DEFENSIN GENE-EXPRESSION IN THE CORNEA

Purpose. To determine whether defensin genes are expressed in human co rneas and bovine corneal keratocytes. Methods. in situ hybridization a nd immunohistochemistry were used to localize defensin mRNA and protei n in normal and diseased human corneas. Cultured bovine keratocytes we re stimulated with IL-1 alpha or TNF alpha to determine whether defens in mRNA production occurred. Reverse transcription polymerase chain re action (RT-PCR) was performed to amplify defensin cDNA from cytokine-i nduced keratocytes, and Southern blots were used to verify the specifi city of RT-PCR amplification products. Results. Defensin mRNA and prot ein were not detected in normal human corneal stroma, but were readily detectable in the corneal stroma in cases of rejected transplants and postinfectious keratitis. IL-1 alpha was a potent inducer of defensin gene expression in keratocytes, which began 12 h after challenge and peaked at 18 to 24 h. TNF alpha weakly induced defensin mRNA in kerato cytes at about 18 h. Southern blots of the RT-PCR products probed with an oligonucleotide complementary to internal sequences of defensin de monstrated the appropriately sized products (198 bp) specific for defe nsin. Conclusions. This report demonstrates the presence of defensin i n the human cornea and the capacity of corneal keratocytes to produce defensin mRNA in response to IL-1 alpha and TNF alpha. Release of defe nsins by keratocytes in response to cytokines elaborated in corneal in flammation may contribute to the host defense response in microbial ke ratitis.