Maturation and release of interleukin-1beta by lipopolysaccharide-primed mouse Schwann cells require the stimulation of P2X7 receptors

The P2X7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1beta maturation and releasing processes by Schwann cells. Lipopolysaccharide-primed Schwann cells synthesized large amounts of pro-IL-1beta but did not release detectable amounts of pro or mature IL-1beta. ATP on its own had no effect on the synthesis of pro-IL-1beta, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1beta by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1beta-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1beta in IL-1beta, was activated by P2X7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1beta. In searching for a link between the P2X7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1beta, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1beta release. Taken together, these results show that P2X7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1beta by immune-challenged Schwann cells.     

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.     

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.     

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.     

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.     

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.     

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.     

Pannexin-1 couples to maitotoxin- and nigericin-induced interleukin-1beta release through a dye uptake-independent pathway

Pannexin-1 is a recently identified membrane protein that can act as a nonselective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X7 receptor (P2X7R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1beta (IL-1beta) in response to P2X7R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X7R to provide the conduit for dye uptake and downstream signaling to processing and release of IL-1beta. We have pursued this hypothesis by measuring dye uptake and IL-1beta processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1beta release via the same mechanism. The experiments were carried out over time periods during which no lactate dehydrogenase was released from cells to examine only noncytolytic pathways. P2X7R activation evoked dye uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1-independent phase. Maitotoxin-evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1beta processing and release in response to all three stimuli. Thus, although pannexin-1 is required for IL-1beta release in response to maitotoxin, nigericin, and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.     

Secretion of intracellular IL-1 receptor antagonist (type 1) is dependent on P2X7 receptor activation

Inflammatory mechanisms are critical in the arterial response to injury. Both IL-1 and the naturally occurring inhibitor of IL-1, IL-1R antagonist (IL-1ra), are expressed in the arterial wall, and in particular in the endothelium. Previous studies suggest that endothelial cells only make the intracellular type I isoform of IL-1ra (icIL-1ra1), an isoform known to lack a secretory signal peptide. It is unclear how icIL-1ra is released from the endothelial cell to act as an antagonist on cell surface IL-1 type I receptors. IL-1beta, which also lacks a secretory signal peptide, may be released by ATP stimulation of the P2X(7)R. Therefore, we examined whether icIL-1ra1 release occurs in an analogous manner, using both the mouse macrophage cell line RAW264.7 and HUVECs. P2X(7)R activation caused icIL-1ra1 release from LPS-primed RAW264.7 macrophages and from HUVECs. This release was inhibited in the absence of extracellular calcium, and attenuated by preincubation with oxidized ATP, KN62, and apyrase. Endogenous ATP release, which also facilitated release of icIL-1ra1, was detected during LPS treatment of both RAW264.7 macrophages and HUVECs. Annexin V assays showed that ATP stimulation resulted in a rapid phosphatidylserine (PS) exposure on the cell surface of RAW264.7 macrophages, and that PS-exposed microvesicles contained icIL-1ra1. However, PS flip and microvesicle shedding was not apparent in ATP-treated HUVECs. These data support a general role for the P2X(7)R in the release of leaderless cytokines into the extracellular medium, and indicate how icIL-1ra1 may act upon its extracellular target, the IL-1R.     

Glu496 to Ala polymorphism in the P2X7 receptor impairs ATP-induced IL-1 beta release from human monocytes

Priming of monocytes with LPS produces large quantities of intracellular, biologically inactive IL-1beta that can be processed and released by subsequent activation of the P2X7 receptor by extracellular ATP. We examined whether a loss-of-function polymorphism of the human P2X7 receptor (Glu496Ala) impairs this process. Both ATP-induced ethidium+ uptake and ATP-induced shedding of L-selectin (CD62L) were nearly absent in monocytes from four subjects homozygous for Glu496Ala confirming that this polymorphism impairs P2X7 function. The level of ATP-induced IL-1beta released in 2 h from LPS-activated whole blood from homozygous subjects was 50% of that from wild-type samples. A more marked defect in IL-1beta release was observed from LPS-activated monocytes of homozygous subjects which was only 22% of that released from wild-type monocytes after a 30-min incubation with ATP. However, after a 60-min incubation with ATP, the amount of IL-1beta released from homozygous monocytes was 70% of that released from wild-type monocytes. Incubation of monocytes of either genotype with nigericin resulted in a similar release of IL-1beta. Western blotting demonstrated that ATP induced the release of mature 17-kDa IL-1beta from monocytes, and confirmed that this process was impaired in homozygous monocytes. Finally, ATP-induced 86Rb+ efflux was 9-fold lower from homozygous monocytes than from wild-type monocytes. The results indicate that ATP-induced release of IL-1beta is slower in monocytes from subjects homozygous for the Glu496Ala polymorphism in the P2X7 receptor and that this reduced rate of IL-1beta release is associated with a lower ATP-induced K+ efflux.     

