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

In previous work, it has been shown that in experimental autoimmune uveitis, the peroxynitrite-mediated protein nitration product nitrotyrosine was localized in the degenerating photoreceptors. Subsequently, phagocyte-generated inducible nitric oxide synthase (iNOS) was also found to localize, primarily in the outer retina and to a lesser extent in the anterior segments. This study was intended to determine whether retinal soluble proteins such as S-antigen and interphotoreceptor retinoid-binding protein (IRBP) play a role in the induction of *NO and superoxide by a macrophage cell line and by rat and rabbit peritoneal macrophages.

Cells from the murine macrophage cell line RAW 264.7 and rat and rabbit peritoneal macrophages were incubated in the presence of retinal soluble proteins. The nitrite level in the cultured supernatant was evaluated for *NO production using the Griess reaction. Activation of nuclear transcription factor kappaB (NF-kappaB) was determined by electrophoretic mobility shift assay. Superoxide production was measured by superoxide dismutase-inhibitable reduction of cytochrome C.

Both S-antigen and IRBP induced significant, dose-dependent nitrite production in RAW 264.7 and rat peritoneal macrophages. Induction of iNOS by retinal proteins was inhibited by the iNOS-specific inhibitor aminoguanidine and the tyrosine kinase inhibitor genistein. This iNOS induction was accompanied by the activation of NF-kappaB. S-antigen also induced superoxide production in rabbit peritoneal macrophages, but not in RAW 264.7.

These results show that soluble retinal proteins significantly induce *NO and superoxide production by macrophages. Increased production of reactive oxygen species by macrophages in the presence of these soluble retinal proteins in vivo may accelerate photoreceptor degeneration in uveitis.