Regulated secretion of complement factor H by RPE and its role in RPE migration

Abstract

Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-gamma (IFN-gamma; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1-100 ng/ml). CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-gamma. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-gamma stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-gamma increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH. IFN-gamma promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-gamma have the potential to play a role in the pathogenesis of AMD.