Neoplasia (H. F. Kraybill, C. J. Dawe, J. C. Harshbarger & R. G. Tardi#, eds). Annales of the New York Academy of Sciences 298, 290–304. Wolfe, S. A., Tracey, D. E. & Henney, C. S. (1977). BCG-induced murine e#ector cells. II. Characterization of natural killer cells in peritoneal exudates. Journal of Immunology 119, 1152–1158. Ziskowski, J. & Murchelano, R. (1975). Fin erosion in winter flounder. Marine Pollution Bulletin 6, 26–29.
Effect of in vitro cadmium exposure on natural killer (NK) cells of catfish, Ictalurus melas A. VIOLA, G. PREGNOLATO
AND
V. ALBERGONI
Department of Biology, University of Padova, Via Trieste 75, 35121 Padova, Italy (Received 2 February 1995, accepted in revised form 24 September 1995) The e#ect of in vitro cadmium exposure on catfish natural killer (NK) cells was investigated. Purification of NK cells by nylon-wool columns and density gradient centrifugation indicated that they were a population of nylon-wool adherent cells. Subsequently NK cells were separated only by density gradient centrifugation, where these cells band at the 43/45·5% Percoll interface. In some cytotoxicity assays, catfish NK cells were exposed to 5, 10, 30 and 50 ìM cadmium (CdCl2); 10 ìl of Cd solutions were added to NK cells simultaneously with the target cells. In vitro, 5 ìM Cd inhibited the ability of catfish NK cells to kill the human erythroleukemic cell line K-562. In the presence of 10 ìM Cd, the antibody-independent cytotoxic activity was totally suppressed. ? 1996 Academic Press Limited
Key words:
cadmium, natural killer cells, catfish.
I. Introduction Fish are often exposed to sublethal toxic doses of cadmium, which is an industrial waste product in the aquatic ecosystem. Several studies have documented the toxic e#ects of Cd on the immune system in fish: in vivo Cd causes both suppressive and stimulatory e#ects on humoral and cellular immune responses (Robohm, 1986; Thuvander, 1989); haematological studies report Cd-induced alteration of the percentage distribution of various kinds of white blood cells (Gill & Pant, 1985) and increased numbers of circulating eosinophils (Gardner & Yevich, 1970) and blood lymphocytes (Johansson-Sjöbeck & Larsson, 1978; Sjöbeck et al., 1984). In earlier investigations, it was reported that catfish exposed to 20 ìg l "1 Cd, in vivo, showed a modified immune response against sheep red blood cells (Albergoni & Viola, 1995a) and inhibition of B- and T-cell proliferation (Albergoni & Viola, 1995b). A series of investigations on the e#ects of in vitro exposure to Cd on various functional parameters of the immune response of I. melas were thus initiated. In vitro exposure to 2–40 ìM Cd inhibited catfish lymphocyte proliferation and enhanced superoxide anion production by activated macrophages (Albergoni & Viola, 1995b). 167 1050–4648/96/030167+06 $18.00/0
? 1996 Academic Press Limited
168
A. VIOLA ET AL.
These investigations were extended in the present study, to ascertain whether cadmium a#ects the antibody-independent cytotoxic activity of catfish natural killer (NK) cells in vitro.
II. Methods FISH
Outbred catfish (Ictalurus melas) of both sexes, weighing 100–150 g, were obtained from a local commercial farm. Fish were kept in 150-litre glass aquaria and temperature was maintained at 17) C throughout the experiments. Acclimatisation lasted for two months.
