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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Interleukin-1β has trophic effects in microglia and its release is mediated by P2X7R pore
Mastura Monif1,2, Christopher A Reid3, Kim L Powell4
1Department of Physiology, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, 3010, Australia.
Background:
Enhanced expression of the purinergic P2X7 receptor (P2X7R) occurs in several neuroinflammatory conditions where increased microglial activation is a co-existing feature. P2X7 receptors can function either as a cation channel or, upon continued stimulation, a large pore. P2X7R-over-expression alone is sufficient to drive microglial activation and proliferation in a process that is P2X7R pore dependent, although the biological signaling pathway through which this occurs remains unclear. Once activated, microglia are known to release a number of bioactive substances that include the proinflammatory cytokine interleukin-1β (IL-1β). Previous studies have linked P2X7R stimulation to the processing and release of IL-1β, but whether the channel or pore state of P2X7R is predominant in driving IL-1β release is unknown and is a major aim of this study. In addition, we will determine whether IL-1β has trophic effects on surrounding microglia.
Methods:
Electron microscopy and immunohistochemistry were used to delineate the sub-cellular localization of P2X7R and IL-1β in primary hippocampal rat cultures. FM1-43 fluorescent dye and confocal microscopy were used to quantify vesicular exocytosis from microglia expressing the pore-forming P2X7R versus a non-pore-forming point mutant, P2X7RG345Y. IL-1β in culture was quantified with an enzyme-linked immunosorbent assay (ELISA). IL-1β intracellular processing was blocked with inhibition of caspase 1 (with a synthetic peptide antagonist), and its extracellular form was neutralized with an IL-1β neutralizing antibody. Microglial activation and proliferation was quantified immunohistochemically with confocal microscopy.
Results:
P2X7R and IL-1β were co-localized in lysosomes. Vesicular exocytosis was higher in microglia expressing the pore-forming P2X7R compared to those expressing the non-pore-forming mutant. There was increased IL-1β in cultures expressing the pore-forming P2X7R, and this proinflammatory cytokine was found to mediate the trophic effects of P2X7R pore in microglia. Inhibition of IL-1β production and function resulted in a significant decrease in P2X7R-mediated microglial activation and proliferation.
Conclusions:
IL-1β is a mediator of microglial activation and proliferation, and its release/production is P2X7R pore dependent. Blockade of P2X7R pore could serve as a therapeutic target in alleviating the degree of inflammation seen in neurodegenerative and neoplastic conditions.
Insights
The purinergic P2X7 receptor (P2X7R) pore state drives microglial activation and proliferation by facilitating interleukin-1β (IL-1β) release. Blocking the P2X7R pore may reduce neuroinflammation in neurological conditions.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Enhanced purinergic P2X7 receptor (P2X7R) expression is linked to neuroinflammation and microglial activation.
- P2X7R can function as a cation channel or a large pore, with its pore state potentially driving microglial activation.
- Microglia release proinflammatory cytokines like interleukin-1β (IL-1β) upon activation, but the role of P2X7R states in this process is unclear.
Purpose of the Study:
- To investigate the predominant role of P2X7R channel or pore state in driving IL-1β release.
- To determine if IL-1β has trophic effects on surrounding microglia.
- To elucidate the signaling pathway through which P2X7R pore-dependent microglial activation occurs.
Main Methods:
- Sub-cellular localization of P2X7R and IL-1β was determined using electron microscopy and immunohistochemistry in primary rat hippocampal cultures.
- Vesicular exocytosis was quantified using FM1-43 dye and confocal microscopy in microglia expressing pore-forming vs. non-pore-forming P2X7R mutants.
- IL-1β levels were measured via ELISA, and its processing was inhibited using caspase 1 antagonists; its function was blocked with neutralizing antibodies.
Main Results:
- P2X7R and IL-1β were co-localized within lysosomes.
- Microglia expressing the pore-forming P2X7R exhibited higher vesicular exocytosis compared to those with the non-pore-forming mutant.
- Increased IL-1β in cultures expressing pore-forming P2X7R mediated trophic effects, and its inhibition significantly reduced P2X7R-mediated microglial activation and proliferation.
Conclusions:
- Interleukin-1β (IL-1β) acts as a mediator of microglial activation and proliferation, with its release being dependent on the P2X7R pore state.
- Targeting the P2X7R pore could be a therapeutic strategy for reducing inflammation in neurodegenerative and neoplastic diseases.
- The P2X7R pore-dependent release of IL-1β is a key mechanism driving microglial responses in neuroinflammatory conditions.
