All-trans-Retinal Sensitizes Human RPE Cells to Alternative Complement Pathway–Induced Cell Death

METHODS. RPE cells were treated with atRal and then incubated with a complement-fixing antibody followed by stimulation with C1q-depleted serum to activate AP. Cell viability was assessed by tetrazolium salt and lactate dehydrogenase release assays. Changes in cell surface CD46 and CD59 expression were assessed by flow cytometry. Cells were pretreated with the antioxidant resveratrol, and C1q-depleted serum was incubated with an anti-C5 antibody prior to initiating AP attack to determine the protective effects of antioxidant therapy and complement inhibition, respectively. RESULTS. Both atRal and AP activation independently caused RPE cell death. When AP attack was initiated following atRal treatment, a synergistic increase in cell death was observed. Following 24-hour atRal treatment, CD46 and CD59 expression decreased, corresponding temporally to increased susceptibility to AP attack. Resveratrol and the anti-C5 antibody both protected against AP-induced cell death following atRal exposure and were most effective when used in combination. CONCLUSIONS. atRal sensitizes RPE cells to AP attack, which may be mediated in part by atRalinduced downregulation of CD46 and CD59. Despite increased susceptibility to AP attack following exposure to atRal, resveratrol and anti-C5 antibody effectively prevent AP-mediated cell death.