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P2X7-dependent, but differentially regulated release of IL-6, CCL2, and TNF-α in cultured mouse microglia
Chu-Hsin Shieh1, Annette Heinrich, Tsvetan Serchov
1Department of Psychiatry and Psychotherapy, University Hospital of Freiburg, Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany.
Abstract:
ATP is an important regulator of microglia and its effects on microglial cytokine release are currently discussed as important contributors in a variety of brain diseases. We here analyzed the effects of ATP on the production of six inflammatory mediators (IL-6, IL-10, CCL2, IFN-γ, TNF-α, and IL-12p70) in cultured mouse primary microglia. Stimulation of P2X7 receptor by ATP (1 mM) or BzATP (500 µM) evoked the mRNA expression and release of proinflammatory cytokines IL-6, TNF-α, and the chemokine CCL2 in WT cells but not in P2X7(-/-) cells. The effects of ATP and BzATP were inhibited by the nonselective P2 receptor antagonists PPADs and suramin. Various selective P2X7 receptor antagonists blocked the P2X7-dependent release of IL-6 and CCL2, but, surprisingly, had no effect on BzATP-induced release of TNF-α in microglia. Calcium measurements confirmed that P2X7 is the main purine receptor activated by BzATP in microglia and showed that all P2X7 antagonists were functional. It is also presented that pannexin-1 hemichannel function and potential P2X4/P2X7 heterodimers are not involved in P2X7-dependent release of IL-6, CCL2, and TNF-α in microglia. How P2X7-specific antagonists only affect P2X7-dependent IL-6 and CCL2 release, but not TNF-α release is at the moment unclear, but indicates that the P2X7-dependent release of cytokines in microglia is differentially regulated.
Insights
Adenosine triphosphate (ATP) regulates microglia, key immune cells in the brain. ATP stimulation via the P2X7 receptor triggers the release of specific inflammatory mediators like IL-6 and TNF-α, but the exact mechanisms remain under investigation.
Area of Science:
- Neuroimmunology
- Cellular and Molecular Neuroscience
- Inflammation Research
Background:
- Microglia are central to neuroinflammation and brain disease pathogenesis.
- Adenosine triphosphate (ATP) is increasingly recognized as a critical modulator of microglial function.
- The precise mechanisms by which ATP influences microglial cytokine release are not fully elucidated.
Purpose of the Study:
- To investigate the effects of ATP on the production of six key inflammatory mediators in primary mouse microglia.
- To determine the role of the P2X7 receptor in ATP-mediated microglial inflammatory responses.
- To explore potential involvement of pannexin-1 hemichannels and P2X4/P2X7 heterodimers.
Main Methods:
- Primary mouse microglia cultures were used for experiments.
- Stimulation with ATP and BzATP to activate P2X7 receptors.
- Measurement of mRNA expression and release of inflammatory mediators (IL-6, IL-10, CCL2, IFN-γ, TNF-α, IL-12p70).
- Utilized P2 receptor antagonists (nonselective and selective P2X7 antagonists) and calcium imaging.
Main Results:
- ATP and BzATP stimulation of wild-type microglia induced mRNA expression and release of IL-6, TNF-α, and CCL2, but not in P2X7(-/-) cells.
- Nonselective P2 receptor antagonists (PPADs, suramin) inhibited ATP/BzATP effects.
- Selective P2X7 antagonists blocked IL-6 and CCL2 release but surprisingly did not affect BzATP-induced TNF-α release.
- Pannexin-1 hemichannel function and P2X4/P2X7 heterodimers were not implicated in the observed P2X7-dependent mediator release.
Conclusions:
- The P2X7 receptor is a key mediator of ATP-induced release of IL-6, TNF-α, and CCL2 in microglia.
- Differential regulation exists in P2X7-dependent cytokine release, as evidenced by the distinct effects of antagonists on TNF-α versus IL-6 and CCL2.
- Further research is needed to understand the specific pathways governing P2X7-mediated release of different inflammatory mediators from microglia.
