A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.     

An Arg307 to Gln polymorphism within the ATP-binding site causes loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).     

ATP-stimulated release of interleukin (IL)-1beta and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage

Interleukin (IL)-1beta and IL-18 are structurally similar proteins that require caspase-1 processing for activation. Both proteins are released from the cytosol by unknown pathway(s). To better characterize the release pathway(s) for IL-1beta and IL-18 we evaluated the role of lipopolysaccharide priming, of interleukin-1beta-converting enzyme (ICE) inhibition, of human purinergic receptor (P2X(7)) function, and of signaling pathways in human monocytes induced by ATP. Monocytes rapidly processed and released both IL-1beta and IL-18 after exogenous ATP. Despite its constitutive cytosolic presence, IL-18 required lipopolysaccharide priming for the ATP-induced release. Neither IL-1beta nor IL-18 release was prevented by ICE inhibition, and IL-18 release was not induced by ICE activation itself. Release of both cytokines was blocked completely by a P2X7 receptor antagonist, oxidized ATP, and partially by an antibody to P2X(7) receptor. In evaluating the signaling components involved in the ATP effect, we identified that the protein-tyrosine kinase inhibitor, AG126, produced a profound inhibition of both ICE activation as well as release of IL-1beta/IL-18. Taken together, these results suggest that, although synthesis of IL-1beta and IL-18 differ, ATP-mediated release of both cytokines requires a priming step but not proteolytically functional caspase-1.     

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.     

ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.     

Lysophospholipids and ATP mutually suppress maturation and release of IL-1 beta in mouse microglial cells using a Rho-dependent pathway

The P2X7 receptor (P2X7R), an ATP-gated ion channel, plays essential roles in the release and maturation of IL-1beta in microglial cells in the brain. Previously, we found that lysophosphatidylcholine (LPC) potentiated P2X7R-mediated intracellular signals in microglial cells. In this study, we determined whether the lysophospholipids, i.e., LPC and sphingosylphosphorylcholine (SPC), modulate the ATP-induced release and processing of IL-1beta mediated by P2X7R in mouse MG6 microglial cells. LPC or SPC alone induced the release of precursor (pro-IL-1beta) and mature IL-1beta (mIL-1beta) from LPS-primed MG6 cells, possibly due to lytic functions. However, these lysophospholipids inhibited ATP-induced caspase-1 activation that is usually followed by the release of mIL-1beta. Conversely, ATP inhibited the release of pro-IL-1beta and mIL-1beta induced by LPC/SPC. This suggests that lysophospholipids and ATP mutually suppressed each function to release IL-1beta. P2X7R activation resulted in microtubule reorganization in the MG6 cells that was blocked in the presence of LPC and SPC. LPC/SPC reduced the amount of activated RhoA after stimulation with ATP, implying that these lysophospholipids block ATP-induced microtubule reorganization by interfering with RhoA activation. In addition, the microtubule inhibitor colchicine inhibited ATP-induced release of mIL-1beta similar to that of LPC and SPC. This suggests that the impairment of the microtubule reassembly may be associated with the inhibitory effects of LPC/SPC on ATP-induced mIL-1beta release. Mutual suppression by ATP and LPC/SPC on the maturation of IL-1beta was observed in LPS-primed primary microglia. Collectively, these data suggest opposing functions by lysophospholipids, either proinflammatory or anti-inflammatory, in regard to the maturation and release of IL-1beta from microglial cells.     

Altered cytokine production in mice lacking P2X(7) receptors

The P2X(7) receptor (P2X(7)R) is an ATP-gated ion channel expressed by monocytes and macrophages. To directly address the role of this receptor in interleukin (IL)-1 beta post-translational processing, we have generated a P2X(7)R-deficient mouse line. P2X(7)R(-/-) macrophages respond to lipopolysaccharide and produce levels of cyclooxygenase-2 and pro-IL-1 beta comparable with those generated by wild-type cells. In response to ATP, however, pro-IL-1 beta produced by the P2X(7)R(-/-) cells is not externalized or activated by caspase-1. Nigericin, an alternate secretion stimulus, promotes release of 17-kDa IL-1 beta from P2X(7)R(-/-) macrophages. In response to in vivo lipopolysaccharide injection, both wild-type and P2X(7)R(-/-) animals display increases in peritoneal lavage IL-6 levels but no detectable IL-1. Subsequent ATP injection to wild-type animals promotes an increase in IL-1, which in turn leads to additional IL-6 production; similar increases did not occur in ATP-treated, LPS-primed P2X(7)R(-/-) animals. Absence of the P2X(7)R thus leads to an inability of peritoneal macrophages to release IL-1 in response to ATP. As a result of the IL-1 deficiency, in vivo cytokine signaling cascades are impaired in P2X(7)R-deficient animals. Together these results demonstrate that P2X(7)R activation can provide a signal that leads to maturation and release of IL-1 beta and initiation of a cytokine cascade.     

Inhibitory effects of chloride on the activation of caspase-1, IL-1beta secretion, and cytolysis by the P2X7 receptor

The P2X7 receptor (P2X7R) is an ATP-gated cation channel that activates caspase-1 leading to the maturation and secretion of IL-1beta. 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-1beta 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-1beta 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-1beta 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-1beta; and 3) a facilitative effect of Cl- substitution on the cytolytic release of unprocessed pro-IL-1beta that occurs with sustained activation of P2X7R. This cytolysis was repressed by the cytoprotectant glycine, permitting dissociation of P2X7R-regulated secretion of mature IL-1beta from the lytic release of pro-IL-1beta. 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.     

A novel P2X7 receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1 beta processing and release

The release of IL-1 beta is a tightly controlled process that requires induced synthesis of the precursor pro-IL-1 beta and a second stimulus that initiates cleavage and secretion of mature IL-1 beta. Although ATP as a second stimulus potently promotes IL-1 beta maturation and release via P2X(7) receptor activation, millimolar ATP concentrations are needed. The human cathelicidin-derived peptide LL37 is a potent antimicrobial peptide produced predominantly by neutrophils and epithelial cells. In this study, we report that LL37 stimulation of LPS-primed monocytes leads to maturation and release of IL-1 beta via the P2X(7) receptor. LL37 induces a transient release of ATP, membrane permeability, caspase-1 activation, and IL-1 beta release without cell cytotoxicity. IL-1 beta release and cell permeability are suppressed by pretreatment with the P2X(7) inhibitors oxidized ATP, KN04, and KN62. In the presence of apyrase, which hydrolyzes ATP to AMP, the effect of LL37 was not altered, indicating that LL37 rather than autocrine ATP is responsible for the activation of the P2X(7) receptor. We conclude that endogenous LL37 may promote IL-1 beta processing and release via direct activation of P2X(7) receptors.     

An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X7 receptor

The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X(7) receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into various lymphocyte subsets and was decreased to values of approximately 25% of normal. The expression of the P2X(7) receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X(7) carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-gamma up-regulated P2X(7) function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X(7) receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.     

P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases,caspases, and IL-1 beta release

In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1 beta from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1 beta, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1 beta release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct "tethered" blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations > or = 3 mM, but optimal IL-1 beta release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1 beta release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1 beta. Our observations that blebbing and IL-1 beta release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.     

Rottlerin inhibits P2X(7) receptor-stimulated phospholipase D activity in chronic lymphocytic leukaemia B-lymphocytes

Phospholipase D (PLD) is a ubiquitous enzyme that can be activated by extracellular adenosine 5'-triphosphate (ATP) or phorbol 12-myristate 13-acetate (PMA) in B-lymphocytes from subjects with chronic lymphocytic leukaemia (CLL). In this study, ATP- but not PMA-induced PLD stimulation in CLL B-lymphocytes was abolished in the presence of an anti-P2X(7) receptor monoclonal antibody, as well as in B-lymphocytes from CLL subjects homozygous for the Glu(496) to Ala loss-of-function P2X(7) polymorphism. Rottlerin, an inhibitor of novel protein kinase C (PKC) isoforms, but not GF 109203X, an inhibitor of conventional PKC isoforms, impaired the ATP-stimulated PLD activity in CLL B-lymphocytes. In contrast, both inhibitors impaired PLD activity stimulated by PMA, a known mediator of PKC activation. The inhibition of P2X(7)-stimulated PLD activity by rottlerin was attributed to a target downstream of P2X(7) activation, as the ATP-mediated (86)Rb(+) efflux from CLL B-lymphocytes was not altered in the presence of rottlerin. Our results indicate a possible role for novel PKC isoforms in the regulation of P2X(7)-mediated PLD activity.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Antimicrobial peptides initiate IL-1 beta posttranslational processing: a novel role beyond innate immunity