EFFECTOR CELLS
Fish were killed by anaesthetic overdose (MS222). The head kidney was aseptically dissected from each fish by ventral incision and transferred to RPMI 1640 medium supplemented with 50 ng ml "1 gentamycin sulphate and 2 g l "1 NaHCO3. Cell suspensions, prepared by macerating the kidneys, were layered on Histopaque-Ficoll and centrifuged at 500 g for 30 min at 17) C. Cells were removed manually, washed twice in RPMI-HEPES for 10 min, then resuspended in RPMI 1640 medium and counted; their viability (95%) was determined by trypan blue exclusion. Collection of non-NK cells Columns (Polyscience Europe) containing 0·5 g of nylon wool fibre were pre-equilibrated with RPMI 1640 plus 10% foetal bovine serum (FBS) for 1 h at 26) C. The column was then charged with 1–2#108 anterior kidney cells, in 2 ml of medium, and incubated for 1 h at 26) C. Nonadherent cells were collected using two washes in RPMI 1640 supplemented with 0·05 ìg ml "1 gentamycin sulphate and 2 g l "1 NaHCO3. Collection of NK cells See Evans et al., 1984. RPMI 1640 plus 10% FBS was adjusted to 250 mOsm by diluting with distilled water. Percoll (Sigma) was adjusted to 250 mOsm using PBS. The osmolarity adjusted Percoll and the osmolarity adjusted RPMI were mixed in appropriate ratios to obtain 41, 43, 45·5, 47·5 and 53% Percoll. Each solution was layered in 2 ml volumes and 1–2#108 cells (obtained after Ficoll centrifugation) were layered on top, in a 1 ml volume. Gradients were centrifuged at 700 g for 30 min, and cells at each interface were collected, washed twice in RPMI 1640 plus 10% FBS and used for the cytotoxicity assay.
TARGET CELLS
K-562 cells, a human erythroleukemic cell line, were used as targets for catfish NK activity.
CATFISH NATURAL KILLER CELLS
169
CYTOTOXICITY ASSAY
Target and e#ector cells were suspended in RPMI 1640 medium supplemented with 10% FBS, 0·05 ìg ml "1 gentamycin sulphate and 2 g l "1 NaHCO3 (Sigma). One million target cells were labelled with 200 ìl sodium 51chromate (Na251CrO4, 1 mCi ml "1, Amersham) for 1 h at 37) C. After washing, 5#103 target cells in 100 ìl of medium were distributed in round-bottomed 96-well plastic microtitre plates. E#ector cells in 100 ìl of medium were added to triplicate wells to give E:T ratios of 400:1, 200:1, 100:1, 50:1, 25:1, 12:1 and 6:1. Each plate also comprised three wells of spontaneous release (SR) (100 ìl target cells+100 ìl medium) and three wells of total release (TR) (100 ìl target cells+10 ìl HCl 2N). After incubation in a 5% CO2 atmosphere for 3 h at 26) C, the plates were centrifuged at 300 g for 10 min at 4) C. Then, 100 ìl medium from each well were carefully removed and counted in a gamma counter. Percent cytotoxicity was determined as follows: % cytotoxicity =
cpm test release"cpm SR cpm TR"cpm SR
# 100.
IN VITRO CD TREATMENT
Cd solutions were prepared by dissolving CdCl2 in double distilled water and sterilising by filtration. Cd concentrations were measured on an atomic absorption spectrophotometer (Perkin Elmer 4000). In some cytotoxicity assays, catfish NK cells were exposed to 5 and 10 ìM cadmium, by addition of 10 ìl of Cd solutions added to the NK cells simultaneously with the target cells. STATISTICS
Data were compared for statistical significance by Student’s t-test (P values obtained on the sum of the treatment results).
III. Results Ictalurus melas NK cells were nylon-wool adherent, in fact, the cytotoxic activity of head kidney cells (separated using Percoll or unfractionated) was absent in nonadherent cells collected by washing the columns. Nevertheless, catfish NK cells could be separated from the other head kidney cells by density gradient centrifugation where they banded at the 45·5% Percoll interface. Gradient-purified NK cells showed a five-fold increase in cytotoxic activity compared with unfractionated cells (Fig. 1). Figure 2 shows the e#ect of exposure to 5 or 10 ìM Cd on the cytotoxic activity of I. melas NK cells. In vitro, 5 ìM Cd significantly inhibited the antibody-independent cytotoxic activity of both unfractionated head kidney cells (not shown) and purified natural killer cells (P<0·01). Higher doses caused loss of the response.
