Stromal interleukin-1 expression in the cornea after haze-associated injury

Abstract

The purpose of this study was to determine whether myofibroblasts or other cells in the stroma in the cornea produce interleukin (IL)-1alpha or IL-1beta that could modulate myofibroblast viability in corneas with haze after photorefractive keratectomy (PRK). Twenty-four female rabbits had haze-generating PRK for 9 diopters of myopia and were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were removed, frozen in OCT at -80 degrees C, and analyzed by immunocytochemistry using primary antibodies to IL-1alpha, IL-1beta and alpha smooth muscle actin (SMA). Double immunostaining was performed for the co-localization of SMA with IL-1alpha or IL-1beta. Central dense haze and peripheral slight haze regions of each cornea were analyzed. SMA+ cells that expressed IL-1alpha protein were detected in both regions of the corneas at most time points following PRK. However, in the haze region at the 1, 3 and 4 week time points, significantly more (p<0.01) SMA+ cells did not express IL-1alpha. Also, in the haze region at all three time points, significantly more (p<0.01) SMA- cells than SMA+ cells expressed interleukin-1alpha protein. IL-1beta expression patterns in SMA+ and SMA- stromal cells was similar to that of IL-1alpha after PRK. Previous studies have demonstrated that IL-1alpha or IL-1beta triggers myofibroblast apoptosis in vitro, depending on the available concentration of apoptosis-suppressive TGFbeta. This study demonstrates that SMA- cells such as corneal fibroblasts, keratocytes, or inflammatory cells may produce IL-1alpha and/or IL-1beta that could act in paracrine fashion to regulate myofibroblast apoptosis--especially in the region where there is haze in the cornea after PRK was performed and SMA+ myofibroblasts are present at higher density. However, some SMA+ myofibroblasts themselves produce IL-1alpha and/or IL-1beta, suggesting that myofibroblast viability could also be regulated via autocrine mechanisms.

Fig. 1

Slit lamp photo of haze in rabbit cornea at one month after -9D PRK. Arrows not edge of dense haze (†) that corresponds to the edge of ablation by the excimer laser. Peripheral to the ablated zone (††) haze is far less pronounced but the corneal stroma is not as clear as unwounded control corneas (not shown). Magnification 40X.

Fig. 2

Double immunocytochemistry for α-smooth muscle actin (SMA) and IL-1α in the region of the cornea with dense haze (* in Fig. 1) at 4 weeks after −9 D PRK in the rabbit. A. Cell nuclei of all cells stained blue with DAPI. B. SMA+ myofibroblasts (arrows) are present in the anterior stroma. C. IL-1α+ cells (arrows) are also detected in the anterior stroma. Note that IL-1α is also detected in the epithelium (e). D. The overlay of A thru C demonstrates that all of the SMA+ myofibroblasts in this section also express IL-1α, but that there are adjacent SMA− cells that also express IL-1α protein (arrowheads). Note that there is an artifactual separation (*) between the epithelium and stroma that is typically formed in an area with severe haze when corneas with prominent myofibroblasts are sectioned for immunocytochemistry. Panels E to H are corresponding −9D PRK cornea sections in which primary antibody to SMA (F, H) or IL-1α (G, H) was omitted. Mag. 300X.

Fig. 3

Double immunocytochemistry for α-smooth muscle actin (SMA) and IL-1α in the peripheral cornea outside of the dense haze zone (** in Fig. 1) at 4 weeks after −9D PRK and in control unwounded corneas in the rabbit. A to D are from a −9D PRK cornea. A. Cell nuclei of all cells stained blue with DAPI. e is the epithelium. B. SMA+ myofibroblasts (arrows) are present in the anterior stroma. C. IL-1α+ cells (arrows) are also detected in the anterior stroma. D. The overlay of A thru C demonstrates that many (arrows), but not all, of the SMA+ myofibroblasts in this section also express IL-1α. There are also many adjacent SMA− cells that also express IL-1α protein (arrowheads). E to H are corresponding tests performed on unwounded control corneas. Note that no SMA+ cells were detected in the unwounded corneal stroma (F). Very light staining in the epithelium in F is background staining. Very few IL-1α+ cells were detected in the stroma of unwounded corneas (G). In this particular section, only one was noted (arrow, G). Note the heavy IL-1α expression in apical cells (arrowheads) of the unwounded corneal epithelium. This pattern of epithelial expression had not recovered by 1 month after −9D PRK (C). I to L are representative −9D PRK cornea sections in which primary antibody to SMA and IL-1α were omitted. Magnification 300X.

Fig. 4

Higher magnification localization of (A) α-smooth muscle actin (SMA) and IL-1α in the region of the cornea with dense haze (* in Fig. 1) at 4 weeks after −9 D PRK and (B) SMA and IL-1α in the peripheral cornea outside of the dense haze zone (** in Fig. 1) at 4 weeks after −9D PRK. Arrowheads in (A) and (B) show SMA+ myofibroblasts that have simultaneous IL-1α. Arrowheads in (A) and (B) indicate SMA− stromal cells near myofibroblasts that express IL-1α. Note that the IL-1α in many SMA+ cells appeared to be compartmentalized within these cells, perhaps within vesicles or organelles. This is perhaps best seen in B where the arrow with the asterisk is a part of the SMA+ cell also indicated by the nearest adjacent arrow. In this cell the IL-1α is clearly within organelles of some type, probably vesicles. This was best seen in real-time at the microscope. Magnification 1000X.

Fig. 5

Double immunocytochemistry for α-smooth muscle actin (SMA) and IL-1β in the peripheral cornea at the junction of the ablated dense haze zone (* in Fig. 1) and the low haze zone (** in Fig. 1) at 4 weeks after −9D PRK and in control unwounded corneas in the rabbit. A to D are from a −9D PRK cornea. Note that there is an artifactual separation (*) between the epithelium and stroma of A to D that is typically formed in an area with severe haze when corneas with prominent myofibroblasts are sectioned for immunocytochemistry. A. Cell nuclei of all cells stained blue with DAPI. e is the epithelium (in C, G and K). B. SMA+ myofibroblasts (arrows) are present in the anterior stroma to the right where haze was present. C. IL-1β+ cells (arrows) are also detected in the anterior stroma. D. The overlay of A thru C demonstrates that many (arrows), but not all, of the SMA+ myofibroblasts in this section also express IL-1β. There are also many adjacent SMA− cells that also express IL-1β protein (arrowheads). E to H are the corresponding tests performed on unwounded control corneas. In G, there is heavy IL-1β production in the unwounded corneal epithelium. Note that the IL-1β production in epithelium had not returned to normal at one month after PRK in panel C. A few cells in the anterior stroma of the unwounded cornea (G) express IL-1β (arrows). I to L are corresponding −9D PRK cornea sections in which primary antibody to SMA (J, L) or IL-1β (K, L) was omitted. Magnification 300X