K. Shimizu et al., Stimulation of macrophages by retinal proteins: Production of reactive nitrogen and oxygen metabolites, INV OPHTH V, 40(13), 1999, pp. 3215-3223
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.