Differential requirement of P2X7 receptor and intracellular K+ for caspase-1 activation induced by intracellular and extracellular bacteria

Interleukin-1beta (IL-1beta) is a pro-inflammatory cytokine that plays an important role in host defense and inflammatory diseases. The maturation and secretion of IL-1beta are mediated by caspase-1, a protease that processes pro-IL-1beta into biologically active IL-1beta. The activity of caspase-1 is controlled by the inflammasome, a multiprotein complex formed by NLR proteins and the adaptor ASC, that induces the activation of caspase-1. The current model proposes that changes in the intracellular concentration of K(+) potentiate caspase-1 activation induced by the recognition of bacterial products. However, the roles of P2X7 receptor and intracellular K(+) in IL-1beta secretion induced by bacterial infection remain unknown. Here we show that, in response to Toll-like receptor agonists such as lipopolysaccharide or infection with extracellular bacteria Staphylococcus aureus and Escherichia coli, efficient caspase-1 activation is only triggered by addition of ATP, a signal that promotes caspase-1 activation through depletion of intracellular K(+) caused by stimulation of the purinergic P2X7 receptor. In contrast, activation of caspase-1 that relies on cytosolic sensing of flagellin or intracellular bacteria did not require ATP stimulation or depletion of cytoplasmic K(+). Consistently, caspase-1 activation induced by intracellular Salmonella or Listeria was unimpaired in macrophages deficient in P2X7 receptor. These results indicate that, unlike caspase-1 induced by Toll-like receptor agonists and ATP, activation of the inflammasome by intracellular bacteria and cytosolic flagellin proceeds normally in the absence of P2X7 receptor-mediated cytoplasmic K(+) perturbations.     

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.     

MSU Crystals induce sterile IL-1β secretion via P2X7 receptor activation and HMGB1 release

BackgroudThe mechanism by which monosodium urate (MSU) crystals induce inflammation is not completely understood. Few studies have shown that MSU is capable of stimulating the release of IL-1β in the absence of LPS treatment. The purinergic P2X7 receptor is involved in the release of IL-1β in inflammatory settings caused by crystals, as is the case in silicosis.MethodsWe investigated the role of P2X7 receptor in sterile MSU-induced inflammation by evaluating peritonitis and paw edema. In in vitro models, we performed the experiments using peritoneal macrophages and THP-1 cells. We measured inflammatory parameters using ELISA and immunoblotting. We measured cell recruitment using cell phenotypic identification and hemocytometer counts.ResultsOur in vivo data showed that animals without P2X7 receptors generated less paw edema, less cell recruitment, and lower levels of IL-1β release in a peritonitis model. In the in vitro model, we observed that MSU induced dye uptake by the P2X7 receptor. In the absence of the receptor, or when it was blocked, MSU crystals induced less IL-1β release and this effect corresponded to the concentration of extracellular ATP. Moreover, MSU treatment induced HMGB1 release; pre-treatment with P2X7 antagonist reduced the amount of HMGB1 in cell supernatants.ConclusionsIL-1β secretion induced by MSU depends on P2X7 receptor activation and involves HMGB1 release.General significanceWe propose that cell activation caused by MSU crystals induces peritoneal macrophages and THP-1 cells to release ATP and HMGB1, causing IL-1β secretion via P2X7 receptor activation.     

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.     

Lysophosphatidylcholine stimulates IL-1beta release from microglia via a P2X7 receptor-independent mechanism

