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Julie Sanderson, Sofia Habib, Matthew Felgate, Phillip Wright, Leanne Stokes, Nuwan Niyadurupola, David C Broadway; Regulation of Interleukin-1β by Purinergic Receptors in Müller and Microglial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):234.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown previously that death of retinal ganglion cells (RGCs) can be caused by activation of P2X7 receptors in the human retina, together with a P2X7–mediated increase in interleukin-1β (IL-1β) expression and release. The purpose of this research was to investigate purinergic receptor-mediated induction of IL-1β in glial cells using Müller and microglial cell lines.
Two immortalised cell lines were used: MIO-M1 (human retinal Müller cells) and BV2 (mouse brain microglial cells). Initial experiments investigated cell death in response to ATP and BzATP (24h). Cell viability and death were evaluated using MTS and LDH assays respectively. To investigate IL-1β expression and release, cells were incubated with a sub-lethal concentration of ATP or BzATP for 24h. Induction of IL-1β mRNA and release were evaluated using RT-PCR and ELISA respectively.
BV2 cells exhibited a dose-dependent decrease in cell viability/increase in cell death in response to ATP (10μM - 5mM) with significant changes from 50μM and 3mM respectively (n=4;p<0.05). BzATP (1 - 500μM) showed similar results, with significant changes from 5μM (viability) and 300μM (death) BzATP (n=4;p<0.05). AZ10606120 (200nM), a P2X7 antagonist, significantly protected against decreased viability with ATP (3mM) and BzATP (100μM and 500μM) (n=4;p<0.05). MIO-M1 cells showed no significant change in cell viability or death at the ATP and BzATP concentrations used (n=4). ATP (300µM) significantly increased IL-1β mRNA expression in BV2 cells by 5-fold compared with control at 24h (n=4;p<0.05), although no significant increase in secretion was seen at this timepoint (n=4). BzATP (30μM) caused no significant changes in IL-1β expression or release in BV2 cells (n=4). No significant changes in IL-1β expression or release were observed in MIO-M1 cells following 24h exposure to ATP (300µM) or BzATP (30μM) (n=4).
Müller and microglial cells exhibited differential responses to ATP and BzATP. Inhibition of P2X7 receptors demonstrated the involvement of P2X7 in purinergic-mediated cell death of microglial cells. Microglial cells showed an increase in IL-1β mRNA with ATP but not BzATP which may indicate the involvement of a different purinergic receptor in regulating expression. Understanding the pathway of ATP-mediated IL-1β regulation in microglia may provide insight into the mechanisms of RGC death in glaucoma.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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