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Spontaneous Cytotoxic Activity of Neutrophilic Granulocytes In ‹›•aŒ¤‹† Fish Pathology, 31 (1),51-52,1996.3 Spontaneous Cytotoxic Activity of separated from the head kidney on discontinuous Neutrophilic Granulocytes in Ginbuna density gradients of Percoll (Pharmacia, Sweden) according to the method previously described3,4). Crucian Carp and Channel Catfish The head kidney cells were applied to the dis continuous density gradients (1.065, 1.080 and 1.090 Osamu Kurata*1,*3,Satoshi Hasegawa*1, Nobuaki Okamoto*1,*4,Teruyuki Nakanishi*2 g/ml for ginbuna crucian carp and common carp, 1.065, 1.070 and 1.080 g/ml for channel catfish) and and Yayoi Ikeda*1 centrifugated at 4•Ž for 20 min at 350 •~ g for gin *1Department of Aquatic Biosciences, Tokyo University of buna crucian carp and common carp, and 300 •~ g for Fisheries, Konan 4, Minato-ku, Tokyo 108, Japan channel catfish. Neutrophilic granulocytes of gin *2National Research Institute of Aquaculture , buna crucian carp and common carp were in the Tamaki, Mie 519-04, Japan interface of 1.080-1.090 g/ml and that of channel (Received December 1,1995) catfish were in the interface of 1.070-1.080 g/ml. The effector cells were stained with Giemsa and Key words:ginbuna crucian carp, channel catfish, neu Sudan black B, and observed under light microscopy trophilic granulocytes, spontaneous cytotoxic activity to identify the cell types. The 51Cr release assay was adopted to determine the cytotoxicity of the effector As in mammals fish neutrophilic granulocytes mi cells. K562 (human chronic myelogenous leukemia) grate more quickly than macrophages during the cells, which were often used for cytotoxicity assay of acute phase of inflammation1), and phagocytose fish leucocytes5), were used as target cells. The microorganisms2). Recent research demonstrated mixture of effector and 51Cr-labeled target cells were that neutrophilic granulocytes from common carp incubated for 4 h at 25•Ž in 96 well microplates with Cyprinus carpio showed spontaneous cytotoxic activ round bottom (Corning Labs. Inc.). After incuba ity against human leukemic cells as well as phagocyt tion, supernatants were taken and the radioactivities ic activity3). This capability of carp neutrophilic were measured. The percent cytotoxicity was calcu granulocytes is interesting as fish defense mecha lated as previously described3). nisms, since no such spontaneous cytotoxic activity Percentages of neutrophilic granulocytes in the has been known in mammals. Therefore, it is neces effector cells of ginbuna crucian carp, channel catfish sary to examine whether other fish species has spon and common carp were 85.5%, 86.3% and 95.0%, taneous cytotoxic activity of neutrophilic granulo respectively, with others mainly comprised of lym cytes similar to common carp. phocytes. Cytotoxicities of neutrophilic granulo In this study, spontaneous cytotoxic activity of cytes from three species were shown in Fig.1.In neutrophilic granulocytes in ginbuna crucian carp ginbuna crucian carp, effector cells composed of Carassius auratus langsdorfii and channel catfish Ict neutrophilic granulocytes showed cytotoxicity alurus punctatus were investigated, and compared to against K562 target cells (approximately 64% at an those of common carp as control. Four ginbuna effector : target cells (E:T) ratio of 50:1), while crucian carp (weighing 80-95g) obtained from Na lymphocyte-rich fraction (1.070-1.080g/ml includ tional Research Institute of Aquaculture, four chan ing 73.9% lymphocytes) expressed low cytotoxicity nel catfish (weighing 150-300g) from a private farm (approximately 15% at an E : T ratio of 50 : 1). and four common carp (weighing 130-180g) from These results indicate that neutrophilic granulocytes Yoshida Research and Experimental Station of of ginbuna crucian carp also possess spontaneous Tokyo University of Fisheries were ketp in 40l tanks cytotoxic activity as that cells of common carp. On with running water at 25 •} 1•Ž. Fish were fed to the contrary, effector cells composed of neutrophilic satiation twice a day with commercial pellet. Neu granulocytes from channel catfish showed extremely trophilic granulocytes were used as effector cells for low or no cytotoxicity against K562 target cells. cytotoxicity assay. Neutrophilic granulocytes were Lymphocytes fraction (•… 1.065g/ml) of channel catfish including small agranular lymphocytes which *3 Present address; Division of Fish Diseases , Nippon Veter are called nonspecific cytotoxic cells (NCC) showed inary and Animal Science University, Kyonan-cho 1-7-1, Musashino, Tokyo 180,Japan* cytotoxicity (data not shown). These results suggest 4 To whom correspondence should be addressed . the different cytotoxic activities of neutrophilic gran 52 . Kurata, S. Hasegawa, N. Okamoto, T. Nakanishi and Y. Ikeda ulocytes among fish species. Spontaneous cytotoxic activities of neutrophilic granulocytes of the present species to other target cells as well as spontaneous cytotoxic activities of neutrophilic granulocytes of other fish species need to be investigated for a better comprehension of this activity in fish. Acknowledgements We wish to thank Dr. M. Nakayasu, National Cancer Center Research Institution, Japan, for kindly providing K562 cells. This study was sup ported in part by Grant-in-Aid for Scientific Re search from the Ministry of Education, Science and Culture, Japan (No. 07660239). References 1) Suzuki, Y. and T. Iida (1992): Ann. Rev. Fish Dis., 2, Fig. 1. Cytotoxicity of effector cells from ginbuna 149-160. 2) Ainsworth, A. J. (1992): Ann. Rev. Fish Dis., crucian carp (•£), channel catfish (•¡) and 2, 123-148. 3) Kurata, O., N. Okamoto and Y. Ikeda common carp (•œ) against K562 target cells. (1995): Dev. Comp. Immunol., 19, 315-325. 4) Waterst Results are expressed as mean •} SD of cyto rat, P. R., A. J. Ainsworth and G. Capley (1988): J. Fish toxicity from individual experimental fish. Dis., 11, 289-294. 5) Evans, D. L. and L. Jaso-Friedmann (1992): Ann. Rev. Fish Dis., 2, 109-121. O.
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