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P. Yang, J. Tyrrell, J. J. Peairs, G. J. Jaffe; Effect of Cytokines and Hydroquinone on Complement Regulatory Proteins in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2511.
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There is increasing evidence that complement, inflammation, and oxidant injury contribute to age-related macular degeneration (AMD). Complement regulatory proteins (CRPs) such as membrane cofactor protein (MCP, CD46), decay-accelerating factor (DAF, CD55), and membrane inhibitor of reactive lysis (MIRL, CD59), protect normal host cells from complement attack. The factors that regulate RPE cell expression of these molecules are not well understood. In this study, we determined whether pro-inflammatory cytokines and hydroquinone (HQ), an important oxidant in cigarette smoke, affect expression of CRPs in human RPE cells.
Two groups of cultured human RPE cells were stimulated as follows: group 1: cells treated with TNF-α (22ng/ml) or IL-1ß (5U/ml) for 24h, 48h, and 72h; group 2: cells treated with HQ (100µM) for multiple 6-hour periods. mRNA and cell surface protein expression of CD46, CD55 and CD59 were evaluated by real-time RT-PCR and flow cytometry, respectively. Protein expression of these molecules from freshly isolated native human RPE cells was also examined by Western blot.
CD46 and CD59 protein was detected in freshly isolated human RPE cells from 3 different donors. In cultured human RPE cells, after stimulation with TNF-α for 72 hours, CD59 mRNA expression was increased an average of 2-fold. Similarly, CD59 mRNA expression was increased nearly 2-fold 48h and 72h following IL-1ß exposure. TNF-α and IL-1ß up-regulated cell surface CD46 and CD59 protein expression in a time dependent manner. Cell surface CD46, CD55 and CD59 protein expression was significantly increased by multiple 6-hour exposures to HQ.
RPE cell surface CRPs are up-regulated by inflammatory cytokines and repetitive non-lethal oxidant exposure. Increased cell surface CRPs may help to protect RPE cells from complement-mediated injury in diseases such as AMD.
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