IL-1β production is dependent on the activation of purinergic receptors and NLRP3 pathway in human macrophages

The Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU). We investigated the role of P2 purinergic receptors in the activation of NLRP3-inflammasome pathway after MSU treatment of primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 mu g/ml), a 6 h treatment withMSUcrystals (250, 500, and 1000 mu g/ml) induced theMDMs to release IL-1 beta, IL-1 alpha, and IL-6 in adose-dependent manner. Moreover, the caspase 1 inhibitor Z-YVAD-FMK and the cathepsin B inhibitor CA-074Me reduced production of IL-1 beta in a dose-dependent manner after LPS + MSU treatment. We used real-time reverse transcription-quantitative PCR to show that treatment with LPS and MSU (500 mu g/ml) induced significantly greater expression of NLRP3 and IL-1 beta than after treatment with LPS. We also found that MSU treatment induced P2X purinergic receptor 7 (P2X7R) mRNA and protein expression. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1 beta production and pro-IL-1 beta cleavage after treatment with LPS + MSU. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited the release of IL-1 beta and other M1 macrophage cytokines (such as IL-1 alpha, IL-6, and TNF-a) fromMDMs stimulated with LPS + MSU. Taken as a whole, our results show that P2 purinergic receptors and the NLRP3 inflammasome pathway are involved in the secretion of IL-1 beta from MSU-stimulated human macrophages. This pathway may constitute a novel therapeutic target for controlling the inflammatory process in several associated pathologies.