IL-1beta released from activated macrophages contributes significantly to tissue damage in inflammatory, degenerative, and autoimmune diseases. In the present study, we identified a novel mechanism of IL-1beta release from activated microglia (brain macrophages) that occurred independently of P2X(7) ATP receptor activation. Stimulation of LPS-preactivated microglia with lysophosphatidylcholine (LPC) caused rapid processing and secretion of mature 17-kDa IL-1beta. Neither LPC-induced IL-1beta release nor LPC-stimulated intracellular Ca(2+) increases were affected by inhibition of P2X(7) ATP receptors with oxidized ATP. Microglial LPC-induced IL-1beta release was suppressed in Ca(2+)-free medium or during inhibition of nonselective cation channels with Gd(3+) or La(3+). It was also attenuated when Ca(2+)-activated K(+) channels were blocked with charybdotoxin (CTX). The electroneutral K(+) ionophore nigericin did not reverse the suppressive effects of CTX on LPC-stimulated IL-1beta release, demonstrating the importance of membrane hyperpolarization. Furthermore, LPC-stimulated caspase activity was unaffected by Ca(2+)-free medium or CTX, suggesting that secretion but not processing of IL-1beta is Ca(2+)- and voltage-dependent. In summary, these data indicate that the activity of nonselective cation channels and Ca(2+)-activated K(+) channels is required for optimal IL-1beta release from LPC-stimulated microglia.     

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.     

Regulation of P2X7-dependent inflammatory functions by P2X4 receptor in mouse macrophages

Activation of the P2X7 receptor of macrophages plays an important role in inflammation. We recently reported that co-expression of P2X4 receptor with P2X7 receptor facilitates P2X7 receptor-mediated cell death via Ca(2+) influx. However, it remained unclear whether P2X4 receptor is involved in P2X7 receptor-mediated inflammatory responses, such as cytokine production. Here, we present evidence that P2X4 receptor modulates P2X7 receptor-dependent inflammatory functions. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced high mobility group box 1 (HMGB1) release and IL-1β production via activation of P2X7 receptor. Knockdown of P2X4 receptor or removal of extracellular Ca(2+) suppressed ATP-induced release of both HMGB1 and IL-1β. On the other hand, knockdown of P2X4 receptor or removal of extracellular Ca(2+) enhanced P2X7-dependent LC3-II expression (an index of autophagy), suggesting that P2X4 receptor suppresses P2X7-mediated autophagy. Since LC3-II expression was inhibited by pretreatment with antioxidant and NADPH oxidase inhibitor, we examined P2X7-mediated production of reactive oxygen species (ROS). We found that activation of P2X7 receptor-mediated production of ROS was significantly facilitated in P2X4-knockdown cells, suggesting that co-expression of P2X4 receptor with P2X7 receptor may suppress anti-inflammatory function-related autophagy via suppression of ROS production. We conclude that co-expression of P2X4 receptor with P2X7 receptor enhances P2X7-mediated inflammation through both facilitation of release of cytokines and suppression of autophagy.     

CD39 modulates IL-1 release from activated endothelial cells

The activation of endothelial cells (EC) and monocyte-macrophages (Mφ) by lipopolysaccharide (LPS) is considered an important element of the vascular injury observed in endotoxemia. Interleukin-1 (IL-1) beta release from Mφ in response to LPS, appears to be mediated by the autocrine/paracrine release of ATP via P2X7 receptor activation. In EC, similar nucleotide-mediated signaling pathways may be influenced by high levels of expression of CD39, the vascular nucleoside triphosphate diphosphohydrolase (NTPDase; ENTPD I). To determine whether CD39 modulates ATP-mediated release of IL-1 from EC, we stimulated human EC with LPS and measured levels of ATP secretion and IL-1 release. LPS triggered ATP secretion from EC that was soon followed by IL-1alpha release. Overexpression of CD39 following infection with recombinant CD39 adenoviral vectors (AdCD39) abrogated the initial phase of ATP secretion and inhibited IL-1alpha release; comparable results were obtained with soluble NTPDase. These data demonstrate that CD39/NTPDase modulates IL-1alpha release from LPS stimulated human EC.     

The P2X7 receptor: a key player in IL-1 processing and release

Human IL-1 family proteins are key mediators of the host response to infections, injury, and immunologic challenges. The mechanism by which IL-1 activates proinflammatory responses in target cells, and the plasma membrane receptors involved, is fairly well known. This has led to the development of innovative drugs that block IL-1 downstream to its synthesis and secretion. On the contrary, the mechanism of IL-1 and other IL-1 family members (e.g., IL-18) maturation and release is incompletely understood. Accruing evidence points to a plasma membrane receptor for extracellular ATP, the P2X(7) receptor, as a key player in both processes. A deeper understanding of the mechanism by which the P2X(7) receptor triggers IL-1 maturation and exteriorization may suggest novel avenues for the treatment of inflammatory diseases and provide a deeper insight in the fundamental mechanism of protease activation and cellular export of proteins lacking a leader sequence.     

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.