Lymphocyte Function-Associated Antigen-1-Dependent Inhibition of Corneal Wound Healing

Abstract

Abrasion of murine corneal epithelium induces neutrophil emigration through limbal vessels into the avascular corneal stroma, peaking within 12 to 18 hours after wounding. A central corneal wound closes within 24 hours by epithelial cell migration and division, and during wound closure corneal epithelial cells express intercellular adhesion molecule (ICAM)-1 (CD54). We investigated the contributions of lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) by analyzing wound closure in mice with targeted deletions of CD11a (CD11a ؊/؊ ) or CD11b (CD11b ؊/؊ ). In contrast to CD11a ؊/؊ mice, CD11b deficiency revealed a much greater delay in epithelial wound closure with >90% inhibition of epithelial cell division at a time when neutrophil accumulation in the cornea was approximately threefold higher than normal. Treating CD11b ؊/؊ mice with anti-CD11a monoclonal antibody at the time of epithelial abrasion resulted in significant reductions in neutrophils and significant increases in corneal epithelial cell division and migration. Treating CD11b ؊/؊ mice with anti-ICAM-1 significantly increased measures of healing but marginally reduced neutrophil influx. In conclusion, wound healing after corneal epithelial abrasion is disrupted by the absence of CD11b. The disruption is apparently linked to excessive neutrophil accumulation at a time when epithelial division is essential to wound repair, and neutrophils appear to be detrimental through processes involving LFA-1 and

γδ T Cells Are Necessary for Platelet and Neutrophil Accumulation in Limbal Vessels and Efficient Epithelial Repair after Corneal Abrasion

“…Central epithelial abrasion without stromal injury results in three general phases of healing. 22,24,30,31 The initial lag phase lasts ϳ6 hours. The migration phase in which epithelial cells (basal and suprabasal) crawl over the provisional matrix on the stromal surface to close the wound lasts ϳ24 hours.…”

“…22 Accumulation of ␥␦ T cells was analyzed in two strains of C57BL/6 mice with targeted deletions. CD11a Ϫ/Ϫ mice exhibited no significant reduction at 18 hours in GL3 ϩ cell accumulation after central corneal abrasion ( Figure 6A).…”

“…22 In brief, mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight), and the central corneal epithelium was demarcated with a 2-mm trephine and then removed using a diamond blade for refractive surgery (Accutome, Malvern, PA) under a dissecting microscope. Wound closure was assessed using fluorescein staining of the ocular surface and digital analysis of the stained area.…”

“…Taking care to include the limbus, wounded corneas were dissected, fixed, permeabilized, and incubated with the following labeled monoclonal antibodies as described 6,7,22,24 : anti-Gr-1-fluorescein isothiocyanate (FITC) (clone RB6-8C5), anti-TCR␦-phycoerythrin (PE) (clone GL3), anti-CD41-PE (clone MWReg30), and anti-CD31-FITC (clone MEC 13.3) (BD PharMingen), which were selected for staining neutrophils, ␥␦ T cells, platelets, and endothelial cells of the limbal vessels, respectively. Radial cuts were made in the cornea so that it could be flattened by a coverslip, and the cornea was mounted in Airvol (Celenase, Ltd., Dallas, TX) containing 1 mol/L 4Ј,6-diamidino-2-phenylindole (DAPI; Sigma Chemical, St. Louis, MO) to assess nuclear morphology.…”

“…A standard pattern for morphometric analysis was used throughout the study as we described before (shown in Figure 3, inset). 6,7,22,24 Whole mounts were evaluated using a ϫ40 oil immersion lens to assess each field of view across the cornea from limbus to limbus. For neutrophils, a central frame covering 8% of the field of view was used to count neutrophils throughout the depth of the cornea from the epithelial to endothelial surfaces (a range of ϳ90 m).…”