Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages fo r Salmonella typhimurium was studied by using peritoneal phagocytes from C5 7BL/6, congenic gp91phox(-/-), iNOS(-/-), and doubly immunodeficient phox(- /-)iNOS(-/-) mice. The respiratory burst and NO radical (NO) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase -dependent killing is confined to the first few hours after phagocytosis, w hereas iNOS contributes to both early and late phases of antibacterial acti vity. NO-derived species initially synergize with oxyradicals to kill S. ty phimurium, and subsequently exert prolonged oxidase-independent bacteriosta tic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxi de anion (O-2.(-)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-) pro duction. However, immunofluorescence microscopy and killing assays using th e scavenger uric acid suggest that peroxynitrite is not responsible for mac rophage killing of wild-type S. typhimurium. Rapid oxidative bacterial kill ing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon gamma appears to augment antibacterial activit y predominantly by enhancing NO . production, although a small iNOS-indepen dent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires th e generation of both reactive oxidative and nitrosative species.