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Claire H Mitchell, Jonathan M Beckel, Wennan Lu, Jason Lim, Alan M Laties; TLR3 stimulation leads to ATP release from both RPE cells and optic nerve head astrocytes; a common pathway to escalate the inflammatory response in ocular cells?. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6344.
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© ARVO (1962-2015); The Authors (2016-present)
An overactive immune and/or inflammatory response is a common factor in several retinal disorders such as macular degeneration and glaucoma. Identifying the signaling pathways that link insults to cellular responses is critical. The toll like receptor 3 (TLR3) is activated by endogenous and viral dsRNA and has been implicated in ocular cell death, although effects on chronic pathology are unclear. As ATP is a major recruitment signal for microglial cells, we asked whether stimulation of TLR3 led to ATP release from RPE cells or optic nerve head astrocytes.
Experiments were performed in rat optic nerve head astrocytes, mouse RPE cells and the human ARPE-19 cell line. ATP was determined using the luciferase method, and lactose dehydrogenase (LDH) with standard colormetric assay.
TLR3 agonist poly(I:C)(10 µg/ml) caused ATP release from rat optic nerve astrocytes, mouse RPE cells and ARPE19 cells. Extracellular levels of LDH were not altered in astrocytes or RPE cells after 1 or 24 hrs exposure to poly(I:C), suggesting the rise in extracellular ATP was not due to a generalized cell rupture. The time course of ATP release differed between the cell types; release from astrocytes was transient with peak response after 15 min, while in RPE cells extracellular ATP increased with continued poly(I:C) exposure up to 24 hrs. The release of ATP triggered by poly(I:C) was blocked in both cell types by N-ethylmaleimide but not pannexin blocker probenicid, consistent with ATP release from a lysosomal source. Serum starvation prevented ATP release by poly(I:C) from both astrocytes and RPE cells.
The release of ATP in response to TLR3 stimulation is a generalized response in both astrocytes and RPE cells. The ability of this secreted ATP to attract microglial cells and escalate the response, and the role of lysosomes in this release, remain to be determined.
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