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Alexa Klettner, Timothy Hamann, Sebastian Wilker, Christin Berg, Karen Schlüter, Ralph Lucius, Mentlein Rolf, Johann Roider; Differential effects of TLR-3 activated RPE on retinal microglia and blood-derived monocytes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4519.
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Toll-like receptor 3 (TLR-3) is a receptor of the innate immune system recognizing double stranded RNA. RPE cells express TLR-3 receptor and react to TLR-3 stimulation. As RPE cells may function as sentinels of the innate immune system in the retina, we investigated the effect of TLR-3 activation of the RPE on retina-based microglia and blood-based monocytes.
Primary porcine RPE and primary porcine microglia cells were prepared from porcine retina. Primary porcine monocytes were obtained from porcine blood. TLR-3 activation was induced by polyinolinic acid:polycytidylic acid (Poly I:C). RPE cells were stimulated with Poly I:C in different concentrations for 24 h. Filtered cell culture supernatant was applied to microglia and monocytes, respectively, for various time peroids. mRNA was prepared using Trizol lysis. Expression of IL-6, IL-1 beta, TNF alpha, Cox2, and iNOS was evaluated in Real Time PCR, using TaqMan Gene expression arrays. Expression of FasL was monitored by Flow Cytometry. Phagocytosis was determined in a phagocytosis assay using FITC-labelled latex beads.
Supernatant of Poly I:C-stimulated RPE cells induces a concentration dependent activation of IL-6 IL-1 beta, and Cox2, but not TNF alpha or iNOS in microglia. The activation exceeds the activation induced by Poly I:C alone. Phagocytotic activity did not appear to be affected. In contrast, monocytes do not display an upregulation of inflammatory cytokines induced by Poly I:C or supernatant of Poly I:C activated RPE cells. However, contact with RPE supernatant, irrespective of TLR-3 activation, induces FasL expression on monocytes. RPE supernatant also induced a reduction of iNOS expression.
TLR-3 stimulated RPE cells activate retinal microglia, inducing IL-6, IL-1 beta and Cox2 expression. This is not seen in blood-derived monocytes where RPE cells induce FasL expression and a reduction of iNOS expression. These results indicate an activating function of RPE cells on retinal microglia but not on monocytes.
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