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Nicholas J. Ward, Rebecca M. Sappington; Pressure-sensitive Modulation of Chemokine CCL5/RANTES by Retinal Glia. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3093.
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© ARVO (1962-2015); The Authors (2016-present)
Pressure-induced cytokine release by retinal glia alters retinal ganglion cell (RGC) survival. Here we performed a cytokine array that identified increased secretion of the chemotactic cytokine CCL5/RANTES by retinal microglia exposed to elevated hydrostatic pressure. We further examined pressure-sensitive CCL5 secretion using primary cultures of retinal microglia and determined retinal localization of this chemokine in C57 and DBA/2J mice.
Immunomagnetic separation by cell surface antigen OX18/RT1a produced purified cultures of rat retinal microglia. Microglial cultures were exposed to elevated hydrostatic pressure (70 mmHg) for up to 48 hours. A multi-plex protein array was used to quantify cytokine levels in cell culture supernatants. Cytokines examined by the array were CCL5, CX3CL1/fractalkine, IL-2, IL-4, IL-6, IL-10, TGF-β1, and TNF-α. Pressure-induced changes in CCL5 secretion by microglia were confirmed by ELISA. We determined CCL5 localization in cell culture and retinal whole mounts by co-immunolabeling for CCL5 and the RGC marker SMI-31, astrocyte marker GFAP, or microglia marker Iba-1.
From candidates screened by cytokine array, only IL-6 and CCL5 exhibited increased secretion from microglia exposed to 24 hours of elevated pressure. We confirmed this two-fold increase in CCL5 secretion by ELISA (p<0.05). Immunocytochemistry of pressure-treated microglia demonstrated increased intracellular CCL5 levels when compared to control microglia. In C57 mouse retina, CCL5 localized to the ganglion cell layer, nerve fiber layer, inner nuclear layer, and both plexiform layers. In C57 retina, CCL5 co-localized with cell markers for microglia, astrocytes and RGCs. The intensity of CCL5 labeling in aged DBA/2J whole-mounted retinas was greater in astrocytes, but lower in RGCs when compared to C57 retinas.
Our data suggest that pressure induces modulation of retinal CCL5 localization and secretion profiles. Expression of this chemokine by microglia, astrocytes, and RGCs suggests that CCL5 signaling may function as a crosstalk mechanism for glial cells and RGCs that is modulated by pressure during glaucoma pathogenesis.
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