Ocular surface epithelia express mRNA for human beta defensin-2

Human skin, lung and trachea produce human beta defensin-2 (hBD-2), an indu cible, transcriptionally regulated antibiotic peptide with activity against gram negative bacteria which may explain the unusual resistance of these t issues to infection. Since an intact corneal epithelium is also highly resi stant to infection, we examined whether human ocular surface epithelia migh t produce hBD-2. Conjunctival epithelial cells were obtained from a human c adaver eye, while corneal epithelial cells were obtained from both a cadave r eye and the eye of a living human patient. Using reverse transcription-po lymerase chain reaction and custom primers for hBD-2, a 257 bp sequence was amplified from both human corneal and conjunctival epithelial cell cDNA, a nd the amino acid sequence of this DNA band was computer-matched with the k nown gene sequence of hBD-2 available through GenBank (Z71389). To determin e whether bacterial by-products upregulate hBD-2 mRNA expression, we stimul ated confluent SV 40-immortalized human corneal epithelial cells with bacte rial culture supernatant prepared from either wild-type P. aeruginosa strai n PAO1 or two different lipopolysaccharide (LPS) mutants of PAO1. Both of t hese mutants, strains AK1012 and PAO1algC::tet, are deficient in phosphoman nomutase activity which is required for the synthesis of both a complete po lysaccharide core and the O side chain structures of the LPS molecule. Neit her of these mutations affects the lipid A portion of LPS. Cells treated wi th P. aeruginosa wild-type PAO1 bacterial culture supernatant demonstrated strong upregulation of hBD-2 mRNA expression, whereas cells stimulated with culture supernatant produced by either of the LPS mutants showed little or no change in hBD-2 gene expression. LPS extracted from the bacterial cultu re supernatant was used to demonstrate that upregulation of hBD-2 is caused by LPS. Genistein blocked this upregulation suggesting that protein tyrosi ne kinase activity is involved. Thus, both human corneal and conjunctival e pithelium express mRNA for hBD-2, and this expression is upregulated by bac terial LPS. Data obtained from LPS mutants suggest that lipid A, which is r esponsible for initiating a number of the pathophysiological manifestations induced by endotoxin in mammals, is not required. Stimulation of endogenou s hBD-2 production via the active portion of LPS might have therapeutic pot ential. (C) 1999 Academic Press.