Interleukin-1 receptor role in the viability of corneal myofibroblasts

Abstract

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p < 0.001) in the IL-1RI knockout group than in the control group at one month, three and six months after irregular PTK. Mean TUNEL+ stromal cells in the anterior 50 μm of stroma was significantly lower in the IL-1RI knockout group compared to the control group at six months after irregular PTK (p = 0.04). These results corroborate the findings of recent in vitro work that demonstrated an antagonistic effect of TGFβ and IL-1 on myofibroblast viability, and found that IL-1-triggered myofibroblast apoptosis was suppressed by TGFβ. Thus, IL-1 is an important modulator of myofibroblast viability during corneal wound healing.

Fig. 1

Irregular PTK was performed by positioning a fine mesh screen in the path of laser over the mouse cornea for the final 50% of the pulses.

Fig. 2

Immunohistochemistry for α-smooth muscle actin (SMA) in the central corneal stroma of control (CO) and IL-1 receptor knockout (KO) mouse corneas with haze at one month, three months or six months following irregular PTK. SMA+ myofibroblast density was significantly higher in the knockout than control mice, especially at the six-month time point, where myofibroblast density had markedly declined in the control cornea compared to three-month time point after irregular PTK. In the IL-1 receptor knockout mice, the SMA+ myofibroblast density has not declined and actually appears increased in this individual section. In unwounded corneas, SMA+ cells were not detected in either unwounded IL-1 receptor knockout (unwounded) or unwounded control (not shown) corneas. Representative SMA+ myofibroblasts are indicated by arrows. The bracket and e indicates the corneal epithelium in each panel. Magnifications 400X.

Fig. 3

Quantitative analysis of α-smooth muscle actin-positive myofibroblasts in central cornea stromal cells/400× microscope field at one month, three months, or six months after irregular PTK in the IL-1 receptor knockout and control groups. Error bars represent SEM. * indicates that the density of SMA+ cells was significantly different (p < 0.001) in the knockout that had irregular PTK compared to the control that had irregular PTK at one month, three months or six months after surgery. SMA+ cell density in the central corneal stroma did not significantly decrease over time in the KO corneas from one month to three months after surgery or from three months to six months after irregular PTK. Conversely, SMA+ myofibroblast density in the central corneal stroma significantly decreased between one and three months (**) or between three and six months (***) after irregular PTK in control corneas.

Fig. 4

Representative TUNEL+ stromal cells (arrows) in the anterior 50 µm of stroma in the IL-1 receptor knockout group (A) and control group (B) at three and six months after irregular PTK. Arrows indicate rare TUNEL+ cells in the anterior 50 µm of stroma in each group. e indicates the epithelium. Note there are also normal TUNEL+ cells in the apical epithelium of each cornea. Magnifications 400X.

Fig. 5

Quantitative analysis of TUNEL+ stromal cells per slide in the anterior 50 µm of stroma at one month, three months, and six months after irregular PTK in the IL-1 receptor knockout and control groups. Error bars represent SEM. Note that mean apoptotic cells in the anterior stroma was less in the IL-1 receptor knockout group than the control group at each time point, although the difference was only statistically significant (asterisk, p = 0.04) at six months after irregular PTK.