Studies proposed here are aimed at evaluating the local contribution of injured corneal tissues in the pathogenesis of ocular surface inflammations. Our hypothesis is that injury of the corneal epithelium results in the release of inflammatory mediators such as leukocyte chemotactic factors (LCF) which activate and recruit leukocytes into injured sites. We also hypothesize that recruited leukocytes upon activation will induce corneal cell damage by releasing proteolytic enzymes and oxygen metabolites such as hydrogen peroxide (H2O2). Our preliminary results support this hypothesis since we have demonstrated that the addition of H2O2 to epithelial surfaces of isolated rabbit corneas and cultured rabbit corneal epithelial cells (CEpC) results in both epithelial cell injury and release of high levels of LCF specific for rabbit peritoneal neutrophils. Currently we propose to further test our hypothesis by: 1. establishing the optimum release of LCF by isolated corneas and cultured CEpC in response to H2O2 injury. 2. isolating and biochemically characterizing cornea-derived LCF using traditional chromatography and high-performance liquid chromatography (HPLC). 3. determining the homogeneity and/or heterogeneity of LCF released by both isolated corneas and cultured CEpC in response to H2O2 injury. 4. determining the similarity and/or dissimilarity* of cornea-derived LCF to known LCF derived from the serum (e.g., C5a and C3a) or immune cells (e.g., leukotriene B4). 5. evaluating the ability of purified cornea-derived LCF, generated in vitro, to induce leukocyte infiltration in vivo when injected into rabbit corneas. Results of these studies will, thus, identify LCF locally released by corneal tissues in response to H2O2 injury, and suggest a role of cornea-derived LCF in the initiation and amplification of ocular surface inflammation, in vivo, by recruiting leukocytes (with their toxic products) to the cornea. * Structural characterization of novel factors is beyond the scope of the present application and will be considered in future studies.
