Role of Macrophage Chemotactic Protein–1 and Interleukin–1beta During Inflammatory Neovascularization in the Mouse Cornea

Abstract

Abstract: : Purpose: Accumulating evidence has suggested that corneal neovascularization is a manifestation of an inflammatory response. We used a mouse inflammatory corneal model (corneas cauterized with silver nitrate) and assessed the role of monocyte/macrophage–attracting factors, macrophage chemotactic protein–1 (MCP–1), and a proinflammatory cytokine, IL–1beta, on macrophage recruitment and neovascularization. Methods: Corneal samples were isolated at selected times for enzyme–linked immunosorbent assay (ELISA) and quantitative RT–PCR to quantify the expression levels of MCP–1 and IL–1beta. Eyes were sectioned for in situ hybridization to determine the cellular sources of MCP–1 and IL–1beta. Immunocytochemistry was also used to determine the spatial distribution of macrophages. Corneal pocket assay was used to assess the angiogenic potential of each molecule. Results: Both MCP–1, IL–1beta protein, and mRNA levels increased markedly 12 hours after the chemical cauterization. In situ hybridization showed that MCP–1 was located in corneal epithelial cells, and IL–1beta was located in corneal epithelial cells and infiltrating inflammatory cells. In addition, double staining of corneas with antibodies specific for monocytes/macrophages and IL–1beta revealed that IL–1beta was found in infiltrating monocytes/macrophages at Day 2 after cauterization. Both IL–1beta and MCP–1 induced neovascularization in a rat cornea model, and the cauterization–induced corneal neovascularization was partially inhibited by subconjunctival injection of anti–IL–1beta or anti–MCP–1. Coadministration of two antibodies inhibited corneal neovascularization slightly more than that by the administration of each. Cauterized corneas treated with anti–MCP–1 antibody had significantly fewer monocytes/macrophages than control. Conclusions: The results indicate the existence of distinct monocyte/macrophage–involved angiogenic pathways in mouse cornea, in which MCP–1 released from corneal epithelial cells attracts monocytes/macrophages into the cornea, where they release IL–1beta leading to inflammatory neovascularization. In addition, the IL–1beta and MCP–1 released from the corneal epithelial cells may directly induce corneal neovascularization.

Keywords: neovascularization • wound healing • cornea: basic science