The P2X7 receptor is an ATP-gated ion channel receptor that acts as both a non-desensitizing cation channel and an inducer of non-selective macropores permeable to ions and small organic molecules. This receptor, which is predominantly expressed in inflammatory cells, such as macrophages, rapidly induces a collapse of normal ionic gradients. Stimulation of the P2X7 receptor in macrophages also triggers the caspase-1 mediated proteolytic maturation and release of the promflammatory cytokines, interleukin-1betsai (IL-1beta) and interleukin-18. Because caspase-1 based signaling is processive and essentially irreversible, stringent regulatory mechanisms have evolved to maintain this protease in an inactive state within resting macrophages. Inflammatory macrophages assemble caspase-1 and other proteins into latent signaling complexes with minimal interleukin converting activity. Despite the critical roles of caspase-1, IL-1beta, and IL-18 in important pathophysiological processes, including septic shock, chronic arthritis, and neurodegenerative disease, the mechanisms for regulating the activation of caspase-1 and release of its cytokine substates remain obscure. We hypothesize that major changes within the ionic milieu of the cytosol, which can be uniquely triggered by P2X7 receptors, provide critical signals to the latent signaling complexes and thereby facilitate the rapid activation of caspase-1. This permits the processing and release of IL-1beta and IL-18 as biologically active cytokines. We additionally hypothesize that IL-1beta and IL-18 are exported from macrophages by non-classical transport mechanisms that involve the packaging of these interleukins and caspase-1 within membrane blebs that rapidly evaginate from the macrophage cell body in response to P2X7 receptor activation.
The specific aims are: (1.) To identify the critical structural and functional characteristics of the P2X7 receptor required for activation of caspase-1 and secretion of IL-10 in mouse macrophages. (2.) To define the temporal and quantitative relationships between P2X7 receptor-induced changes in intracellular ionic milieu, activation of caspase-1, and IL-1beta/IL-18 secretion. (3.) To characterize the effects of P2X7 receptor activation on the assembly and activity of the multi-protein signaling omplexes that mediate activation of caspase-1 and processing of IL-1beta/ IL-18. (4.) To define the mechanisms by which L-1beta, IL-18, and active caspase-1 are exported from macrophages in response to P2X7 receptor activation.
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