Cytokine-induced calgranulin C expression in keratocytes

Abstract

The authors have identified a corneal stromal protein (CO-Ag) that may be involved in the pathogenesis of Mooren's ulcer. The CO-Ag cDNA sequence is identical to that of human neutrophil calgranulin C (CaGC). This study sought to demonstrate expression of the CaGC gene in the human cornea and in corneal keratocytes after cytokine stimulation. In situ hybridization and immunohistochemistry 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-1α or TNF-α, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify CaGC cDNA from cytokine-stimulated keratocytes and unstimulated controls. Southern blotting verified the specificity of the RT-PCR amplification products. In situ hybridization detected human CaGC mRNA in the stroma of corneas with Fuchs' dystrophy, postinfection corneas, and a cornea with Mooren's ulcer. In cultured bovine keratocytes, peak levels of CaGC mRNA were reached 6 h after cytokine stimulation. Southern blots with an oligonucleotide probe specific for CaGC detected the RT-PCR products of expected sizes (273 bp) 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 the human cornea and the ability of stromal keratocytes to produce CaGC (CO-Ag). The up-regulation of CaGC gene expression by corneal keratocytes due to proinflammatory cytokines from trauma or inflammation may induce autoimmunity that ultimately results in Mooren's ulceration. © 1999 Academic Press.