In response to inflammatory mediators or specific pathogens, macrophages as well as neutrophils produce nitric oxide and express the high output cytokine-inducible form of the nitric oxide-producing enzyme nitric oxide synthase-2 (NOS2). Nitric oxide is important in diverse cellular processes including vasodilation, neurotransmission, regulation of cell proliferation, tumor cell cytostasis and non-specific host defense. Recent studies suggest that nitric oxide may also regulate apoptosis. In the present studies we show that nitric oxide mediates apoptosis induced by Fas, a cell surface receptor involved in regulating tissue function and homeostasis. Using the murine macrophage cell line RAW 264.7, we have found that treatment of the cells with lipopolysaccharide (LPS) derived from Salmonella enteritidis (100 nM) in conjunction with anti-Fas antibodies resulted in apoptosis. This process was blocked by the NOS inhibitors n-monomethyl-L-arginine (1 mM) and aminoguanidine (0.1 mM). Stimulation of RAW cells with LPS enhanced binding of FITC-labeled Fas ligand to its receptor, expression of NOS2 and nitric oxide production. Nitric oxide production was also inhibited by the NOS inhibitors. These data suggest that nitric oxide is an important mediator of Fas-induced apoptosis in RAW 264.7 cells. We speculate that during the resolution of inflammation, prolonged exposure to LPS induces expression of Fas receptors on macrophages rendering them susceptible to apoptosis. Diminished nitric oxide production may impair this process and prolong the inflammatory response ultimately resulting in tissue injury.
