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MP 1 Intracellular calcium after mitogenic stimulation of head kidney and spleen cells of rainbow trout
Abstracts of the 7th Congress of the ISDCI: Session M
191
MPl
INTRACELLULAR CALCIUM AFTER MITOGENIC STIMULATION HEAD KIDNEY AND SPLEEN CELLS OF R41NBOW TROUT
OF
EddaSiegl.HeidrunBlunk.’ BarbaraNebe.’JoachimRychly,_Ulrike Blohm.UniversititRestock.FB Biologieand ’ Klinik f. lnnereMedizin. 18055Restock.Universitfitsplatz 2. Germany Flow cytometric analysisofthe forward/side light scatter (FSC/SSC) of density gradient separated head kidney cells of the rainbow trout revealed three distinctly separated populations. In spleen cells, population 1 and 2 were identical but population 3 was absent. An analysisof the populations regarding their sIg’ showed : Population 2 and 3 contained 56,5% and 46,0% of slg + cells. A minor population of 5,6 % was found in population 1. In spleen the population 2 represented the predominant sIg’ population with 65 %.To investigate the activation of distinct population of leukocytes we examined the intracellular calcium response after treatment with mitogens and other cellular stimulants. Incubation of Fluo-3 loaded head kidney and spleen cells with different concentrations of ConA (25 - 200 p&ml) and PHA (10 - 40 &ml) induced a pronounced .intracellular calcium increase, but only in cells of population 2, whereas population 1 and 3 showed no response. This reaction was concentration dependent. The same reaction was observed in the absence of extracellular calcium. FMLP, a chemotactic peptide, which induces a calcium signal in mammalian granulocytes, had no effect on intracellular calcium response in all three populations. Also the stimulation with PMA (5 or 50 rig/ml) had no effect. The conclusion, sIg--lympho-cytes are mainly pre-B-cells and T-lymphocytes and sIg’-cell B-lymphocytes is in general accepted. Population 2 of head kidney as well and spleen cells is the unique population which contains (beside B-lymphocytes) lymphocytes stimulated by PHA and ConA. Therefore the conclusion should be admitted that only this contains preferentially T-cells. The detection of increasing cytosolic Ca*’ content may be a tool to characterize T-lymphocytes in fish and, otherwise faster than with other methods it allowes to detect a biological effect of mitogens and possibly cytokines. MP2
PRODUCTION OF CYTOKINES KERATINOCYTES
FROM
CULTURED
CHICKEN
Masaf’iuniMukamoto’, Itsuko Kitamural, Tomohiro ~shibel, Hidebito Ohyal, Hiroshi Kcdamal, Tsuyoshi BabaZLLaboratoryof VeterinaryImmunology,Departmentof VeterinaryScience,Collegeof Agriculture,Osaka PrefecmeUniversity,Sakai,Osaka593, Japan,and *Laboratoryof VeterinaryPublicHealth,Departmentof VetermaryScience,Collegeof Agriculture,KagoshimaUniversity,Kagoshima,Kagoshima890,Japan
Keratinocytes (KC) comprise the major subpopulation of cells in the epidermis. Human and mouse KC have been recently recognized to produce a variety of cytokines that are biochemically and functionally similar to those produced by lymphocytes. We separated and cultured keratinocytes from the skin of 20&y chick embryos. The cultured cells were positive for keratin and MHC class II antigen. Proliferation of spleen lymphocytes was observed when the cells were cultured in the presence of culture supematants from KC, and were enhanced by culture supematants from lipopolysaccharide (LPS)-stimulated KC. Culture supematnts from KC exhibited cytotoxic activity for I-XC-EW lyrnphoid tumor cells which were sensitive for tumor necrosis factor (TNF). KC expressed interferon type 1 (IFN-1) mRNA, but not interferon type 2 (IFN-2) mEWA, regardless of LI?S-stimulation. The production of T cell growth factor (IL-l), TNF like factor, and IFN-1 and the expression of MHC class II antigen suggest that chicken KC play an important role in the induction and regulation of immune responsesin the epidermis.