A. VIOLA ET AL.
170
60
% Cytotoxicity
50
40
30
20
10
400:1 200:1 100:1
50:1 E:T
25:1
12:1
6:1
Fig. 1. Antibody-independent cytotoxic activity of catfish lymphocytes. Cells purified by density gradient centrifugation (-) show a five-fold increased cytotoxicity compared with unfractionated cells (4). (The results are expressed as means (& S.D.) of 10 fish determinations.)
To control for direct toxicity of cadmium to cells, the survival of e#ector cells after Cd treatment was measured. The viability of e#ector cells was determined by trypan blue exclusion. Unexposed e#ector cells showed a mortality of 5%, but this mortality rose to 13% after 5 ìM Cd treatment and to 17% after 10 ìM Cd treatment. IV. Discussion Nylon-wool columns have been extensively used for the fractionation of mammalian natural killer cells (Herberman & Holden, 1978). It is now accepted that nylon-wool filtration does not a#ect the cytotoxic activity of lymphoid cells and that NK cells are generally nonadherent to this fibre (Stutman et al., 1980). However, some NK activity has also usually been found in the nylon adherent population, although to a lesser extent (Kiessling et al., 1976; Wolfe et al., 1977). In fish, natural killer cells are known to be involved in the rejection of tumours and transplanted tissues (Anderson, 1990) and they share properties with mammalian NK cells (Evans et al., 1992). In the present experiments, Ictalurus melas natural killer cells were found to be nylon-wool adherent and no sign of cytotoxic activity was evident in the
CATFISH NATURAL KILLER CELLS
171
60
% Cytotoxicity
50 40 30 20 10
0
5 [Cd] µ M
10
Fig. 2. In vitro CdCl2 inhibition of catfish natural killer cell activity. E:T ratios: 400:1 (/); 200:1 (.). (The results are expressed as means (& S.D.) of 10 fish determinations.)
nonadherent cell fractions. I. melas NK cells were able to kill the human erythroleukemic cell line K562, and a five-fold increase in cytotoxicity of target cells was produced by purified cytotoxic cells compared with unfractionated ones. The ability of I. melas NK cells to kill transformed target cells was partially inhibited by in vitro exposure to 5 ìM Cd and totally suppressed by exposure to 10 ìM Cd. In vitro, Cd has been shown to inhibit human NK activity at a postbinding (e#ector-target) level (Cifone et al., 1990). Several studies report that Cd can block calcium channels in several cell types (Nelson, 1986; Hinkle et al., 1987; Verbost et al., 1987): the e#ect of Cd on NK activity may be due to the impaired ability of NK cells to accumulate optimal concentrations of Ca2+ . Considering that NK cells seem to play a leading role in host defence against tumours and infectious diseases (Herberman, 1982), the demonstration that heavy metals alter NK cell activity provides insight into the enhanced susceptibility to bacterial, fungal or viral infections observed in fish exposed to sublethal levels of toxicants (Ziskowski & Murchelano, 1975; Mearns & Sherwood, 1977; Welling et al., 1977). Supported by a grant from the ‘Murst-Sistema Lagunare Veneziano’.
References Albergoni, V. & Viola, A. (1995a). E#ects of cadmium on catfish, Ictalurus melas, humoral immune response. Fish & Shellfish Immunology 5, 89–95. Albergoni, V. & Viola, A. (1995b). E#ects of cadmium on lymphocyte proliferation and macrophage activation in catfish, Ictalurus melas. Fish & Shellfish Immunology 5, 301–311. Anderson, D. P. (1990). Immunological indicators: e#ects of environmental stress on immune protection and disease outbreaks. In Biological Indicators of Stress in Fish (S. M. Adams, ed.) pp. 38–50. American Fisheries Society, Symposium 8.
