Effect of Defensins on Corneal Cell Migration and Cytokine Production

Abstract

Abstract: : Purpose: Defensins are antimicrobial peptides that may also modulate corneal wound healing. The goals of this study were to investigate whether human ß–defensin–2 (hBD–2) and human neutrophil peptide–1 (HNP–1) stimulate migration and cytokine expression in cultured human corneal epithelial cells (HCEC) and fibroblasts. Methods: The effect of defensins (0.1–5µg/ml) on fibronectin (2µg/ml) stimulated migration of HCEC or fibroblasts was quantitated using blind well chambers with polycarbonate membranes (8–10µm pore size). Chambers were incubated over night, membranes stained and the number of migrated cells counted by light microscopy. To study cytokine expression, cells were exposed to defensins (0.05–5µg/ml) or 10ng/ml IL–1α (positive control) for 6 hours. The cells were then harvested for cytokine RT–PCR (IL–1ß, IL–6, IL–8, TNFα) and culture media was collected for quantitation of secreted cytokine (IL–8) by enzyme immunoassay. Results: hBD–2 and HNP–1 (1–5 µg/ml) each enhanced fibronectin stimulated migration of HCEC by 17 to 56% (n=2–3). The effects of the defensins were additive and were chemotactic rather than chemokinetic. In contrast, hBD–2 (3µg/ml) inhibited fibronectin stimulated fibroblast migration by 41% (n=3), whereas HNP–1 (3µg/ml) had no effect (n=3). hBD–2 and HNP–1, did not significantly stimulate expression of IL–1ß, IL–6, IL–8 or TNFα mRNA by HCEC or fibroblasts (n=1–3). In contrast, IL–1α, the positive control, markedly upregulated (1.5 to 20 fold) expression of all cytokines tested. The defensins did not stimulate IL–8 release from HCEC, but induced a 7 fold increase in IL–8 secretion by fibroblasts (n=3). In comparison, IL–1α induced a >50 fold increase in IL–8 secretion by both cell types (n=3). Conclusions: Defensins have differential effects on HCEC and fibroblasts. The data suggest that defensins secreted at the ocular surface may promote corneal epithelial wound healing by enhancing fibronectin dependent cell migration. Our data support a direct defensin effect rather than a secondary effect resulting from cytokine secretion. Our results do not support a role for defensins in stromal wound healing per se, however defensin mediated IL–8 secretion by fibroblasts may contribute to neutrophil recruitment after injury.

Keywords: wound healing • cornea: epithelium • cornea: stroma and keratocytes