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S.G. Elner, D. Yang, Z.–M. Bian, V.M. Elner; Pro–Inflammatory Cytokines Increase Retinal Pigment Epithelial Cell Reactive Oxygen Species Production Through Mitochondria and NADPH Oxidase . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2072.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammatory mediators play critical roles in various ocular diseases. However, the underlying mechanisms are poorly understood. The purpose of this study was to assess: 1) if human retinal pigment epithelial (RPE) cells produced reactive oxygen species (ROS) when stimulated by pro–inflammatory cytokines, TNF–α, IL–1ß or IFN–γ ; and 2) if so, the cellular source of ROS production induced by those cytokines.
Primary human RPE cell cultures were used. The intracellular ROS production was measured by using 5–(and 6)–chloromethyl–2`,7`–dichlorodihydrofluorescence diacetate, acetyl ester (CM–H2DCFDA) assay. The source of intracellular ROS production was examined by using pharmacological reagents that affect ROS production pathways.
TNF–α, IL–1ß or IFN–γ increased intracellular ROS production in a time– and dose–dependent manner. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial respiratory chain, blocked TNF–α– and IFN–γ–, but not IL–1ß–induced ROS, whereas other two mitochondrial respiratory chain inhibitors, rotenone and antimycin A, had no effect. The ROS production induced by IL–1ß or IFN–γ, but not by TNF–α, was abolished by NADPH oxidase inhibitor (diphenylene iodinium), whereas 6–aminonicotinamide (6AN), an inhibitor of the hexose monophosphate shunt (HMS) had no statistically significant effect on the ROS induced by all three cytokines. Furthermore, ROS scavengers, pyrrolidinedithiocarbamate (PDTC) and N–acetyl–cysteine (NAC), abolished TNF–α–, IL–1ß– or IFN–γ–induced ROS Production.
Collectively, these results demonstrate that TNF–α, IL–1ß and IFN–γ increase RPE cell ROS through mitochondria and NADPH oxidase. These findings may help understand the possible roles of ROS in the RPE responses to cytokines in age–related macular degeneration (AMD).
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