Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium.
OBJECTIVE
To examine the expression of integrin vitronectin receptors (integrins alphavbeta3 and alphavbeta5) in native human fetal retinal pigment epithelium (RPE) and cultured hunman fetal RPE and to examine the role of RPE VnRs in the phagocytosis of photoreceptor rod outer segments (ROS).


METHODS
Monoclonal antibodies against human integrin subunit alphav and heterodimers alphavbeta3 (LM609) and alphavbeta5 (P1F6) were used to label freshly isolated human fetal RPE explant and cultured human fetal RPE and to immunoprecipitate membrane proteins from cultured human RPE. Effects of antibodies and peptides that inhibit integrin vitronectin receptors on phagocytosis of ROS by RPE were determined using cultured human fetal RPE and fluorescein-labeled bovine outer segments.


RESULTS
Antibodies against integrin subunit alphav and against alphavbeta5 (P1F6) labeled the RPE apical membrane in both native tissue and cultured RPE, while anti-alphavbeta3 antibody (LM609) labeled less than 1% of cultured RPE and did not label native RPE. Antibodies against alphav and alphavbeta5 P1F6 also immunoprecipitated 2 protein bands corresponding to integrin subunits alphav and beta5 from a membrane extract of cultured human RPE. The peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP, 1 mM) inhibited the total ROS uptake (externally bound and ingested) by 48% (P < 0.001) and ingestion of ROS by 37% (P < 0.001). Antibody PIF6 (50 microg/ml) inhibited the total ROS uptake and the ingestion of ROS by 63% (P < 0.001) and 43% (P < 0.01), respectively.


CONCLUSIONS
The integrin alphavbeta5 vitronectin receptor is expressed on the apical membrane of human RPE and participates in the binding of photoreceptor ROS during phagocytosis by cultured human RPE.
Neuroscience Research Institute and Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara 93106, USA.
H
H
Lin
D O
DO
Clegg
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005333:Fetus; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010587:Phagocytosis; D010786:Photoreceptor Cells / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D011233:Precipitin Tests; D019030:Receptors, Vitronectin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016023:Integrins; D009842:Oligopeptides; D019030:Receptors, Vitronectin; C064660:integrin alphaVbeta5; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline