Macrophages in Corneal Epithelial Wound Healing

Purpose: After corneal epithelial injury, the ensuing inflammatory response is necessary for efficient wound healing. While beneficial healing effects are attributed to recruited neutrophils and platelets, little is known regarding the relative distribution of macrophage phenotypes within the cornea and whether macrophages contribute to the inflammatory cascade that is so important for corneal wound healing. The objectives of this research were: (1) To characterize macrophages in normal and wounded mouse corneas; (2) To determine if macrophage-derived IL-20 plays a beneficial role in corneal wound healing; (3) To determine if oncomodulin, a potent stimulus for nerve regeneration known to be present in macrophages and neutrophils, plays a beneficial role in corneal wound healing.

Methods: In all wounded corneas, a 2mm diameter central epithelial region was mechanically debrided with a gulf-club spud. (1) Fluorescently tagged antibodies raised against mouse macrophage markers (F4/80, CD115, CX3CR1 and CD206) together with known M1 (CD80, CD86) and M2 (CD301) pro- and anti-inflammatory markers, respectively, were used to identify and localize macrophages within corneal wholemounts of wildtype mice before and after injury (24, 48, 72h and 7days). To enumerate different macrophage phenotypes within the cornea, whole mounts were sub-divided into five regions: (a) limbus (L), (b) paralimbus (PL), (c) parawound (PW), (d) wound (W) and (e) wound center (WC). Within each region, a single camera field (40X, 150 x 150 µm) was recorded and cells staining positively for macrophage markers were counted. (2) Injured female wildtype C57BL/6 mouse corneas were topically treated every 4h up to 24h with 10l of neutralizing IL-20 antibody (200g/ml) or control antibody (non-immune isotype matched IgG). To examine the effects of recombinant IL-20 (rIl-20) on epithelial wound healing, wildtype mice, neutrophil-depleted wildtype mice (anti-Ly6G antibody pre-treatment) , and mutant mice known to have reduced neutrophil infiltration (ɣδ T cell deficient mice (TCR-/-) and CD11a deficient mice (CD11a-/-)) received 10µL of rIL-20 dissolved in phosphate buffered saline (200ng/mL) every 4h up to 24h, while appropriate control mice received buffer only. The rate of corneal wound closure, the numbers of dividing basal epithelial cells, infiltrating neutrophils, and platelets, and the density of epithelial nerves were evaluated. Some corneas were prepared for immunofluorescence microscopy to localize IL-20 and its receptor, IL-20R1. (3) Some wildtype mice receiving corneal epithelial abrasions were topically treated with oncomodulin-specific blocking peptide P1 (100ng/5ul) or control peptide P3 (100ng/5ul), respectively. The process was repeated every 6 hours for 24 hours. The rate of corneal wound closure, epithelial nerve density, and number of dividing epithelial cells were evaluated to assess the functional contribution of oncomodulin on epithelial wound healing. Some dissected mouse corneas were immunostained with anti-oncomodulin antibody together with CD301 (M2 macrophage marker), Ly6G (neutrophil marker), and FITC-avidin (mast cell marker) to assess the cellular localization of oncomodulin. Non-immune isotype matched antibody served as a control for non-specific staining.

Results: (1) CD301+ CD80- cells were present at the limbus and showed a strong association with limbal vessels. CD301+ cells were negative for CX3CR1 but positive for all other macrophage markers examined (F4/80, CD115, and CD206), suggesting these cells are CD301+ M2 macrophages. Within the uninjured mouse cornea, macrophages failed to stain with antibodies against M1 markers (CD86 and CD80). However, CD80+ cells were observed at the limbus and additional staining with FITC-avidin established their identity as mast cells. Prior to injury, CD301+ macrophages were found at the L and PL. 24h after wounding, CD301+ macrophage numbers declined by 50% at the L (P0.05) and were elevated at the PL, PW, W and WC (P0.05). By 48h and 72h post-injury, CD301+ macrophage numbers returned to baseline at the L while their numbers remained elevated at the PL, PW, W and WC. Seven days after wounding, CD301+ macrophage numbers showed a second decline to 67% of baseline at the L while their numbers in the PL and PW returned to baseline values and remained elevated at the W and WC. Mast cell numbers at the limbus remained unchanged after wounding. (2) Corneal epithelium and keratocytes showed positive staining for IL-20. CD301+ macrophages and CD11C+ dendritic cells were negative for IL-20. Immunostaining confirmed IL-20R1 was expressed on wildtype corneal epithelial cells prior to injury. Topical application of neutralizing IL-20 antibody markedly delayed epithelial wound closure between 8 and 24h post-injury (P≤0.05) and, unlike control mice, the wound remained open at 24h. In addition, fewer dividing epithelial cells were detected across the cornea (P≤0.05) and epithelial nerve recovery was also markedly depressed by anti-IL-20 treatment, while neutrophil infiltration increased (P≤0.05). Topical administration of rIL-20 accelerated wound closure, increased epithelial cell division and enhanced nerve recovery; similar findings were also observed in injured neutropenic, TCR-/- and CD11a-/- mice. The already low neutrophil infiltration in TCR-/- mice was inhibited further by rIL-20 treatment (P≤0.05). In wildtype mice, topical application of rIL-20 decreased neutrophil infiltration and platelet accumulation while the overall rate of wound closure was not affected. Further investigation revealed these mice had 39% more dividing epithelial cells at the PL and a higher nerve density at the WC at 24h after rIL-20 treatment. (3) Oncomodulin staining was predominantly found in mast cell granules, while weaker staining was observed in the infiltrating neutrophils. Corneal CD301+ macrophages showed no evidence of oncomodulin staining. During the first 24h after corneal abrasion, topical application of the oncomodulin receptor blocking peptide P1 did not affect the epithelial wound closure rate. However, nerve regeneration was significantly depressed at the PL and W (P≤0.05) when compares to control peptide P3 treated corneas.

Conclusions: In summary, macrophages with an anti-inflammatory phenotype (M2, CD301+) were detected at the limbus and within the corneal stroma. Pro-inflammatory macrophages (M1) were not detected, even after corneal epithelial abrasion. CD301+ macrophage infiltration into the wounded cornea is biphasic and the corneal epithelium and keratocytes appear to be the source of IL-20, a cytokine that is necessary for efficient corneal epithelial wound closure, cell division and nerve recovery. The fact that IL-20 also inhibits neutrophil and platelet recruitment identifies IL-20 as a possible beneficial therapeutic agent capable of sustaining wound healing while limiting neutrophil and platelet infiltration. Oncomodulin, a potent stimulus for nerve regeneration, was detected in the cornea but not in macrophages. Oncomodulin staining was observed in mast cells and infiltrating neutrophils (after corneal injury) and oncomodulin appears to contribute to nerve regeneration as blocking the binding of oncomodulin to its receptor using the P1 peptide delayed nerve regeneration during the first 24h after abrasion. Mast cells, unlike neutrophils, are resident cells at the limbus and the observation that oncomodulin staining is found in mast cells raises the possibility that these cells play an important role in maintaining the health of epithelial nerves in the uninjured cornea.