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.

Fig. 1

Myofibroblast apoptosis following epithelial scrape in corneas with haze. Sections underwent double TUNEL assay (red) and immunocytochemistry for α-smooth muscle actin (green). Coverslips were mounted with DAPI to stain cell nuclei (blue). A. At one month after -9 diopter PRK without epithelial scrape, myofibroblasts stained for α smooth muscle actin were detected in the anterior stroma (arrows). TUNEL-positive cells are noted in the epithelium (e), but not in the myofibroblasts in this section. When many sections from unscraped corneas with haze were examined, rare myofibroblasts were noted to be TUNEL+, but the majority were TUNEL-. The asterisks indicate an artifactual separation between the epithelium and stroma that occurred during preparation of this section. B. At one month after -9 diopter PRK, the epithelium was scraped gently with a scalpel blade and 1 hour later the cornea was processed for immunocytochemistry. Myofibroblast cells (arrows) in the anterior stroma double stained for the myofibroblast marker α-smooth muscle actin (green) and the TUNEL assay for apoptosis. Heavy scraping of the epithelium with a scalpel completely removed the myofibroblasts and associated extracellular matrix material (not shown). Magnification 500X.

Fig. 2

Alpha smooth muscle actin (green) staining in myofibroblasts cultured in RSF with 1% FBS and 2 ng/ml TGF β1. Blue is DAPI stained nuclei. Magnification 200X.

Fig. 3

Effect of IL-1α and IL-1β on apoptosis of corneal myofibroblasts. Representative TUNEL and immunocytochemistry for alpha smooth muscle actin staining of myofibroblasts in consecutive slides exposed to vehicle (A, D), 5 ng/ml IL-1α (B, E) or 5 ng/ml IL-1 β (C, F). Red is TUNEL assay staining. Green is α-smooth muscle actin staining. Blue is DAPI staining of nuclei. Magnification 400X.

Fig. 4

Dose response study showing % apoptosis in myofibroblasts in vitro in response to one hour of exposure to 1, 5, or 10 ng/ml of IL-1α (a) or IL-1β (b). The error bars represent the mean ± the standard error of the mean. *, **, and *** indicate that the means are different from one another (ANOVA, p <0.01).

Fig. 5

Percent apoptosis in treated with 5 ng/ml IL-1α (A) or IL-1β (B), along with increasing dosages of TGF β1 from 0 to 10 ng/ml. The error bars represent the means ± standard error of the mean. *, **, and *** indicate that the means are significantly different from MEM alone and are significantly different compared to each other (ANOVA p < 0.01)

Fig. 6

Schematic diagram of hypothesized interactions between epithelial TGF β and PDGF and stromal IL-1 in modulating myofibroblast viability and death. A. After corneal injuries that lead to structural and/or functional defects in the epithelial basement membrane (BM), TGF β and PDGF from the epithelium gain access to the stroma at sufficient concentrations to stimulate differentiation of myofibroblast precursors. B. The myofibroblast developmental sequence includes the development of a pro-apoptotic autocrine IL-1 cycle that, however, is blocked by TGF β. Alternatively, paracrine IL-1 could be released by other stromal cells such as keratocytes (not shown). C. Eventually, repair of the epithelial basement membrane (BM) leads to a fall in epithelium-derived TGF β and PDGF in the corneal stroma and the IL-1 effect on myofibroblasts is unchecked. D. IL-1 unopposed by TGF β triggers apoptosis of myofibroblasts.