CHARACTERIZATION OF MACROPHAGES AND NEUTROPHILIC GRANULOCYTES FROM THE PRONEPHROS OF CARP (CYPRINUS CARPIO)

To analyse the functional activity of different leucocyte types, carp pronephros cells were separated on Percoll density gradients and by use of fluorescence-activated cell sorting. Cell populations were characterised by light and electron microscopy and by flow cytometry. Fractions enriched in macrophages and neutrophilic granulocytes were subsequently analysed for phagocytic activity in vitro by quantification of the uptake of Escherichia coli bacteria or yeast cells, and for respiratory burst response by measurement of the production of the reactive oxygen intermediates O2G and H2O2. Both cell types showed very active in vitro phagocytosis and production of both O2G and H2O2. When activated with phorbol myristate acetate or bacteria, it was evident that the neutrophilic granulocytes were significantly more active than the macrophages. Analysis of single-cell respiratory burst activity in fish phagocytes was investigated after preloading of cells with dihydrorhodamine123. Cells were subsequently separated and analysed for fluorescence using flow cytometry. Both the macrophage-enriched fraction and the granulocyte-enriched fraction appeared to consist of active and inactive subpopulations. In comparison with the inactive populations, active populations had characteristic high forward/sideward scatter profiles