Action of intracellular IL-1Ra (Type 1) is independent of the IL-1 intracellular signalling pathway

The balance between IL-1 and its naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra) is critical in determining the inflammatory response. Four splice variants of the IL-1ra gene have been identified; one secreted (sIL-1ra) and three intracellular (icIL-1ra1-3). The biological roles of the intracellular isoforms remain largely unclear. We wished to determine whether icIL-1ra1 had intracellular functions regulating IL-1 signalling. Signalling was determined using an NF-kappaB reporter assay measuring induction of the IL-8 promoter in transfected cells. Over-expression of icIL-1ra1 in HeLa cells had no effect on IL-1 stimulated IL-8 activity. In contrast over-expression of sIL-ra significantly attenuated IL-1 activity. In addition, transfection of icIL-1ra1 in HeLa cells did not cause inhibition of IL-8 promoter activity following over-expression of the IL-1 signalling components MyD88, IRAK-1, TRAF-6, Ikappakappabeta or RelA. This implies that icIL-1ra1 does not act to alter IL-1 mediated intracellular signalling in this system. We investigated whether ATP and/or over-expression of the P2X7 receptor caused icIL-1ra1 inhibition of IL-1beta mediated IL-8 reporter activation, by permitting its release. In HeLa cells, no effect of icIL-1ra1 was observed in ATP stimulated and/or P2X7 transfected cells, compared to a significant inhibition in sIL-1ra transfected cells. However, in endothelial cells stimulated with ATP, the released fraction was effective in attenuating IL-1beta activation of the IL-8 reporter. These results suggest that icIL-1ra1 does not act at an intracellular level to alter IL-1 mediated signalling, and is effective in inhibiting IL-1 responses only when released in an ATP-dependent and cell type specific manner.     

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.     

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.     

Involvement of P2X4 receptor in P2X7 receptor-dependent cell death of mouse macrophages

Interaction of P2X7 receptor with P2X4 receptor has recently been suggested, but it remains unclear whether P2X4 receptor is involved in P2X7 receptor-mediated events, such as cell death of macrophages induced by high concentrations of extracellular ATP. Here, we present evidence that P2X4 receptor does play a role in P2X7 receptor-dependent cell death. Treatment of mouse macrophage RAW264.7 cells with 1mM ATP induced Ca(2+) influx, non-selective large pore formation, activation of extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), and cell death via activation of P2X7 receptor. P2X4-knockdown cells, established by transfecting RAW264.7 cells with two short hairpin RNAs (shRNAs) targeting P2X4 receptor, showed a decrease of the initial peak of intracellular Ca(2+) after treatment with ATP, though pore formation and the P2X7-mediated activation of ERK1/2 and p38 MAPK were not affected. Intriguingly, P2X4 knockdown resulted in significant suppression of cell death induced by ATP or P2X7 agonist BzATP. In conclusion, our results suggest that P2X4 receptor is involved in P2X7 receptor-mediated cell death, but not pore formation or MAPK signaling.     

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.     

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.     

Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome

The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IkappaB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1alpha-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1alpha-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.     

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.     

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.     

Intracellular IL-1 receptor antagonist is elevated in human dermal fibroblasts that overexpress intracellular precursor IL-1 alpha.

Cultured dermal fibroblasts from systemic sclerosis patients express higher levels of intracellular IL-1 alpha than fibroblasts from healthy controls. In this study, we found that systemic sclerosis dermal fibroblasts also express higher levels of the intracellular isoform of IL-1 receptor antagonist (icIL-1Ra) than normal fibroblasts after stimulation with IL-1 beta or TNF-alpha. A possible relationship between elevated precursor IL-1 alpha (preIL-1 alpha) and elevated icIL-1Ra was investigated by transducing normal dermal fibroblasts to overexpress preIL-1 alpha, preIL-1 beta, or icIL-1Ra. Fibroblasts that overexpressed icIL-1Ra did not have elevated levels of IL-1 alpha. On the other hand, fibroblasts that overexpressed preIL-1 alpha had at least 4-fold higher basal levels of icIL-1Ra than control fibroblasts and 4-fold higher levels of icIL-1Ra after induction with IL-1 beta or TNF-alpha. Fibroblasts overexpressing preIL-1 beta did not exhibit elevated icIL-1Ra. The differences in icIL-1Ra protein levels were reflected in differences in mRNA. In contrast, IL-1-stimulated levels of MCP-1 and IL-6 were not different in control and preIL-1 alpha-transduced fibroblasts. Addition of neutralizing anti-IL-1 alpha Abs to fibroblast cultures did not diminish basal or stimulated levels of icIL-1Ra in the preIL-1 alpha-transduced cells, supporting an intracellular site of action of preIL-1 alpha. This is the first report of an association between intracellular levels of these IL-1 family members. We hypothesize that intracellular preIL-1 alpha participates in the regulation of icIL-1Ra.     

Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 alpha precursor on cell motility

Interleukin (IL)-1alpha, a proinflammatory cytokine, is produced as a 33 kDa protein precursor (preIL-1alpha) which is cleaved to generate the 17 kDa C-terminal mature IL-1alpha (mIL-1alpha) and the 16kDa N-terminal IL-1alpha propiece (NIL-1alpha). The biological effect of IL-1alpha is regulated by the IL-1 receptor antagonist (IL-1Ra), its naturally occurring inhibitor. Four different isoforms of the IL-1Ra have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Whether the icIL-1Ra1 isoform can antagonize some of the biological effects of intracellular IL-1alpha is still unknown. The aim of this study is to investigate effects of preIL-1alpha and icIL-1Ra1 on cell motility in stably transfected ECV304 cells. We show that expression of preIL-1alpha in ECV304 cells significantly increases cell motility. Furthermore, transfection with NIL-1alpha propiece also increases cell motility whereas this stimulatory effect was not observed by addition of exogenous mIL-1alpha, suggesting an intracellular effect of preIL-1alpha mediated by NIL-1alpha propiece. Co-transfection of ECV304 cells with icIL-1Ra1 completely antagonizes the stimulatory effect of preIL-1alpha and NIL-1alpha propiece on cell motility. In conclusion, NIL-1alpha propiece increases ECV304 cell motility and icIL-1Ra1 exerts intracellular functions regulating this stimulatory effect.     

Regulatory responses of hepatocytes,macrophages and vascular endothelial cells to magnesium deficiency

The liver is the organ that responds to nutritional disturbances including magnesium deficiency. The present study evaluated cellular responses to magnesium deficiency using model cells of the liver, namely, HepG2 cells as hepatocytes, RAW264.7 cells as Kupffer cells and human umbilical vein endothelial cells (HUVECs) as vascular endothelial cells; we examined effects of culture with magnesium deficient medium on cell responses in individual types of cells as well as interactive responses among cells. Metabolomic analyses indicated that magnesium deficiency differentially affected the cellular content of metabolites among HepG2 cells, RAW264.7 cells and HUVECs. The cellular content of the metabolites in HepG2 cells and HUVECs was also affected by the conditioned medium from RAW264.7 cells cultured with the magnesium-deficient media. The changes in HUVECs partly resembled those of the livers of magnesium-deficient rats previously described. RNA-seq analyses indicated that magnesium deficiency modulated the expression levels of molecules related to the ubiquitin-proteasome pathway and oxidative stress/antioxidant response in HepG2 cells and RAW264.7 cells, respectively. Furthermore, when HUVECs were co-cultured with RAW264.7 cells, lipopolysaccharide-induced expression of interleukin (IL)-1β and IL-6 was enhanced by magnesium deficiency, depending on the presence of RAW264.7 cells. The present study reveals that magnesium deficiency affects cellular metabolism in HepG2 liver cells, RAW264.7 macrophages and HUVECs, and that the modulation of cellular responses to extracellular magnesium deficiency in HUVECs depends on the presence of RAW264.7 cells. The complex responses in individual cells and through cell interactions partly explain the regulatory reaction to magnesium deficiency in the liver.     

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.     

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.     

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.     

Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3'-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.     

Effect of the purinergic receptor P2X7 on Chlamydia infection in cervical epithelial cells and vaginally infected mice

Ligation of the purinergic receptor, P2X7R, with its agonist ATP has been previously shown to inhibit intracellular infection by chlamydiae and mycobacteria in macrophages. The effect of P2X7R on chlamydial infection had never been investigated in the preferred target cells of chlamydiae, cervical epithelial cells, nor in vaginally infected mice. In this study, we show that treatment of epithelial cells with P2X7R agonists inhibits partially Chlamydia infection in epithelial cells. Chelation of ATP with magnesium or pretreatment with a P2X7R antagonist blocks the inhibitory effects of ATP. Similarly to previous results obtained with macrophages, ATP-mediated inhibition of infection in epithelial cells requires activation of host-cell phospholipase D. Vaginal infection was also more efficient in P2X7R-deficient mice, which also displayed a higher level of acute inflammation in the endocervix, oviduct, and mesosalpingeal tissues than in infected wild-type mice. However, secretion of IL-1beta, which requires P2X7R ligation during infection by other pathogens, was decreased mildly and only at short times of infection. Taken together, these results suggest that P2X7R affects Chlamydia infection by directly inhibiting infection in epithelial cells, rather than through the ability of P2X7R to modulate IL-1beta secretion.