Activated αvβ3 Integrin Regulates αvβ5 Integrin–Mediated Phagocytosis in Trabecular Meshwork Cells

Abstract

To investigate the roles of αvβ3 and αvβ5 integrins in phagocytosis in human trabecular meshwork (HTM) cells. Immunofluorescence microscopy and FACS analysis were used to determine levels of αvβ3 and αvβ5 integrins in TM tissue and cultures of normal and immortalized TM cells. Phagocytosis was measured using pHrodo-labeled S. aureus bioparticles followed by FACS analysis. The role of αvβ5 integrin in phagocytosis was evaluated by knocking down αvβ5 integrin expression with siRNA against the human β5 gene. Signaling from focal adhesion kinase (FAK) was blocked using FAK inhibitor 14. The role of αvβ3 integrins in phagocytosis was determined by treating HTM cells with dexamethasone (DEX) or ethanol (EtOH) and by generating stable cell lines that overexpressed either wild type (WT) or constitutively active (CA) β3 integrin subunit. Both TM tissue and cell lines expressed αvβ3 and αvβ5 integrins. Knockdown of αvβ5 integrin reduced phagocytosis by ∼60% and FAK inhibition significantly reduced phagocytosis up to 84%, in a dose-dependent manner. DEX treatment increased αvβ3 integrin expression in HTM cells but reduced phagocytosis by ∼50% compared with untreated and EtOH-treated cells. The CA β3 integrin-expressing cell line showed increased αvβ3 integrin levels and decreased phagocytosis by ∼50% compared with the control. The αvβ5 integrin-FAK-mediated pathway regulates phagocytosis in TM cells and this pathway is inhibited by activation of αvβ3 integrins. This suggests that changes in integrin expression and activity may be responsible for alterations in phagocytosis observed in steroid induced glaucoma.