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

Abstract

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 β1 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 α-smooth muscle actin (α-SMA) and the TUNEL assay to detect apoptosis. Rabbit corneal fibroblasts were cultured with 2 ng/ml of transforming growth factor β1 (TGF β1) to induce myofibroblast differentiation confirmed by monitoring α-SMA expression. Fluorescence-based TUNEL assay was performed to analyze the apoptotic response of myofibroblasts to IL-1α or IL-1β, in the presence or absence of TGF β1. Dose response experiments were performed after withdrawal of TGF β1 and exposure to 1, 5, or 10 ng/ml of IL-1α or IL-1β for 1 h. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml of IL-1α or IL-1β in conjunction with 0, 1, 5, or 10 ng/ml of TGF β1. Corneal epithelial scrape with a scalpel blade produced myofibroblast apoptosis. Exposure to TGF β1 in vitro resulted in greater than 99% transformation of corneal fibroblasts to α-SMA+ myofibroblasts. There was a statistically significant dose-dependent increase in the percentage of TUNEL+ cells with either IL-1α or IL-1β initiated at concentrations as low as 1 ng/ml. For example, after withdrawal of TGF β1, the % TUNEL+ cells at 1 h after exposure to IL-1α 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-1β. The differences between the means of apoptotic myofibroblasts for the different concentrations of cytokine for either IL-1α or IL-1β were significantly different (ANOVA, p < 0.001). When myofibroblasts were exposed to 5 ng/ml of IL-1α or IL-1β, the % TUNEL+ cells at 1 h were reduced in a significant dose-dependent manner when TGF β1 at a concentration of 5 ng/ml or 10 ng/ml was present in the medium (ANOVA p < 0.01). IL-1α or IL-1β triggers the death of myofibroblasts in vitro and TGF β1 reduces the IL-1 effect on cell death. TGF β1 and IL-1 have opposing effects on myofibroblast viability and likely interact to modulate haze generation after corneal injury.