Corneal myofibroblast viability: Opposing effects of IL-1 and TGF β1

The purpose of this study was to test the effect of corneal epithelial scrape on myofibroblasts associated with haze and elucidate the effect of interleukin-1 and transforming growth factor beta 1 on corneal stromal myofibroblasts viability and death in vitro. Corneal epithelial scrape was performed in rabbit eyes with severe haze at one month after -9 diopter photorefractive keratectomy. Corneas were processed for immunocytochemistry for myofibroblast marker alpha-smooth muscle actin (alpha-SMA) and the TUNEL assay to detect apoptosis. Rabbit corneal fibroblasts were cultured with 2 ng/ml of transforming growth factor beta 1 (TGF beta 1) to induce myofibroblast differentiation confirmed by monitoring a-SMA expression. Fluorescence-based TUNEL assay was performed to analyze the apoptotic response of myofibroblasts to IL-1 alpha or IL-1 beta, in the presence or absence of TGF 1. Dose response experiments were performed after withdrawal of TGF beta 1 and exposure to 1, 5, or 10 ng/ml of IL-1 alpha or IL-1 beta for 1 h. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml of IL-1 alpha or IL-1 beta in conjunction with 0, 1, 5, or 10 ng/ml of TGF beta 1. Corneal epithelial scrape with a scalpel blade produced myofibroblast apoptosis. Exposure to TGF beta 1 in vitro resulted in greater than 99% transformation of corneal fibroblasts to alpha-SMA+ myofibroblasts. There was a statistically significant dose-dependent increase in the percentage of TUNEL+ cells with either IL-1 alpha or IL-1 beta initiated at concentrations as low as 1 ng/ml. For example, after withdrawal of TGF beta 1, the % TUNEL+ cells at 1h after exposure to IL-1 alpha increased significantly with increasing concentration (0 ng/ml, 2.4 +/- 0.8% [S.E.M.]; 1 ng/ml, 15.4 +/- 1.8%; 5 ng/ml, 47.4 +/- 3.9%; or 10 ng/ml, 70.3 +/- 3.2%). Similar results were obtained with IL-1 beta. The differences between the means of apoptotic myofibroblasts for the different concentrations of cytokine for either IL-1 alpha or IL-1 beta were significantly different (ANOVA, p < 0.001). When myofibroblasts were exposed to 5 ng/ml of IL-1 alpha or IL-1 beta, the % TUNEL+ cells at 1 h were reduced in a significant dose-dependent manner when TGF beta 1 at a concentration of 5 ng/ml or 10 ng/ml was present in the medium (ANOVAp < 0.01). IL-1 alpha or IL-1 beta triggers the death of myofibroblasts in vitro and TGF beta 1 reduces the IL-1 effect on cell death. TGF beta 1 and IL-1 have opposing effects on myofibroblast viability and likely interact to modulate haze generation after corneal injury. (C) 2009 Elsevier Ltd. All rights reserved.