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D. Yang, S. G. Elner, A. L. Kindezelskii, X. Chen, A. J. Clark, H. R. Petty, V. M. Elner; Role of Monocyte-Induced Ca2+ Signaling in Human Retinal Pigment Epithelial Cell Reactive Oxygen Metabolite Production and Apoptosis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2204.
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Low-grade inflammation is now recognized to play a role in age-related macular degeneration (AMD). The inflammatory response in AMD lesions is characterized by mononuclear phagocyte and lymphocyte infiltration of the outer blood-retina barrier that is formed by the retinal pigment epithelium (RPE). A key mechanistic element in AMD progression is RPE dysfunction and apoptotic loss. In this study, we evaluated the role of monocyte (Mϕ)-induced sequential RPE Ca2+ signaling, and reactive oxygen metabolite (ROM) production in RPE apoptosis.
Primary human RPE cell cultures, freshly-isolated human peripheral Mϕ, and U937 monocytic line were used. Interferon-γ (IFN-γ) or monocyte chemoattractant protein-1 (MCP-1) was used to activate Mϕ. High-speed fluorescence microscopy was used to visualize Ca2+ signaling in RPE cells loaded with indo-1. Intracellular RPE ROM production was measured by using 5-(and 6)-chloromethyl-2`,7`-dichlorodihydrofluorescence diacetate, acetyl ester (CM-H2DCFDA) assay. Apoptosis was evaluated by caspase-3 detection.
Upon binding, activated Mϕ rapidly induced substantial increases in cytosolic Ca2+ concentrations in RPE cells that were initiated adjacent to sites of Mϕ binding, as visualized by high-speed fluorescence microscopy. Increases in Ca2+ concentration propagated through the cytoplasm and spread from RPE cells bound by activated Mϕ to contiguous surrounding unbound RPE cells by propagating spatial Ca2+ waves. Activated Mϕ-RPE binding subsequently induced high levels of intracellular RPE ROM within 1 hr and apoptosis within 6 hr in the bound and contiguous unbound RPE cells. ROM production was inhibited by the Ca2+ modulator, 8-Br-cADPR, and ROM scavengers, pyrrolidinedithiocarbamate (PDTC) and N-acetyl-cysteine (NAC) which also significantly inhibited apoptosis. Correlations among the extent of Ca2+ signaling wave propagation, ROM deposition, and caspase-3 activation were observed.
These results show that rapidly propagating RPE Ca2+ concentration fluctuations are important signals leading to increased RPE ROM generation and apoptosis upon direct contact with activated Mϕ. The findings also suggest that leukocyte infiltration of the RPE layer may initiate Ca2+ signaling which may promote damage of large areas of RPE monolayer that are not in direct contact with leukocytes.
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