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.
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