Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins

Abstract

Purpose: To determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury.

Methods: Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18(-/-)) 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(-/-) mice. PMN contact with collagen was similar in wild-type and CD18(-/-) 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.

FIGURE 1

Transmission electron micrographs of wild-type paralimbal stroma 6 hours after injury, illustrating the method used for morphometric analysis of PMN (white asterisk) contacts with keratocytes and collagen. The initial image (A) is rotated (B) to match the cycloid grid orientation. (C) Enlarged view of (B). The grid points and lines intersecting PMN contacts are colored according to the nature of the contact. Ep, corneal epithelium. Black arrows: keratocytes. Red dots: PMN body. Yellow dots: PMN close contact with keratocyte. Blue dots: PMN close contact with collagen. Scale bar, 2 µm.

FIGURE 2

Transmission electron micrographs of collagen fibrils in the paralimbal stroma of a wild-type cornea 6 hours after injury. For morphometric analysis, collagen fibrils were photographed in cross-section (A). Higher magnification (B, C) reveals collagen fibrils arranged in a hexagon around a central fibril. Collagen fibril diameters (B) and the distances between the central and outer fibrils (C) were measured. Scale bars: 200 nm (A), 30 nm (B, C).

FIGURE 3

Transmission electron micrographs of wild-type cornea, 6 hours after injury. In the central abraded region (A), the Bowman membrane remains intact (black arrow), whereas injured keratocytes (white arrow) are present beneath the wound. Apoptotic bodies (white arrows, B) are frequently detected within injured keratocytes. Ep, corneal epithelium. Scale bars: 5 µm (A), 2 µm (B).

FIGURE 4

Transmission electron micrograph of CD18^−/− cornea 24 hours after injury. Keratocyte networks (arrows) remain intact under uninjured epithelium (Ep) overlying paralimbal stroma. Scale bar, 5 µm.

FIGURE 5

Light micrograph of wild-type cornea in cross-section, 6 hours after injury. PMNs migrate preferentially in the anterior half of the paralimbal corneal stroma (arrows, anterior PMNs; arrowheads, posterior PMNs). Dashed line divides corneal stroma into anterior and posterior halves. Scale bar, 15 µm.

FIGURE 6

Transmission electron micrographs of injured corneas in wild-type (A, B) and CD18^−/− (C, D) mice. (A) Wild-type PMN (white asterisk) in close association with a keratocyte (black arrowheads). (B) Enlarged view of (A) shows that the PMN makes extensive close contact with collagen (white arrowheads) and the keratocyte (black arrowheads). (C) Migrating CD18^−/− PMN (white asterisk) within the stroma beneath the paralimbal epithelium (Ep). A nearby keratocyte (black arrowhead) is surrounded by electron translucent space (edema; black arrow). (D) Enlarged view of (C). Note how the PMN establishes close contact with collagen (white arrowheads), whereas close contacts with the keratocyte are less evident, and the distance between opposing PMN and keratocyte surfaces is typically more than 25 nm (black arrowheads). Scale bars: 2 µm (A), 0.5 µm (B), 5 µm (C), 1 µm (D).

FIGURE 7

Paralimbal keratocyte surface–volume ratios within the anterior and posterior corneal stroma. Data expressed as mean ± SEM.

FIGURE 8

Collagen fibril spacing (distance) in anterior and posterior aspects of corneal stroma. Data expressed as mean ± SEM.