The proinflammatory cytokines IL-1β and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-κB, as pretreatment of cells with the proteasome inhibitor MG132, or the IκB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.

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The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1β. Because previous studies indicated that extracellular Cl− exerts a negative allosteric effect on ATP-gating of P2X7R channels, we tested whether Cl− attenuates the P2X7R→caspase-1→IL-1β signaling cascade in murine and human macrophages. In Bac1 murine macrophages, substitution of extracellular Cl− with gluconate produced a 10-fold increase in the rate and extent of ATP-induced IL-1β processing and secretion, while reducing the EC50 for ATP by 5-fold. Replacement of Cl− with gluconate also increased the potency of ATP as an inducer of mature IL-1β secretion in primary mouse bone marrow-derived macrophages and in THP-1 human monocytes/macrophages. Our observations were consistent with actions of Cl− at three levels: 1) a negative allosteric effect of Cl−, which limits the ability of ATP to gate the P2X7R-mediated cation fluxes that trigger caspase-1 activation; 2) an intracellular accumulation of Cl− via nonselective pores induced by P2X7R with consequential repression of caspase-1-mediated processing of IL-1β; and 3) a facilitative effect of Cl− substitution on the cytolytic release of unprocessed pro-IL-1β that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1β from the lytic release of pro-IL-1β. These results suggest that under physiological conditions P2X7R are maintained in a conformationally restrained state that limits channel gating and coupling of the receptor to signaling pathways that regulate caspase-1.

1 Periodate-oxidized ATP (oATP), which covalently modifies nucleotide-binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X 7 nucleotide receptor (P2X 7 R) is irreversibly antagonized by oATP, it is unclear whether anti-inflammatory actions of oATP are predominantly mediated via its actions on P2X 7 R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X 7 R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes. 2 oATP decreased by 40 -70% the secretion of interleukin (IL)-8 stimulated by tumor necrosis factor-a (TNF-a) in all three cell types, by IL-1b in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF-a-stimulated IL-8 secretion by oATP was similar in wild-type HEK cells or HEK cells stably expressing recombinant P2X 7 R. 3 oATP also attenuated cytokine-stimulated expression of nuclear factor-kB-luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IkB. 4 oATP had no effect on uridine triphosphate-induced activation of native P2Y 2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y 1 receptors. However, inhibition of P2Y 1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF-a-stimulated IL-8 secretion. 5 Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto-ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP. 6 In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by G s -coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by G s -coupled 5-HT 7A receptors and G q -coupled 5-HT 2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT 2A receptormediated activation of the two pathways was found to be Ca 2+ -dependent. In contrast, 5-HT 7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca 2+ ], while activation of ERK was inhibited by Ca 2+ . The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT 2A and 5-HT 7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT 7A receptor agonists in enhancing 5-HT 2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT 2A and 5-HT 7A receptors may also be relevant to the interaction of other neuronally expressed G q -and G s -coupled receptors.

HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca24 mobilization in response to ATP or UTP. We examined the expression of functional P2UR and P2UR mRNA levels during in vitro differentiation of HL-60 cells by dibutyryl-cAMP (Bt2cAMP), which induces a granulocyte/neutrophil phenotype, or by phorbol-12-myristate-13-acetate (PMA), which induces a monocyte/macrophage phenotype. Both P2UR function and P2UR mRNA levels were only modestly attenuated during granulocytic differentiation by Bt2cAMP. In contrast, P2UR function, as assayed by either Ca2+ mobilization or inositol trisphosphate generation, was greatly reduced in PMA-differentiated cells. This inhibition of P2UR function was strongly correlated with PMA-induced decreases in P2UR mRNA levels, as assayed by Northern blot analysis or reverse transcription-polymerase chain reaction-based quantification. Although PMA induced an early, transient up-regulation of P2UR mRNA, this was rapidly followed by a sustained decrease in P2UR mRNA to a level 5-10-fold lower than that in undifferentiated HL-60 cells. The half-life of the P2UR transcript in HL-60 cells was approximately 60 min, and this was not affected by acute exposure (< or = 4 hr) to Bt2cAMP or PMA. PMA down-regulated P2UR mRNA in THP-1 monocytes and HL-60 granulocytes but not in A431 human epithelial cells or human keratinocytes. P2UR mRNA was also down-regulated in THP-1 monocytes differentiated into inflammatory macrophages by gamma-interferon and endotoxin. These data indicate that myeloid leukocytes possess tissue-specific mechanisms for the rapid modulation of P2UR expression and function during differentiation and inflammatory activation.

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+influx/mobilization demonstrated that the platelet P2X1receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.

The RIP Kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between the kinase domains of the four major RIPK family members. We find that while RIPK1, RIPK2 and RIPK4 are similar in that they can all activate NF-kB and induce NEMO ubiquitination, only RIPK2 is a dual specificity kinase. Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual specificity kinase and is essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2’s kinase domain with RIPK4’s did not complement a NOD2 signaling or gene expression induction defect in RIPK2−/− macrophages. These findings suggest that RIPK2’s kinase domain is functionally unique compared to other RIPK family members, and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs.