Abstract
Purpose: :
The ocular surface epithelia express the antimicrobial peptide LL–37. We have previously shown that LL–37 induces human corneal epithelial cell (HCEC) migration (Huang LC, et al. IOVS; 45:ARVO E–Abstract 4940). Here we studied whether LL–37 stimulate cytokine production and further investigated intracellular pathways involved in LL–37 modulated cellular activities in HCEC.
Methods: :
Migration assays were performed using blind well chambers at 37oC overnight. LL–37 (5ug/ml) was placed in the bottom chambers; primary cultured or SV40–transformed HCEC were placed in the top chambers. Some HCEC were pre–treated with various inhibitors: PD98059 (20uM), SP600125 (25uM), SB203580 (5uM), H–7 (100uM), Genistein (50uM), LY294002 (50uM), AG1478 (10 or 50uM), or WRW4 (10 or 50uM) for 15 minutes. For cytokine studies, cells were exposed to LL–37 (0.0001–5ug/ml) for 24hours. In some experiments, cells were pre–incubated with selected inhibitors for 15 minutes prior to addition of LL–37. ELISAs were performed to assess the ability of LL–37 to stimulate secretion of IL–8, IL–6, IL–1beta, and TNF–alpha by HCEC.
Results: :
Of the inhibitors tested, PD98059 (ERK1/2 inhibitor), Genistein (tyrosine kinase, TK inhibitor), WRW4 (formyl peptide receptor–like 1, FPRL–1 specific peptide antagonist) all partially (37–60% inhibition), and LY294002 (PI3K inhibitor), SP203580 (c–JNK inhibitor), and AG1478 (epidermal growth factor receptor, EGFR–TK inhibitor) almost completely (78–92% inhibition) attenuated LL–37 mediated HCEC migration whilst SB600125 (p38 MAP kinase inhibitor) showed minimal (up to 20%) inhibition, and H–7 (PKC inhibitor) showed no effect (n=2–3). LL–37 induced IL–8 (2–10 fold), IL–6 (8–23 fold), IL–1beta (4–16 fold) and TNF–alpha (2–16 fold) secretion in HCEC (n=4–7). While SP203580 and Genistein partially (51–60% inhibition), and LY294002, PD98059 and AG1478 all almost completely (73–88% inhibition) blocked LL–37 mediated cytokine production, WRW4 had no inhibitory effect (n=2–3).
Conclusions: :
LL–37 secreted at the ocular surface may modulate wound healing by inducing cell migration and cytokine secretion. Our data indicate that the stimulatory effects of LL–37 on HCEC are mediated through the FPRL–1 and/or EGFR which likely operate in vivo to help promote corneal wound repair following epithelial injury.
Keywords: cornea: epithelium • signal transduction • wound healing