PURPOSE: To determine whether defensin genes are expressed in human corneas and bovine corneal keratocytes. METHODS: In situ hybridization and immunohistochemistry were used to localize defensin mRNA and protein in normal and diseased human corneas. Cultured bovine keratocytes were stimulated with IL-1alpha or TNFalpha to determine whether defensin mRNA production occurred. Reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify defensin cDNA from cytokine-induced keratocytes, and Southern blots were used to verify the specificity of RT-PCR amplification products. RESULTS: Defensin mRNA and protein 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-1alpha was a potent inducer of defensin gene expression in keratocytes, which began 12 h after challenge and peaked at 18 to 24 h. TNFalpha weakly induced defensin mRNA in keratocytes at about 18 h. Southern blots of the RT-PCR products probed with an oligonucleotide complementary to internal sequences of defensin demonstrated the appropriately sized products (198 bp) specific for defensin. CONCLUSIONS: This report demonstrates the presence of defensin in the human cornea and the capacity of corneal keratocytes to produce defensin mRNA in response to IL-1alpha and TNFalpha. Release of defensins by keratocytes in response to cytokines elaborated in corneal inflammation may contribute to the host defense response in microbial keratitis.
PURPOSE: To determine whether defensin genes are expressed in human corneas and bovine corneal keratocytes. METHODS: In situ hybridization and immunohistochemistry were used to localize defensin mRNA and protein in normal and diseased human corneas. Cultured bovine keratocytes were stimulated with IL-1alpha or TNFalpha to determine whether defensin mRNA production occurred. Reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify defensin cDNA from cytokine-induced keratocytes, and Southern blots were used to verify the specificity of RT-PCR amplification products. RESULTS: Defensin mRNA and protein were not detected in normal humancorneal stroma, but were readily detectable in the corneal stroma in cases of rejected transplants and postinfectious keratitis. IL-1alpha was a potent inducer of defensin gene expression in keratocytes, which began 12 h after challenge and peaked at 18 to 24 h. TNFalpha weakly induced defensin mRNA in keratocytes at about 18 h. Southern blots of the RT-PCR products probed with an oligonucleotide complementary to internal sequences of defensin demonstrated the appropriately sized products (198 bp) specific for defensin. CONCLUSIONS: This report demonstrates the presence of defensin in the human cornea and the capacity of corneal keratocytes to produce defensin mRNA in response to IL-1alpha and TNFalpha. Release of defensins by keratocytes in response to cytokines elaborated in corneal inflammation may contribute to the host defense response in microbial keratitis.