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

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus

Activation of the IL-3 receptor is neuroprotective. (A) Immunoblot showing presence of IL-3 receptor α (IL-3Rα) in immunopanned ganglion cells, but not astrocytes. Bands for IL-3Rα were also detected on material from spleen but not macrophages/microglia (M’Phage). (B) Staining for IL-3Rα (green) was found primarily in the RGC layer in adult retinas. Blue—4′,6-diamidino-2-phenyindole dilactate (DAPI). Neg- no primary antibody, RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ONH, optic nerve head; ON, optic nerve. (C) Staining for cytokine IL-3 (red) was also found predominantly in the RGC layer of the retina. (D) Percentage of ganglion cells decreases with time in culture. Numbers normalized to levels 2 h after plating (n = 3). (E) Images of fluorescently labeled neurons (ex 360 nm). Neurons in mixed culture had been incubated with growth medium (Control), IL-3 (10 ng/ml), IL-3 + AG490 (20 μM) or AG490 alone for 24 h to determine the effects of IL-3 on neuronal survival. (F) Quantification of effect of IL-3 on cell survival. IL-3 increased the number of surviving RGCs (labeled with fluorescent marker fluorogold) after 24 h in culture (*p = 0.003 IL-3 vs. control; #p < 0.05); the number of surviving cells decreases over this period in untreated conditions. The addition of JAK2 inhibitor AG490 reduced the neuroprotective effects of IL-3 (p = 0.027 IL-3 vs. AG490 + IL-3, n = 5–11, normalized to levels in IL-3).
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Figure 3: Activation of the IL-3 receptor is neuroprotective. (A) Immunoblot showing presence of IL-3 receptor α (IL-3Rα) in immunopanned ganglion cells, but not astrocytes. Bands for IL-3Rα were also detected on material from spleen but not macrophages/microglia (M’Phage). (B) Staining for IL-3Rα (green) was found primarily in the RGC layer in adult retinas. Blue—4′,6-diamidino-2-phenyindole dilactate (DAPI). Neg- no primary antibody, RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ONH, optic nerve head; ON, optic nerve. (C) Staining for cytokine IL-3 (red) was also found predominantly in the RGC layer of the retina. (D) Percentage of ganglion cells decreases with time in culture. Numbers normalized to levels 2 h after plating (n = 3). (E) Images of fluorescently labeled neurons (ex 360 nm). Neurons in mixed culture had been incubated with growth medium (Control), IL-3 (10 ng/ml), IL-3 + AG490 (20 μM) or AG490 alone for 24 h to determine the effects of IL-3 on neuronal survival. (F) Quantification of effect of IL-3 on cell survival. IL-3 increased the number of surviving RGCs (labeled with fluorescent marker fluorogold) after 24 h in culture (*p = 0.003 IL-3 vs. control; #p < 0.05); the number of surviving cells decreases over this period in untreated conditions. The addition of JAK2 inhibitor AG490 reduced the neuroprotective effects of IL-3 (p = 0.027 IL-3 vs. AG490 + IL-3, n = 5–11, normalized to levels in IL-3).

Mentions: While the results above suggest the mechanosensitive release of IL-3 depended upon a P2X7R-dependent pathway, additional experiments probed the possible targets and consequences of IL-3. Western blot analysis revealed that the ligand-binding alpha subunit of the IL-3 receptor (IL-3Rα) was expressed by RGCs, but not by ONH astrocytes (Figure 3A). Immunohistochemistry identified RGCs as the primary of location for both IL-3Rα (Figure 3B) and IL-3 (Figure 3C) staining in the posterior eye of adult rats. Together, this implied the IL-3 released by ganglion cells autostimulated their own receptors.


