Activated avb3 Integrin Regulates avb5 Integrin-Mediated Phagocytosis in Trabecular Meshwork Cells

PURPOSE. To investigate the roles of avb3 and avb5 integrins in phagocytosis in human trabecular meshwork (HTM) cells. METHODS. Immunofluorescence microscopy and FACS analysis were used to determine levels of avb3 and avb5 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 avb5 integrin in phagocytosis was evaluated by knocking down avb5 integrin expression with siRNA against the human b5 gene. Signaling from focal adhesion kinase (FAK) was blocked using FAK inhibitor 14. The role of avb3 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) b3 integrin subunit. RESULTS. Both TM tissue and cell lines expressed avb3 and avb5 integrins. Knockdown of avb5 integrin reduced phagocytosis by~60% and FAK inhibition significantly reduced phagocytosis up to 84%, in a dose-dependent manner. DEX treatment increased avb3 integrin expression in HTM cells but reduced phagocytosis by~50% compared with untreated and EtOH-treated cells. The CA b3 integrin-expressing cell line showed increased avb3 integrin levels and decreased phagocytosis by~50% compared with the control. CONCLUSIONS. The avb5 integrin-FAK-mediated pathway regulates phagocytosis in TM cells and this pathway is inhibited by activation of avb3 integrins. This suggests that changes in integrin expression and activity may be responsible for alterations in phagocytosis observed in steroid induced glaucoma

Neutrophil Interactions with Keratocytes during Corneal Epithelial Wound Healing: A Role for CD18 Integrins

PURPOSE. To determine the role of keratocytes and leukocyte ␤ 2 (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. METHODS. Using C57BL/6 wild-type and CD18 Ϫ/Ϫ mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18 Ϫ/Ϫ ) after central corneal epithelial abrasion, time points determined previously to have similar levels of emigrated PMNs. Corneas were prepared for ultrastructural morphometric analysis of PMNs, keratocyte networks, and collagen. RESULTS. Transmission electron microscopy revealed intact keratocyte networks within the paralimbus that were morphometrically similar, regardless of epithelial injury or mouse genotype. Secondary to epithelial abrasion, extravasated PMNs within the paralimbus developed close contacts with keratocytes and collagen. In wild-type mice, 40% of the PMN surface was in contact with the keratocyte surface, and this value decreased to 10% in CD18 Ϫ/Ϫ mice. PMN contact with collagen was similar in wild-type and CD18 Ϫ/Ϫ mice, with approximately 50% of the PMN surface contacting the collagen fibrils. Since corneal edema resulting from scrape injury was similar, regardless of genotype and did not involve structural changes in collagen fibrils, these data favor a direct role for CD18 in mediating PMN contact with keratocytes. CONCLUSIONS. The data show that in response to epithelial scrape injury, PMN migration in the corneal stroma involves close contact between keratocytes and collagen. Although PMN-keratocyte contacts require CD18 integrins, contact with collagen is CD18 independent. Fundamentally, PMN migration along keratocyte networks constitutes the beginning of a new experimental concept for understanding leukocyte migration within the wounded cornea. (Invest Ophthalmol Vis Sci. 2007; 48:5023-5029

Dexamethasone increases alpha v beta3 integrin expression and affinity through a calcineurin/NFAT pathway

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p<0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6days of treatment and then an additional 10days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1day of DEX treatment to increase levels for 4days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7h from 22.5h in control cultures (p<0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway.Jennifer A. Faralli, Debjani Gagen, Mark S. Filla, Tania N. Crotti, Donna M. Peter