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 O₂^-, 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-κB 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 O₂^- generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhabited by antioxidative enzymes, but not by SOD. Exogenous H₂O₂ induced NF-κB activation in macrophages, which was inhibited by catalase and pyrroline dithaocarbamate (PDTC). H₂O₂ enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-γ, 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-κB activation and that NO is a negative feedback inhibitor of iNOS protein expression.