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 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 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 TGFβ1. Dose response experiments were performed after withdrawal of TGFβ1 and exposure to 1, 5, or 10 ng/ml of IL-1α or IL-1β for 1 hour. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml IL-1α or IL-1β in conjunction with 0, 1, 5, or 10 ng/ml TGFβ1. 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 TGFβ1, the % TUNEL+ cells at 1 hour 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 IL-1α or IL-1β, the % TUNEL+ cells at 1 hour 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.

Myofibroblasts

“…IL-1 is well studied in the context of corneal myofibroblasts where it was shown not only to antagonize TGF-β1 but to induce myofibroblast apoptosis when 'TGF-β1 protection' is lost (Barbosa et al, 2010b;Kaur et al, 2009a). Concomitantly, myofibroblast apoptosis was reduced and cell numbers augmented in the cornea after phototherapeutic keratectomy in mice deficient for the IL-1 receptor 1 (Barbosa et al, 2012).…”

Cytokine Expression in Keratoconus and its Corneal Microenvironment: A Systematic Review

“…78 Interestingly, TGF-β and IL-1 have antagonistic effect on corneal myofibroblasts; high levels of TGF-β1 increase myofibroblast viability, while IL-1 induces apoptosis in these cells. 79 Thus, low levels of TGF-β1 increase the susceptibility of myofibroblasts to apoptotis mediated by IL-1. Indeed, reduced expression of TGF-β1 has been observed in KC.…”

The role of cardiac fibroblasts in the transition from inflammation to fibrosis following myocardial infarction

“…Further evidence for the counterbalancing effects of proinflammatory cytokines and profibrotic cytokines comes from 13 studies using fibroblasts of non-cardiac origin. For example, IL-1-induced apoptosis in corneal myofibroblasts was prevented by TGF1 (Kaur et al, 2009). In a co-culture of keratinocytes and dermal fibroblasts, TGF-mediated SMA induction was suppressed by IL-1 (Shephard et al, 2004).…”