Abstract
Abstract: :
Purpose: We have previously identified that after injury and in response to cytokines, human corneal epithelium expresses the cationic antimicrobial peptide LL–37 (Huang, L, et al. IOVS; 44:ARVO E–Abstract #1335). The purpose of this study was to examine the activity of LL–37 against Pseudomonas aeruginosa (PA, ATCC #27853) and investigate the effect of LL–37 on corneal epithelial cell migration. Methods: PA (105cfu) were incubated with LL–37 (0.05–100µg/ml) in the absence or presence of 150mM NaCl or human tears. Samples were spread on agar plates and the number of colonies counted 24 hours later. Migration assays were performed using blind well chambers with 12µm pore size polycarbonate membranes. LL–37 (0.1–20µg/ml) was diluted in serum–free culture media and placed in the bottom chambers. Media containing fibronectin (2µg/ml) acted as a positive control. 105 Primary cultured or SV40–transformed human corneal epithelial cells (HCEC) were placed in the top chambers. In some experiments, HCEC were pre–treated with pertussis toxin (100–250ng/ml). The chambers were incubated at 37oC overnight, then the membranes were stained and the number of migrated cells counted. RT–PCR was performed to detect expression of formyl peptide receptor–like 1 (FPRL1), a known LL–37 receptor. Results: LL–37 inhibited PA growth in a concentration dependent manner (EC50 =2.3+0.9µg/ml, n=3), but when tested in the presence of NaCl, the activity of LL–37 was moderately reduced (EC50 =11.6+3.4µg/ml, n=3). LL–37 (100µg/ml) was 100% effective at killing PA in the presence of tears (n=2). Fibronectin stimulated HCEC migration (197+10 HCEC per 400x field) whilst culture media did not (n=3). LL–37 (5µg/ml) also induced HCEC migration (66+6 HCEC per 400x field), and this effect was inhibited by pertussis toxin (n=3). Additionally, HCEC constitutively expressed FPRL1 mRNA (n=4). Conclusions: LL–37 inhibits the growth of PA and induces HCEC migration in vitro. The latter is mediated by a Gi linked receptor, likely FPRL1. Our data show that LL–37 likely acts as a multifunctional mediator at the ocular surface that helps protect the cornea from infection and promotes wound healing.
Keywords: cornea: epithelium • cornea: basic science • wound healing