191
MPl
INTRACELLULAR CALCIUM AFTER MITOGENIC STIMULATION HEAD KIDNEY AND SPLEEN CELLS OF R41NBOW TROUT
OF
EddaSiegl.HeidrunBlunk.’ BarbaraNebe.’JoachimRychly,_Ulrike Blohm.UniversititRestock.FB Biologieand ’ Klinik f. lnnereMedizin. 18055Restock.Universitfitsplatz 2. Germany Flow cytometric analysisofthe forward/side light scatter (FSC/SSC) of density gradient separated head kidney cells of the rainbow trout revealed three distinctly separated populations. In spleen cells, population 1 and 2 were identical but population 3 was absent. An analysisof the populations regarding their sIg’ showed : Population 2 and 3 contained 56,5% and 46,0% of slg + cells. A minor population of 5,6 % was found in population 1. In spleen the population 2 represented the predominant sIg’ population with 65 %.To investigate the activation of distinct population of leukocytes we examined the intracellular calcium response after treatment with mitogens and other cellular stimulants. Incubation of Fluo-3 loaded head kidney and spleen cells with different concentrations of ConA (25 - 200 p&ml) and PHA (10 - 40 &ml) induced a pronounced .intracellular calcium increase, but only in cells of population 2, whereas population 1 and 3 showed no response. This reaction was concentration dependent. The same reaction was observed in the absence of extracellular calcium. FMLP, a chemotactic peptide, which induces a calcium signal in mammalian granulocytes, had no effect on intracellular calcium response in all three populations. Also the stimulation with PMA (5 or 50 rig/ml) had no effect. The conclusion, sIg--lympho-cytes are mainly pre-B-cells and T-lymphocytes and sIg’-cell B-lymphocytes is in general accepted. Population 2 of head kidney as well and spleen cells is the unique population which contains (beside B-lymphocytes) lymphocytes stimulated by PHA and ConA. Therefore the conclusion should be admitted that only this contains preferentially T-cells. The detection of increasing cytosolic Ca*’ content may be a tool to characterize T-lymphocytes in fish and, otherwise faster than with other methods it allowes to detect a biological effect of mitogens and possibly cytokines. MP2
PRODUCTION OF CYTOKINES KERATINOCYTES
FROM
CULTURED
CHICKEN
Masaf’iuniMukamoto’, Itsuko Kitamural, Tomohiro ~shibel, Hidebito Ohyal, Hiroshi Kcdamal, Tsuyoshi BabaZLLaboratoryof VeterinaryImmunology,Departmentof VeterinaryScience,Collegeof Agriculture,Osaka PrefecmeUniversity,Sakai,Osaka593, Japan,and *Laboratoryof VeterinaryPublicHealth,Departmentof VetermaryScience,Collegeof Agriculture,KagoshimaUniversity,Kagoshima,Kagoshima890,Japan
Keratinocytes (KC) comprise the major subpopulation of cells in the epidermis. Human and mouse KC have been recently recognized to produce a variety of cytokines that are biochemically and functionally similar to those produced by lymphocytes. We separated and cultured keratinocytes from the skin of 20&y chick embryos. The cultured cells were positive for keratin and MHC class II antigen. Proliferation of spleen lymphocytes was observed when the cells were cultured in the presence of culture supematants from KC, and were enhanced by culture supematants from lipopolysaccharide (LPS)-stimulated KC. Culture supematnts from KC exhibited cytotoxic activity for I-XC-EW lyrnphoid tumor cells which were sensitive for tumor necrosis factor (TNF). KC expressed interferon type 1 (IFN-1) mRNA, but not interferon type 2 (IFN-2) mEWA, regardless of LI?S-stimulation. The production of T cell growth factor (IL-l), TNF like factor, and IFN-1 and the expression of MHC class II antigen suggest that chicken KC play an important role in the induction and regulation of immune responsesin the epidermis.