ESTABLISHMENT AND CHARACTERIZATION OF A MACROPHAGE CELL-LINE FROM THE GOLDFISH

A long-term macrophage cell line from the goldfish was established, which has been maintained in culture for more than 2 years. The macrophages were originally isolated by Percoll(R) density gradient centrifugation from the kidneys of goldfish and were grown in specially formulated medium. The macrophages used for the characterization of the cell line have undergone 50 bi-weekly passages. Fish macrophages grew spontaneously in vitro and this property was used to establish the cell line by long-term in vitro selection. The doubling time for long-term cultured macrophages was 82.4 +/- 4.8 h under optimal conditions, Karyotype analysis showed a typical tetraploid model, with 100 chromosomes per cell, indicating that cultured macrophages were normal fish cells. Morphologically and functionally, cultured fish macrophages resembled mammalian and avian macrophages. Virtually all (>99% of macrophages) stained positive for non-specific esterase. Long-term cultured macrophages engulfed sheep red blood cells and both amastigotes and promastigotes of Leishmania major. After stimulation with phorbol esters (PMA) and/or lipopolysaccharide (LPS), cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The production of reactive oxygen intermediates was induced by either PMA or LPS and was greatly enhanced following both PMA and LPS treatment. The production of nitric oxide was induced by LPS alone, and was inhibited by 1000 mu M N-G-monomethyl-L-arginine. The inhibition of nitric oxide response by N-G-MMLA, suggests that the metabolic pathways for the production of nitric oxide may be similar to that of mammalian macrophages. The macrophage cell line from the goldfish will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts. (C) 1995 Academic Press Limited