title: Corneal myofibroblast viability: opposing effects of IL-1 and TGF beta1.
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 beta1 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 beta1 (TGF beta1) to induce myofibroblast differentiation confirmed by monitoring alpha-SMA expression. Fluorescence-based TUNEL assay was performed to analyze the apoptotic response of myofibroblasts to IL-1alpha or IL-1beta, in the presence or absence of TGF beta1. Dose response experiments were performed after withdrawal of TGF beta1 and exposure to 1, 5, or 10 ng/ml of IL-1alpha or IL-1beta for 1 h. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml of IL-1alpha or IL-1beta in conjunction with 0, 1, 5, or 10 ng/ml of TGF beta1. Corneal epithelial scrape with a scalpel blade produced myofibroblast apoptosis. Exposure to TGF beta1 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-1alpha or IL-1beta initiated at concentrations as low as 1 ng/ml. For example, after withdrawal of TGF beta1, the % TUNEL+ cells at 1 h after exposure to IL-1alpha 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-1beta. The differences between the means of apoptotic myofibroblasts for the different concentrations of cytokine for either IL-1alpha or IL-1beta were significantly different (ANOVA, p < 0.001). When myofibroblasts were exposed to 5 ng/ml of IL-1alpha or IL-1beta, the % TUNEL+ cells at 1 h were reduced in a significant dose-dependent manner when TGF beta1 at a concentration of 5 ng/ml or 10 ng/ml was present in the medium (ANOVA p < 0.01). IL-1alpha or IL-1beta triggers the death of myofibroblasts in vitro and TGF beta1 reduces the IL-1 effect on cell death. TGF beta1 and IL-1 have opposing effects on myofibroblast viability and likely interact to modulate haze generation after corneal injury.
authors.affiliation: Cole Eye Institute, The Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Harmeet
authors.initials: H
authors.lastname: Kaur
authors.forename: Shyam S
authors.initials: SS
authors.lastname: Chaurasia
authors.forename: Vandana
authors.initials: V
authors.lastname: Agrawal
authors.forename: Chikako
authors.initials: C
authors.lastname: Suto
authors.forename: Steven E
authors.initials: SE
authors.lastname: Wilson
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003318:Corneal Opacity / Q000473:pathology*; D003319:Corneal Stroma / Q000187:drug effects* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000293:injuries; D005260:Female; D005347:Fibroblasts / Q000187:drug effects*; D020287:In Situ Nick-End Labeling; D007375:Interleukin-1 / Q000494:pharmacology*; D018903:Photorefractive Keratectomy; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D007375:Interleukin-1; D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1