TNF-–Induced Inhibition of Gap Junction–Mediated Intercellular Communication Among Human Corneal Fibroblasts in Culture

Abstract

Abstract: : Purpose: Corneal fibroblasts are connected to each other by gap junctions, which mediate intercellular communication and thereby contribute to maintenance of corneal homeostasis. The possible impact of pathological conditions such as inflammation on gap junctional intercellular communication (GJIC) among corneal fibroblasts was investigated by examining the effects of the inflammatory cytokine tumor necrosis factor–α (TNF-α) on such communication in cultured human corneal fibroblasts. Methods: GJIC was assessed by observing the intercellular diffusion of a fluorescent dye. A single cell was injected with Lucifer yellow and the number of cells containing the dye 1 min thereafter was counted. Expression and phosphorylation of the gap junctional protein connexin43 (Cx43) were evaluated by immunoblot and immunocytochemical analyses with a monoclonal antibody to this protein. Results: TNF-α induced a significant decrease in the number of cells to which the dye was transferred. This effect was both dose and time dependent. Immunoblot analysis revealed four specific Cx43 bands, one corresponding to the nonphosphorylated form of the protein and three corresponding to phosphorylated forms. Exposure of the cells to TNF-α (10 ng/ml) for 24 h resulted in a significant decrease in the relative abundance of the three phosphorylated forms of Cx43. Immunocytochemistry also revealed that such treatment reduced the level of specific staining for Cx43 at sites of contact between adjacent cells. Conclusions: TNF-α inhibited GJIC among corneal fibroblasts, presumably by reducing the proportion of phosphorylated forms of Cx43. These data suggest that down-regulation of GJIC among corneal fibroblasts by TNF-α might contribute to the breakdown of corneal homeostasis during corneal inflammation.

Keywords: gap junctions/coupling • cornea: stroma and keratocytes • cytokines/chemokines