Cellular and molecular studies on factors influencing lymphocyte-phagocyte interactions in fish
The molecular biology of macrophage activating and deactivating cytokines and their receptors was discussed. Comparison of IFN-γ amino acid sequences of several mammalian species reveals a low conservation of amino acids. The interaction of IFN-γ with its receptor system is complicated and coherent with the species specificity of IFN-γ. Identification by PCR of an IFN-γ-like gene in the trout genome was not possible. In contrast with IFN-γ, TGF-β is very conserved in its amino acid sequence. The PCR-amplification of a TGF-β fragment from amphibian, Xenopus, and rainbow trout cDNA libraries was possible. Two oligonucleotide primers were used in PCRs to amplify a 360 bp fragment of trout Mhc class II β chain. Using these two oligos, this 360 bp fragment could be amplified from trout spleen cDNA library and HK leucocytes. cDNA synthesized from RNA extracted from ConA/PMA stimulated HK leucocytes was used as template DNA in PCR, and a class II specific fragment was amplified. This class II fragment could not be amplified from HK macrophages treated with a MAF containing supernatant, although HK macrophages treated with a control supernatant did express class II molecules. This could suggest that priming or activation of trout macrophages results in a decreased expression of Mhc class II antigens. A novel method for analysing 5'nucleotidase activity of head kidney macrophages was optimised for use with cell monolayers, with respect to the effect of cell numbers, temperature and substrate concentration. Both lysed and whole cells could be used for determination of 5'nucleotidase activity. Maximal 5'nucleotidase activity was found in the range of 27°C to 33°C and using a substrate concentration of ≥ 1 μmol AMP ml-1 for whole cells and ≥ 1.5 μmol AMP ml-1 for lysed cells. 5'nucleotidase activity was also correlated with respiratory burst activity in cells treated with a variety of supernatants containing MAF activity. A significant inverse relationship between these two activities was found. MAF-treated cells were also found to lose 5'nucleotidase activity faster than control cells in the presence of cycloheximide, suggesting such cells may have a higher membrane turnover of this enzyme.