Injury to the corneal epithelium can occur through mechanical or chemical trauma, as well as through elective refractive surgeries (e.g., LASIK or PRK). Despite our current understanding that neutrophil recruitment to the injured cornea can be important for wound healing, many questions remain regarding the molecular mechanisms regulating neutrophil migration within the corneal stroma. Our working hypothesis is that corneal keratocytes provide an important adhesive substrate for integrin-dependent neutrophil adhesion and motility within the injured cornea. The primary objective of this proposal is to define basic molecular adhesive interactions regulating neutrophil migration in ocular injury and disease.
In Specific Aim 1, we will determine the relative contribution of specific adhesion molecules to neutrophil surface interactions with keratocytes and the extracellular matrix during corneal wound healing. Using electron microscopy, we will evaluate the relative surface densities of neutrophil plasma membrane in contact with keratocyte plasma membrane and ECM (e.g., collagen fibrils) following corneal injury in the mouse. The relative contribution of specific adhesion molecules to these surface interactions will be assessed using antibody blockade and mice deficient in P- and E-selectin, leukocyte integrins (CD11a/GD18, CD11b/CD18, CD18), and members of the IgG superfamily (ICAM-1, PECAM-1 and JAM-C). The relative importance of endothelial versus neutrophil PECAM-1 expression will be assessed by bone marrow transplantation.
In Specific Aim 2, we will determine the relative contribution of specific adhesion molecules to neutrophil motility on corneal keratocytes. We will assess how transendothelial migration and endogenous keratocyte chemokine secretion alter neutrophil motility on cultured mouse and human keratocytes. Specific contributions of adhesion molecules (Beta 1-3 integrins, PECAM-1, ICAM-1, VCAM-1 and JAM-C) will be assessed using mutant mice and antibody blocking strategies. Information gained from these studies will help delineate novel adhesive mechanisms underlying neutrophil migration in the injured cornea and this may define new targets for therapeutic treatment of ocular inflammation associated with injury or infection.
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