Human monocytes stimulated with LPS produce large quantities of prointerleukin-1beta, but little of this cytokine product is released extracellularly as the mature biologically active species. To demonstrate efficient proteolytic cleavage and export, cytokine-producing cells require a secondary effector stimulus. In an attempt to identify agents that may serve as initiators of IL-1beta posttranslational processing in vivo, LPS-activated human monocytes were treated with several individual antimicrobial peptides. Two peptides derived from porcine neutrophils, protegrin (PTG)-1 and PTG-3, promoted rapid and efficient release of mature IL-1beta. The PTG-mediated response engaged a mechanism similar to that initiated by extracellular ATP acting via the P2X(7) receptor. Thus, both processes were disrupted by a caspase inhibitor, both were sensitive to ethacrynic acid and CP-424,174, two pharmacological agents that suppress posttranslational processing, and both were negated by elevation of extracellular potassium. Moreover, the PTGs, like ATP, promoted a dramatic change in monocyte morphology and a loss of membrane latency. The PTG response was concentration dependent and was influenced profoundly by components within the culture medium. In contrast, porcine neutrophil antimicrobial peptides PR-26 and PR-39 did not initiate IL-1beta posttranslational processing. The human defensin HNP-1 and the frog peptide magainin 1 elicited export of 17-kDa IL-1beta, but these agents were less efficient than PTGs. As a result of this ability to promote release of potent proinflammatory cytokines such as IL-1beta, select antimicrobial peptides may possess important immunomodulatory functions that extend beyond innate immunity.     

Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production.

ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages. Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1. The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta. The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP. Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted. Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages. We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release. We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release.     

Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor

In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1beta processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10-30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3'-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, alpha-fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and alpha-fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1beta, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases.     

Probenecid directly impairs activation of the canine P2X7 receptor

The current study aimed to determine if probenecid could directly impair the canine P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate (ATP). Patch clamp measurements demonstrated that probenecid impairs ATP-induced inward currents in HEK-293 cells expressing canine P2X7. Flow cytometric measurements of ethidium⁺ uptake into HEK-293 cells expressing canine P2X7 showed that probenecid impairs ATP-induced pore formation in a concentration-dependent manner, with a half maximal inhibitory concentration of 158 µM. Finally, ELISA measurements revealed that probenecid impairs ATP-induced interleukin-1β release in dog blood. In conclusion, this study reveals that probenecid can directly impair canine P2X7 activation.     

Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages

Several mechanistically distinct models of nonclassical secretion, including exocytosis of secretory lysosomes, shedding of plasma membrane microvesicles, and direct efflux through plasma membrane transporters, have been proposed to explain the rapid export of caspase-1-processed IL-1 beta from monocytes/macrophages in response to activation of P2X7 receptors (P2X7R) by extracellular ATP. We compared the contribution of these mechanisms to P2X7R-stimulated IL-1 beta secretion in primary bone marrow-derived macrophages isolated from wild-type, P2X7R knockout, or apoptosis-associated speck-like protein containing a C-terminal CARD knockout mice. Our experiments revealed the following: 1) a novel correlation between IL-1 beta secretion and the release of the MHC-II membrane protein, which is a marker of plasma membranes, recycling endosomes, multivesicular bodies, and released exosomes; 2) a common and absolute requirement for inflammasome assembly and active caspase-1 in triggering the cotemporal export of IL-1 beta and MHC-II; and 3) mechanistic dissociation of IL-1 beta export from either secretory lysosome exocytosis or plasma membrane microvesicle shedding on the basis of different requirements for extracellular Ca(2+) and differential sensitivity to pharmacological agents that block activation of caspase-1 inflammasomes. Thus, neither secretory lysosome exocytosis nor microvesicle shedding models constitute the major pathways for nonclassical IL-1 beta secretion from ATP-stimulated murine macrophages. Our findings suggest an alternative model of IL-1 beta release that may involve the P2X7R-induced formation of multivesicular bodies that contain exosomes with entrapped IL-1 beta, caspase-1, and other inflammasome components.     

P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage

The proinflammatory IL-1 cytokines IL-1alpha, IL-1beta, and IL-18 are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X(7) receptors (P2X(7)R) by extracellular ATP is a key physiological inducer of rapid IL-1beta release from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X(7)R(-/-) mice and found that release of IL-1alpha, IL-18, as well as IL-1beta, by ATP resulted exclusively from activation of P2X(7)R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1beta release. We compared IL-1-release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage, and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL-1beta independently of panx1 but do not release mature IL-1beta because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1beta that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1beta that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1beta.     

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.