Although the precise mechanism leading to the http://www.selleckchem.com/products/Perifosine.html activation of NLRP3 remains largely unknown, it is proposed that oxidative stress, lysosomal destabilization with cytosolic cathepsin activity and potassium efflux due to the stimulation of ATP-sensitive potassium channels, or pore formation by bacterial toxins, converge into the activation of NLRP3 [17]. In human monocytes, contrary to the two-step signaling system in macrophages and DCs, differential requirements for the activation of the inflammasome were documented [18]. Caspase-1 is constitutively activated in these cells; therefore, a single stimulation event triggers the expression of pro-IL-1�� and mature IL-1�� release. The second signal is dispensable, because monocytes release endogenous ATP after stimulation, which in turn activate the inflammasome, and induces IL-1�� secretion through the P2X7 receptor.
IL-1�� production is still dependent on the inflammasome components and modulated by K+ efflux [19], [20]. In celiac patients, downstream products of NLRP3 inflammasome activation (such as IL-1�� and IL-18) were shown to affect Th1/Th17 responses [7], [21]. However, the mechanism of IL-1�� activation has not yet been elucidated. Here, we analyzed the production of IL-1 cytokine family members in human monocytes and PBMC after stimulation with PDWGF, and investigated the upstream mechanism underlying PDWGF-induced IL-1�� production and release in the PBMC of celiac patients.
In particular, the role of the signaling molecules underlying de novo synthesis of pro-IL-1�� [especially the role of TLRs, MyD88 and Toll-IL-1 receptor domain-containing adaptor-inducing interferon-�� (TRIF); the role of MAPK JNK, ERK and p38 MAPK; the role of NF-��B and the mechanisms of caspase-1 activation culminating in IL-1�� production] were studied. Materials and Methods Abs and Reagents Glybenclamide, KN-62, N-Acetyl-L-cysteine (NAC), quinidine and polymyxin B were from Sigma-Aldrich (St. Louis, MO, USA). Benzyloxycarbonyl-Tyr-Val-Ala-Asp-(OMe) fluoromethylketone (Z-YVAD-fmk) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ��-amylase inhibitor (AI) from Triticum aestivum type I and III were from Sigma. The p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, serine-protease inhibitor N-p-Tosyl-L-phenyl-alanine chloromethyl ketone (TPCK) (all Sigma), and the ERK inhibitor UO126 (Cell Signaling Technology, Danvers, MA, USA) were dissolved in DMSO (Sigma). The FLICA Caspase-1 Assay kit was from ImmunoChemistry Technologies (Bloomington, MN, USA). Anti-human IL-1 ��/IL-1F2 Ab and anti-mouse IL-1��/IL-1F2 Anacetrapib Ab were from R&D Systems (Minneapolis, MN, USA), while anti-human cleaved IL-1�� Ab was purchased from Cell Signaling Technology.