Abstract
To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ.
Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry.
A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury.
Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.