Neuronal Release of Cytokine IL-3 Triggered by Mechanosensitive Autostimulation of the P2X7 Receptor Is Neuroprotective
Activation of the IL-3 receptor is neuroprotective. (A) Immunoblot showing presence of IL-3 receptor α (IL-3Rα) in immunopanned ganglion cells, but not astrocytes. Bands for IL-3Rα were also detected on material from spleen but not macrophages/microglia (M’Phage). (B) Staining for IL-3Rα (green) was found primarily in the RGC layer in adult retinas. Blue—4′,6-diamidino-2-phenyindole dilactate (DAPI). Neg- no primary antibody, RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ONH, optic nerve head; ON, optic nerve. (C) Staining for cytokine IL-3 (red) was also found predominantly in the RGC layer of the retina. (D) Percentage of ganglion cells decreases with time in culture. Numbers normalized to levels 2 h after plating (n = 3). (E) Images of fluorescently labeled neurons (ex 360 nm). Neurons in mixed culture had been incubated with growth medium (Control), IL-3 (10 ng/ml), IL-3 + AG490 (20 μM) or AG490 alone for 24 h to determine the effects of IL-3 on neuronal survival. (F) Quantification of effect of IL-3 on cell survival. IL-3 increased the number of surviving RGCs (labeled with fluorescent marker fluorogold) after 24 h in culture (*p = 0.003 IL-3 vs. control; #p < 0.05); the number of surviving cells decreases over this period in untreated conditions. The addition of JAK2 inhibitor AG490 reduced the neuroprotective effects of IL-3 (p = 0.027 IL-3 vs. AG490 + IL-3, n = 5–11, normalized to levels in IL-3).
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Figure 3: Activation of the IL-3 receptor is neuroprotective. (A) Immunoblot showing presence of IL-3 receptor α (IL-3Rα) in immunopanned ganglion cells, but not astrocytes. Bands for IL-3Rα were also detected on material from spleen but not macrophages/microglia (M’Phage). (B) Staining for IL-3Rα (green) was found primarily in the RGC layer in adult retinas. Blue—4′,6-diamidino-2-phenyindole dilactate (DAPI). Neg- no primary antibody, RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ONH, optic nerve head; ON, optic nerve. (C) Staining for cytokine IL-3 (red) was also found predominantly in the RGC layer of the retina. (D) Percentage of ganglion cells decreases with time in culture. Numbers normalized to levels 2 h after plating (n = 3). (E) Images of fluorescently labeled neurons (ex 360 nm). Neurons in mixed culture had been incubated with growth medium (Control), IL-3 (10 ng/ml), IL-3 + AG490 (20 μM) or AG490 alone for 24 h to determine the effects of IL-3 on neuronal survival. (F) Quantification of effect of IL-3 on cell survival. IL-3 increased the number of surviving RGCs (labeled with fluorescent marker fluorogold) after 24 h in culture (*p = 0.003 IL-3 vs. control; #p < 0.05); the number of surviving cells decreases over this period in untreated conditions. The addition of JAK2 inhibitor AG490 reduced the neuroprotective effects of IL-3 (p = 0.027 IL-3 vs. AG490 + IL-3, n = 5–11, normalized to levels in IL-3).
Mentions: While the results above suggest the mechanosensitive release of IL-3 depended upon a P2X7R-dependent pathway, additional experiments probed the possible targets and consequences of IL-3. Western blot analysis revealed that the ligand-binding alpha subunit of the IL-3 receptor (IL-3Rα) was expressed by RGCs, but not by ONH astrocytes (Figure 3A). Immunohistochemistry identified RGCs as the primary of location for both IL-3Rα (Figure 3B) and IL-3 (Figure 3C) staining in the posterior eye of adult rats. Together, this implied the IL-3 released by ganglion cells autostimulated their own receptors.

View Article: PubMed Central - PubMed

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-&alpha;, CXCL9, VEGF, L-selectin, IL-4, GM-CSF, IL-10, IL-1R&alpha;, 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-3R&alpha; receptor was expressed on RGCs but not astrocytes, and both IL-3R&alpha; 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-3R&alpha; 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.

No MeSH data available.


Related in: MedlinePlus