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Jason C. Lim, Wennan Lu, Jonathan M. Beckel, Margaret Buell, Jingsheng Xia, Edward J. Macarak, Alan M. Laties, Claire H. Mitchell; Stretch-activated IL-3 Release From Retinal Ganglion Cells Is Protective And Involves The P2X7 Receptor. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6594.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated intraocular pressure (IOP) leads to impairment and death of retinal ganglion cells (RGCs) in glaucoma. Inflammatory signals triggered by increased IOP contribute to the pathogenesis, although new evidence suggests they may also have a beneficial role in some situations. While astrocytes are known to release cytokines, this study examines whether RGCs themselves specifically release cytokines in response to mechanical strain and begins to probe their function.
RGCs were isolated by immunopanning and seeded on silicone substrates in stretch chambers or coverslips coated with poly-L-lysine and laminin. Cells were stretched for 15 min (3x 4 min, 4.1% strain then 1 min, 0% strain) or stimulated by a 30 min incubation with P2X7 receptor agonist BzATP. Released interleukin-3 (IL-3) was assayed using a sandwich ELISA. IL-3 receptor expression was investigated by Western blotting. The survival of RGCs back-labeled with FluoroGold exposed in primary culture to IL-3 for 24 hrs was determined by counting fluorescent cells. qPCR was performed on primary rat astrocytes.
IL-3 was released from stretched RGCs. This release of IL-3 was inhibited by P2X7 receptor antagonists A438079 and Brilliant Blue G by 59.0% and 69.9%, respectively. IL-3 was also released after exposure of RGCs to P2X7R agonist BzATP. The BzATP-triggered IL-3 release was reduced 74.3% by removal of extracellular calcium and reduced 62.3% by the P2X7 receptor antagonist A438079. The ligand-binding alpha subunit of the IL-3 receptor was expressed in retinal ganglion cells, but not in optic nerve head astrocytes. The presence of IL-3 enhanced survival of RGCs after 24 hrs in culture but did not affect astrocytes levels of GFAP or NFkB mRNA.
Mechanical strain triggers IL-3 secretion from RGCs. As ATP is released from stretched ganglion cells, autocrine stimulation of the P2X7 receptor likely leads to IL-3 release. The presence of the IL-3 binding receptor subunit on retinal ganglion cells and the ability of IL-3 to promote survival of ganglion cells imply a neuroprotective role for IL-3, although the balance of pro- and anti-survival signals from the P2X7 receptor may be complex.
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