Stimulation of macrophages by retinal proteins: Production of reactive nitrogen and oxygen metabolites

PURPOSE. In previous work, it has been shown that in experimental autoimmun e uveitis, the peroxynitrite-mediated protein nitration product nitrotyrosi ne was localized in the degenerating photoreceptors. Subsequently, phagocyt e-generated inducible nitric oxide synthase (iNOS) was also found to locali ze, primarily in the outer retina and to a lesser extent in the anterior se gments. This study was intended to determine whether retinal soluble protei ns such as S-antigen and interphotoreceptor retinoid-binding protein (IRBP) play a role in the induction of (NO)-N-. and superoxide by a macrophage ce ll line and by rat and rabbit peritoneal macrophages. METHODS. Cells from the murine macrophage cell line RAW 264.7 and rat and r abbit peritoneal macrophages were incubated in the presence of retinal solu ble proteins. The nitrite level in the cultured supernatant was evaluated f or (NO)-N-. production using the Griess reaction. Activation of nuclear tra nscription factor kappa B (NF-kappa B) Was determined by electrophoretic mo bility shift assay. Superoxide production was measured by superoxide dismut ase-inhibitable reduction of cytochrome C. RESULTS. Both S-antigen and IRBP induced significant, dose-dependent nitrit e production in RAW 264.7 and rat peritoneal macrophages. Induction of iNOS by retinal proteins was inhibited by the iNOS-specific inhibitor aminoguan idine and the tyrosine kinase inhibitor genistein. This iNOS induction was accompanied by the activation of NF-kappa B. S-antigen also induced superox ide production in rabbit peritoneal macrophages, but not in RAW 264.7. CONCLUSIONS. These results show that soluble retinal proteins significantly induce (NO)-N-. and superoxide production by macrophages. Increased produc tion of reactive oxygen species by macrophages in the presence of these sol uble retinal proteins in vivo may accelerate photoreceptor degeneration in uveitis.