Roles of Nitric Oxide Radicals in Intracellular Bactericidal Activities on Human Macrophage-like MABS-1 Cells

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We demonstrated examined the roles of specific free radical species on intracellular bactericidal activity of human macrophage-like MABS-1 cells. In an intracellular bactericidal activity assay in which MABS-1 cell phagocytosis and digestion of FITC-conjugated Escherichia coli (E. coli) is detected by FACScan®, almost all phagocytosed E. coli were killed within 3 h, whereas digestion of E. coli was delayed. Inducible nitric oxide synthase (iNOS) was expressed in MABS-1 cell cytosolic fractions, and iNOS inhibitors reduced the intracellular bactericidal activity. Generation of nitric oxide in MABS-1 cells was detected by diaminofluorescent-2 (DAF-2) assay, but not Griess assay, and responded to exposure to tumor necrosis factor and/or interferon-γ. Generation of peroxynitrite, which has strong cytotoxicity, was detected by dihydrorhodamine 123 assay, and N<SUP>G</SUP>-monomethyl-L-arginine, superoxide dismutase and nitrosobenzen, an inhibitor of NADPH oxidase, interfered with peroxynitrite generation. These results suggested that E. coli phagocytosed by MABS-1 cells were killed by nitric oxide (NO) radicals generated by iNOS, and that the killing activity was caused by peroxynitrite rather than NO.
昭和大学・昭和医学会の論文