A New Role for Complement Regulatory Protein CD46 in Retinal Pigment Epithelial Adhesion to Human Bruch’s Membrane

Abstract

Abstract: : Purpose: The complement system may play a role in the pathogenesis of age-related macular degeneration (AMD), as complement proteins have been localized in drusen and drusen formation may involve complement activation. We have characterized a new protein complex in the retinal pigment epithelium (RPE) that includes a complement regulatory protein (CD46), which localizes to the basolateral membrane of RPE and coprecipitates with beta-1 integrin. Here we investigate the role of CD46 and other complement regulatory proteins (CRPs) in the adhesion of RPE from human donor eyes to Bruch’s membrane. Methods: Explants of human Bruch’s membrane were prepared from the periphery of donor (26 explants used; age range = 48 to 80 years) human cadaver eyes obtained from the National Disease Research Interchange within 48 hours of death. 400 ul of a solution containing 60,000 first passage human RPE (51year old donor) was pre-incubated at room temperature with various CRP antibodies (0.1-25ug/ml mouse anti human CD46, CD55 or CD59); mouse anti-human integrin beta-1 (10ug/ml); mouse IgG1; and a non specific kappa MOPC 21 antibody (1: 500) for one hour, then washed three times with Minimum Essential Medium. The proportion of attached cells was determined 24 hours later using a colorimetric assay that estimates the number of live cells by measuring intracellular dehydrogenase activity. Results: In RPE adhesion assays, only CD46 antibodies blocked cell adhesion to human Bruch’s membrane explants tested in a dose-dependent manner; anti-CD55 and CD59 antibodies had no effect. CD46 antibody inhibits the binding of RPE to Bruch’s membrane starting at a concentration of 0.25ug/ml and achieves the maximal inhibition (approximately 50% of binding) at 12.5 ug/ml. Double inhibition studies using both CD46 and beta-1 integrin antibody did not demonstrate synergism. There is no correlation between the maximum inhibition achieved with anti-CD46 and the age of the donor Bruch’s membrane (slope = -0.6186 ± 0.4691; r² = 0.1105; p = 0.2084). Conclusions: This study suggests that CD46, a complement regulatory protein, may play a role in the attachment of RPE to its basement membrane. The magnitude of the effect is independent of donor Bruch’s membrane age over the range tested. To our knowledge, this is the first demonstration of this unique role for CD46 within the eye.

Keywords: age-related macular degeneration • Bruch's membrane • retinal pigment epithelium