Interleukin‐1β has trophic effects in microglia and its release is mediated by P2X7R pore

Abstract

Numerous neuroinflammatory conditions such as multiple sclerosis show enhanced expression of the purinergic P2X7 receptor (P2X7R) in the neuroinflammatory foci, where increased microglial activation is a co-existing feature. Activated microglia are known to release a number of bio-active substances such as interleukin 1β (IL-1β). Previous studies have linked P2X7R stimulation to the processing and release of IL-1β, but whether P2X7R channel or P2X7R pore is the predominant entity driving that release is unknown. Also the effect of released IL-1β on microglial activation and proliferation is unclear. The aim of this study was to characterise IL-1β release dynamics from microglia. Primary hippocampal rat cultures with electron microscopy and immunohistochemistry was used to delineate the sub-cellular localisation of IL-1β. 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. Amount of IL-1β in culture was quantified with enzyme-linked immunosorbent assay (ELISA). IL-1β intracellular processing was blocked with inhibition of caspase 1 and its extracellular form was neutralised with an IL-1β neutralising antibody. Microglial activation and proliferation was quantified immunohistochemically and with aid of confocal microscopy. Immunohistochemistry revealed P2X7R and IL-1β co-localisation in lysosomes. Vesicular exocytosis was higher in microglia expressing the pore forming P2X7R compared to those expressing the non-pore forming mutant. IL-1β levels were raised in cultures expressing the pore forming P2X7R and this 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. The results reveal IL-1β as a mediator of microglial activation and proliferation. Blockade of P2X7R pore could serve as a therapeutic target in alleviating the degree of inflammation seen in conditions such as multiple sclerosis.