Neuronal Release of Cytokine IL-3 Triggered by Mechanosensitive Autostimulation of the P2X7 Receptor Is Neuroprotective

Abstract

Mechanical strain due to increased pressure or swelling activates inflammatory responses in many neural systems. As cytokines and chemokine messengers lead to both pro-inflammatory and neuroprotective actions, understanding the signaling patterns triggered by mechanical stress may help improve overall outcomes. While cytokine signaling in neural systems is often associated with glial cells like astrocytes and microglia, the contribution of neurons themselves to the cytokine response is underappreciated and has bearing on any balanced response. Mechanical stretch of isolated neurons was previously shown to trigger ATP release through pannexin hemichannels and autostimulation of P2X7 receptors (P2X7Rs) on the neural membrane. Given that P2X7Rs are linked to cytokine activation in other cells, this study investigates the link between neuronal stretch and cytokine release through a P2X7-dependent pathway. Cytokine assays showed application of a 4% strain to isolated rat retinal ganglion cells (RGCs) released multiple cytokines. The P2X7R agonist BzATP also released multiple cytokines; Interleukin 3 (IL-3), TNF-α, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1Rα, MIP and CCL20 were released by both stimuli, with the release of IL-3 greatest with either stimuli. Stretch-dependent IL-3 release was confirmed with ELISA and blocked by P2X7R antagonists A438079 and Brilliant Blue G (BBG), implicating autostimulation of the P2X7R in stretch-dependent IL-3 release. Neuronal IL-3 release triggered by BzATP required extracellular calcium. The IL-3Rα receptor was expressed on RGCs but not astrocytes, and both IL-3Rα and IL-3 itself were predominantly expressed in the retinal ganglion cell layer of adult retinal sections, implying autostimulation of receptors by released IL-3. While the number of surviving ganglion cells decreased with time in culture, the addition of IL-3 protected against this loss of neurons. Expression of mRNA for IL-3 and IL-3Rα increased in rat retinas stretched with moderate intraocular pressure (IOP) elevation; BBG blocked the rise in IL-3, implicating a role for the P2X7R in transcriptional regulation in vivo. In summary, mechanical stretch triggers release of cytokines from neurons that can convey neuroprotection. The enhancement of these signals in vivo implicates P2X7R-mediated IL-3 signaling as an endogenous pathway that could minimize damage following neuronal exposure to chronic mechanical strain.

The P2X7 Receptor Primes IL-1β and the NLRP3 Inflammasome in Astrocytes Exposed to Mechanical Strain

“…We hypothesized that the increased expression of following IOP elevation might relate to the release of ATP and autostimulation of P2X7 receptors found in optic nerve head astrocytes exposed to mechanical strain ( Beckel et al, 2014 ). This theory was supported by recent findings implicating the P2X7 receptor in the mechanosensitive upregulation of cytokines IL-3 ( Lim et al, 2016 ) and IL-6 ( Lu et al, 2017 ) in the retina. To determine if the P2X7 receptor was involved in IOP-sensitive priming of IL-1β, the P2X7 receptor antagonist BBG (0.8%) was injected intravitreally 1–3 days before the IOP rise, then retinas were collected 22 h after the IOP returned to baseline.…”

The P2X7 receptor links mechanical strain to cytokine IL‐6 up‐regulation and release in neurons and astrocytes

“…), and recent work demonstrates P2X7 receptor stimulation leads to IL‐3 responses in isolated retinal ganglion cells (Lim et al . ). As stimulation of P2X7 receptors by ATP has been associated with the up‐regulation of IL‐6 in microglia cells (Shieh et al .…”

“…Stretch did not induce release of lactose dehydrogenase. IL-6 levels were determined with the rat antibody cytokine array following manufacturer's instructions (R & D Systems), as described in detail in a recent publication (Lim et al 2016). In brief, IL-6 levels were quantified by incubating the membrane in Streptavidin-horseradish peroxidase followed by chemiluminescent detection reagents (GE Healthcare).…”

“…A considerable body of past work implicates autostimulation of the P2X7 receptor following the mechanosensitive release of ATP in the retina (Zhang et al 2006;Reigada et al 2008;Xia et al 2012;Beckel et al 2014), and recent work demonstrates P2X7 receptor stimulation leads to IL-3 responses in isolated retinal ganglion cells (Lim et al 2016). As stimulation of P2X7 receptors by ATP has been associated with the up-regulation of IL-6 in microglia cells (Shieh et al 2014), the role of the P2X7 receptor in mediating the pressure-dependent rise in IL-6 was examined.…”

Characterization of proliferative, glial and angiogenic responses after a CoCl2‐induced injury of photoreceptor cells in the adult zebrafish retina

“…Microglia activation and proinflammatory signal secretion might induce early mitotic activity in retinal areas adjacent to the optic disc and CMZ (at 55 hpl). It has been demonstrated that cultured RGC from rat samples release cytokines, including TNFα and several interleukins, to the extracellular medium (Lim, Lu, Beckel, & Mitchell, ). Our results showed that TNFα transcriptional expression was increased by 72 hpl, supporting the idea of enhanced TNFα release and its involvement in the induction of multipotent cell proliferation, in addition to its known proinflammatory role.…”