Multifunctional roles of human cathelicidin (LL-37) at the ocular surface.
OBJECTIVE
The goals of this study were to examine the expression of the antimicrobial peptide LL-37 in the corneal epithelium during wound healing and to investigate whether LL-37 stimulates human corneal epithelial cell (HCEC) migration, proliferation, and cytokine production.


METHODS
Expression of LL-37 was determined by RT-PCR and immunostaining in tissue sections and HCECs scraped from corneas before (original) and after (regrown) re-epithelialization. The antimicrobial activity of LL-37 against Pseudomonas aeruginosa (PA) was determined in the presence of NaCl and tears. Blind-well chamber assays were performed to study the effect of LL-37 on migration. Proliferation was determined using calcein-AM, and cytotoxicity was evaluated by MTT assay. ELISA was performed to assess the ability of LL-37 to stimulate HCEC cytokine secretion.


RESULTS
LL-37 peptide was present throughout the corneal epithelium (n=4). All original corneal epithelial samples expressed a low level of LL-37 (n=10). Regrown epithelial samples collected 24 (n=3 of 5) or 48 (n=4 of 5) hours after wounding showed upregulated expression of LL-37. LL-37 killed PA in the presence of NaCl (EC50=10.3+/-2.5 microg/mL) and retained its activity in tears (n=3). LL-37 induced HCEC migration (n=5) and secretion of IL-8, IL-6, IL-1beta, and TNF-alpha (2- to 23-fold, n=4-7). Inhibitor studies indicated that LL-37's effects are mediated through multiple pathways involving a G protein-coupled receptor (formyl peptide receptor-like 1 in migration) and the epidermal growth factor receptor (n=2 to 5). LL-37 did not stimulate HCEC proliferation (n=3) and high concentrations (>10 microg/mL) were cytotoxic (n=3).


CONCLUSIONS
LL-37 expression is upregulated in regenerating human corneal epithelium, has antibacterial activity against ocular pathogens under physiologically relevant conditions, and stimulates HCEC migration and cytokine production. These findings suggest that LL-37 acts as a multifunctional mediator that helps protect the cornea from infection and modulates wound healing.
Huang
Ling C
LC
University of Houston, College of Optometry, Houston, Texas 77204-2020, USA.
Petkova
Tihomira D
TD
Reins
Rose Y
RY
Proske
Rita J
RJ
McDermott
Alison M
AM
D000328:Adult; D000368:Aged; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology / Q000502:physiology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008297:Male; D005810:Multigene Family / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013666:Tears / Q000502:physiology; D014945:Wound Healing / Q000502:physiology*; D054804:Cathelicidins
D023181:Antimicrobial Cationic Peptides; D016207:Cytokines; D054804:Cathelicidins