Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-κB activation:: Role of H2O2 and nitric oxide in inducible nitric oxide synthase expression in macrophages

Reactive molecules O-2(-), H2O2, and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N-G-monomethyl-L-argine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappaB was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O-2(-) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H2O2 induced NF-kappaB activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H2O2 enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H2O2 was inhibited by catalase and PDTC. These findings suggest that H2O2 production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappaB activation and that NO is a negative feedback inhibitor of iNOS protein expression. (C) 2001 Academic Press.