While defending against pathogens that enter its open environment, the eye must protect itself from self cell damage that would impair visual acuity. In earlier work, we found that the decay accelerating factor (DAF or CD55), membrane cofactor protein (MCP or CD46), and the membrane inhibitor of reactive lysis (MIRL or CD59), three surface proteins that play critical roles in protecting blood cells from autologous complement-mediated injury are both ubiquitously expressed intraocularly and present in unusually high levels on the ocular surface. In the previous grant cycle, we showed that functionally active soluble forms of the molecules are present in tears and aqueous humor, and obtained evidence that changes in the cell-associated regulators are clinically relevant in ocular infections. Although these proteins have received limited study to date, an understanding of their physiological importance is beginning to emerge, the most important finding being that in a rat model the localized blocking of certain regulator activities in the conjunctiva or the anterior chamber can result in massive inflammation as well as frank tissue necrosis. One main obstacle to fully studying the regulators has been the lack of appropriate experimental animal models allowing for direct analysis of regulator functions throughout the eye in pathologic processes. In recent work, we have 1) developed a knockout mouse for one of the regulators, DAF, and 2) obtained preliminary data in a bacterial keratitis model in this knockout that neutrophil infiltration and destruction of ocular tissues are dramatically increased. In another line of studies of complement regulation, we have 1) performed exploratory studies in the eye of C5a receptors (C5aR), a newly-described membrane element that activates cellular responses and is operative in multiple brain cell types, 2) unexpectedly found that C5aR are present on retinal pigment epithelial (RPE) cells as well as corneal cells, and 3) shown that C5a ligation of C5aR on RPE cells induces the release of a number of cytokines. In this application we propose to 1) exploit our DAF knockout mouse in conjunction with CD59 knockouts developed by our collaborators to precisely define the physiological importance of the regulators in ocular infections, and 2) fully characterize the functions of C5aR on RPE and corneal cells and examine their role in immunological processes. For this work, in addition to available human and mouse antibodies, mouse knockouts will be utilized. The studies should provide new insights into how both complement regulators and C5aR serve to protect the eye, new knowledge about RPE cells and corneal cells, and new information regarding host defense mechanisms in ocular infections.
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