Macrophage activating factor(s) secreted by mitogen stimulated goldfish kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages

Recent studies in our laboratory demonstrated that fish macrophages produce nitric oxide. To elucidate the mechanisms which regulate nitric oxide production in teleosts, we examined whether macrophage activating factors (MAFs) secreted by mitogen stimulated leukocytes, induced nitric oxide production in a long-term cultured macrophage cell line and in primary cultures of kidney macrophages from the goldfish. The results indicate that both primary and long term cultured goldfish macrophages produce nitric oxide in response to MAF or bacterial lipopolysaccharide (LPS), and co-stimulation with both factors results in a synergistic induction of nitric oxide production. MAF that induced nitric oxide production were present in leukocyte supernatants as early as 24 h after addition of mitogens to cell cultures. The production of MAF was dependent upon the incubation temperature, presence of serum in the culture medium and duration of incubation: maximal MAF activity was detected in 72–96 h supernatants raised in media with serum at 30 °C. MAF-induced nitric oxide production by long term cultured macrophages was inhibited by 1000 μM N G-monomethyl- l-arginine or amino-guanidine, indicating an l-arginine-dependent metabolic pathway for the production of the reactive nitrogen intermediates in teleosts. The biochemical events of cytokine induced nitric oxide production by teleost macrophages appear to be similar to those of mammalian macrophages.