Cytokine-induced calgranulin C expression in keratocytes

The authors have identified a corneal stromal protein (CO-Ag) that may be i nvolved in the pathogenesis of Mooren's ulcer. The CO-Ag cDNA sequence is i dentical to that of human neutrophil calgranulin C (CaGC). This study sough t to demonstrate expression of the CaGC gene in the human cornea and in cor neal keratocytes after cytokine stimulation. In situ hybridization and immu nohistochemistry were used to localize CaGC mRNA and protein in normal and diseased human corneas, including a specimen with Mooren's ulcer. Cultured bovine keratocytes were stimulated with IL-1 alpha or TNF-alpha and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify CaGC cDNA from cytokine-stimulated keratocytes and unstimulated controls. S outhern blotting verified the specificity of the RT-PCR amplification produ cts. In situ hybridization detected human CaGC mRNA in the stroma of cornea s with Fuchs' dystrophy, postinfection corneas, and a cornea with Mooren's ulcer. In cultured bovine keratocytes, peak levels of CaGC mRNA were reache d 6 h after cytokine stimulation. Southern blots with an oligonucleotide pr obe specific for CaGC detected the RT-PCR products of expected sizes (273 b p) and confirmed that the amplified CO-Ag sequence was identical to that of CaGC. These studies are the first to demonstrate the presence of CaGC in t he human cornea and the ability of stromal keratocytes to produce CaGC (CO- Ag). The up-regulation of CaGC gene expression by corneal keratocytes due t o proinflammatory cytokines from trauma or inflammation may induce autoimmu nity that ultimately results in Mooren's ulceration. (C) 1999 Academic Pres s.