title: Interleukin-1β has trophic effects in microglia and its release is mediated by P2X7R pore.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Physiology, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, 3010, Australia.|The Department of Neurology, The Royal Melbourne Hospital, Parkville, Victoria, 3052, Australia.
authors.forename: Mastura
authors.initials: M
authors.lastname: Monif
authors.affiliation: Howard Florey Institute, The University of Melbourne, Melbourne, Victoria, 3010, Australia.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Reid
authors.affiliation: Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Victoria, 3010, Australia.
authors.forename: Kim L
authors.initials: KL
authors.lastname: Powell
authors.affiliation: Department of Surgery, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Victoria, 3010, Australia.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Drummond
authors.affiliation: Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Victoria, 3010, Australia.
authors.forename: Terrence J
authors.initials: TJ
authors.lastname: O'Brien
authors.affiliation: Department of Physiology, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, 3010, Australia. davidaw@unimelb.edu.au.
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D053583:Interleukin-1beta / Q000096:biosynthesis* / Q000378:metabolism*; D017628:Microglia / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D058486:Receptors, Purinergic P2X7 / Q000096:biosynthesis*
chemical_list: D053583:Interleukin-1beta; D058486:Receptors, Purinergic P2X7
keywords: Activation; Interleukin-1β; Microglia; Neuroinflammation; P2X7 receptor; P2X7R pore; Proliferation