The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells.

Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.

Quercetin protects ARPE-19 cells against photic stress mediated by the products of rhodopsin photobleaching.

Although the primary biological function of retina photoreceptors is to absorb light and provide visual information, exposure to intense light could increase the risk of phototoxic reactions mediated by rhodopsin photobleaching products (RPBP) that might accumulate in photoreceptor outer segments (POS). Here we investigated whether quercetin can modify the phototoxic potential of RPBP under in vitro photic stress conditions. ARPE-19 cells or quercetin enriched cultures pre-loaded with rhodopsin-rich POS isolated from bovine retinas were irradiated with green light to photobleach rhodopsin, and subsequently with blue light. Survival of cells was determined by MTT assay and propidium iodide staining. Changes in mitochondrial membrane potential (MMP) were assessed by JC-1 staining. Protein hydroperoxides, formed by photosensitized oxidation, mediated by RPBP, were analyzed in cells and in a model system with bovine serum albumin (BSA), using the coumarin boronic acid fluorogenic probe. The effect of photic stress on specific phagocytosis of RPE cells was determined by flow cytometry. Photoreactivity of POS with and without quercetin was analyzed by EPR oximetry and EPR spin trapping. Cytotoxicity measurements and MMP analyses confirmed that supplementation with quercetin protected ARPE-19 cells against photic stress mediated by rhodopsin-rich POS. Quercetin significantly reduced the inhibitory effect of RPBP-mediated stress on POS phagocytosis and the RPBP ability to photooxidize cellular proteins or BSA. The data support the hypothesis that quercetin may efficiently diminish the phototoxic action of retinoids, necessary for restoring the phagocytic function of ARPE-19 cells.