Human skin, lung and trachea produce human beta defensin-2 (hBD-2), an inducible, transcriptionally regulated antibiotic peptide with activity against gram negative bacteria, which may explain the unusual resistance of these tissues to infection. Since an intact corneal epithelium is also highly resistant to infection, we examined whether human ocular surface epithelia might produce hBD-2. Conjunctival epithelial cells were obtained from a human cadaver eye, while corneal epithelial cells were obtained from both a cadaver eye and the eye of a living human patient. Using reverse transcription-polymerase chain reaction and custom primers for hBD-2, a 257 bp sequence was amplified from both human corneal and conjunctival epithelial cell cDNA, and the amino acid sequence of this DNA band was computer-matched with the known gene sequence of hBD-2 available through GenBank (Z71389). To determine whether bacterial by-products upregulate hBD-2 mRNA expression, we stimulated confluent SV 40-immortalized human corneal epithelial cells with bacterial culture supernatant prepared from either wild-type P. aeruginosa strain PAO1 or two different lipopolysaccharide (LPS) mutants of PAO1. Both of these mutants, strains AK1012 and PAO1 algC::tet, are deficient in phosphomannomutase activity which is required for the synthesis of both a complete polysaccharide core and the O side chain structures of the LPS molecule. Neither of these mutations affects the lipid A portion of LPS. Cells treated with 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 culture supernatant was used to demonstrate that upregulation of hBD-2 is caused by LPS. Genistein blocked this upregulation suggesting that protein tyrosine kinase activity is involved. Thus, both human corneal and conjunctival epithelium express mRNA for hBD-2, and this expression is upregulated by bacterial LPS. Data obtained from LPS mutants suggest that lipid A, which is responsible for initiating a number of the pathophysiological manifestations induced by endotoxin in mammals, is not required. Stimulation of endogenous hBD-2 production via the active portion of LPS might have therapeutic potential.
Copyright 1999 Academic Press.