Numerous retinal and uveal diseases possess a common set of pathologic events that appear to be due in part to the elicitation and activation of leukocytes. These maladies include uveitis, endophthalmitis, proliferative vitreoretinopathy (PVR), diabetic retinopathy, and macular degeneration. Although the initiating insult may vary, the ocular pathologic response commonly includes the presence of an inflammatory cell component. In order to elucidate whether a common mechanistic pathway leads to the recruitment, activation, and proliferation of leukocytes in ocular tissue, this project will examine RPE mRNA expression, and the production of three well defined cytokines-- interleukin-8 (IL-8), monocyte chemotactic protein (MCP), and interleukin-6 (IL-6). These cytokines can either be chemotactic for neutrophils, mononuclear phagocytes, and lymphocytes or cause activation and proliferation of lymphocytes. They are produced by RPE cells when stimulated with the pro-inflammatory cytokines (PIC), interleukin-1-beta (IL-1Beta) and tumor necrosis factor-alpha (TNF-Alpha). A unique aspect of this project is the assessment of the mechanisms regulating the production of RPE IL-8, MCP, and IL-6 at the molecular and cellular levels. Initial studies will characterize stimulus specificity and the regulation of their production by PIC, by examining direct gene induction, stability of mRNA transcripts, and actual cytokine protein production and secretion. Subsequent experiments will assess endogenous lymphokines and exogenous pharmacologic agents that modulate IL-8, MCP, and IL-6 expression. These experiments will utilize nuclear transcription analysis and will indicate the molecular levels at which these agents modulate RPE IL-8, MCP, and IL-6 in vitro. Experiments will then be performed to determine whether findings on cultured RPE cells reflect actual mechanisms in intact human tissue. In situ hybridization and immunohistochemistry will be used to localize either IL-8, MCP, and IL-6 mRNA expression or antigenic protein in cytokine-treated uveoretinal explants, surgically-removed vitreous and periretinal cellular material, and diseased human eyes. ELISA and bioactivity assays for each of these cytokines will be performed on vitreous samples from patients with uveitis, endophthalmitis, PVR, and diabetic retinopathy to determine the actual presence or absence of these cytokines in human disease. Molecular techniques to measure and localize mRNA will include Northern blot, nuclear transcriptional, mRNA stability, and in situ hybridization assays. Bioactivity, ELISA, and immunohistochemical assays will be used to determine cytokine production, localization, and secretion. The successful execution of the these studies will begin to explain common mechanisms regulating the recruitment, activation, and proliferation of leukocytes in retinal and uveal diseases. These studies will also provide an understanding of how inflammatory mediators and pharmacologic agents regulate RPE-derived leukocyte chemotaxins at the blood-eye barrier and help direct the development of therapeutic interventions aimed at the inflammatory cell component of many common and blinding ocular diseases.
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