Phorbol ester/calcium ionophore activate fish leukocytes and induce long-term cultures

This study documents that phorbol ester (TPA) and calcium ionophore (A23187) in combination are potent mitogens for channel catfish peripheral blood leukocytes (PBL), stimulating both catfish T and B cells. Unlike T-cell responses to Concanavalin A (ConA), these responses to TPA/A23187 did not appear to require monocytes and were not strongly inhibited by low culture temperature. These results support the notion that catfish lymphocytes utilize the bifurcating phosphatidylinositol bisphosphate second-messenger system for transmembrane signaling during the activation process, as do mammalian lymphocytes. Furthermore, it was unexpectedly found that TPA/A23187 stimulation of normal catfish PBL reproducibly (greater than or equal to 95%) resulted in the generation of long-term leukocyte cultures that did not require restimulation or the addition of exogenous factors for continued proliferation. These TPA/A23187-induced leukocyte cultures were refractory to cloning and appeared to contain 10-40% monocytes and 50-80% putative T cells with no detectable B cells or neutrophils.