172
A. VIOLA ET AL.
Cifone, M. G., Procopio, A., Napolitano, T., Alesse, E., Santoni, G. & Santoni, A. (1990). Cadmium inhibits spontaneous (NK), antibody-mediated (ADCC) and IL-2stimulated cytotoxic functions of natural killer cells. Immunopharmacology 20, 73–80. Evans, D. L. & Jaso-Friedman, L. (1992). Non specific cytotoxic cells as e#ectors of immunity in fish. Annual Review of Fish Diseases 2, 109–121. Gardner, G. R. & Yevich, P. P. (1970). Histological and hematological responses of an estuarine teleost to cadmium. Journal of Fisheries Research Board of Canada 27, 2185–2196. Gill, T. S. & Pant, J. C. (1985). Erythrocytic and leukocytic response to cadmium poisoning in a freshwater fish, Puntius conchonius Ham. Environmental Research 36, 327–337. Herbermann, R. B. & Holden, H. T. (1978). Natural cell-mediated immunity. Advances in Cancer Research 27, 305–377. Hinkle, P. M., Kinsella, P. A. & Osterhoudt, K. C. (1987). Cadmium uptake and toxicity via voltage-sensitive calcium channels. Journal of Biological Chemistry 262, 16333–16337. Johansson-Sjöbeck, M. L. & Larsson, Å. (1978). The e#ect of cadmium on the hematology and the activity of delta-aminolevulinic acid dehydratase (ALA-D) in blood and hematopoietic tissue of the flounder, Pleuronectes flesus L. Environmental Research 17, 191–204. Kiessling, R., Petranyi, G., Ka¨rre, K., Jondal, M., Tracey, M. & Wigzell, H. (1976). Killer cells: a functional comparison between natural, immune T-cell and antibody-dependent in vitro systems. The Journal of Experimental Medicine 143, 772–780. Mearns, A. J. & Sherwood, M. J. (1977). Distribution of neoplasms and other diseases in marine fishes relative to the discharge of waste water. In Aquatic Pollutants and Biological Effects with Emphasis on Neoplasia (H. F. Kraybill, C. J. Dawe, J. C. Harshbarger & R. G. Tardi#, eds). Annales of the New York Academy of Sciences 298, 210–224. Nelson, M. T. (1986). Interactions of divalent cations with single calcium channels from rat brain synaptosomes. Journal of General Physiology 87, 201–222. Robohm, R. A. (1986). Paradoxical e#ects of cadmium exposure on antibacterial antibody responses in two fish species: inhibition in cunners (Tautogolabrus adspersus) and enhancement in striped bass (Morone saxatilis). Veterinary Immunology and Immunopathology 12, 251–262. Sjöbeck, M. L., Haux, C., Larsson, Å. & Lithner, G. (1984). Biochemical and hematological studies on perch, Perca fluviatilis, from the cadmium-contaminated river Eman. Ecotoxicology and Environmental Safety 8, 303–312. Stutman, O., Figarella, E. F., Paige, C. J. & Lattime, E. C. (1980). Natural cytotoxic (NC) cells against solid tumors in mice: general characteristics and comparison to natural killer (NK) cells. In Natural Cell-Mediated Immunity Against Tumors (R. B. Herbermann, ed.) pp. 187–229. New York: Academic Press. Thuvander, A. (1989). Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: e#ects on immune functions. Journal of Fish Biology 35, 521–529. Verbost, P. M., Senden, M. H. M. N. & van Os, C. H. (1987). Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium. Biochimica et Biophysica Acta 902, 247–252. Welling, S. R., Alpers, C. E., McCain, B. B. & Myers, M. S. (1977). Fish disease in the Bering Sea. In Aquatic Pollutants and Biological Effects with Emphasis on Neoplasia (H. F. Kraybill, C. J. Dawe, J. C. Harshbarger & R. G. Tardi#, eds). Annales of the New York Academy of Sciences 298, 290–304. Wolfe, S. A., Tracey, D. E. & Henney, C. S. (1977). BCG-induced murine e#ector cells. II. Characterization of natural killer cells in peritoneal exudates. Journal of Immunology 119, 1152–1158. Ziskowski, J. & Murchelano, R. (1975). Fin erosion in winter flounder. Marine Pollution Bulletin 6, 26–29.