apyrase and Inflammation

There is increasing evidence for a link between inflammation and thrombosis. Following tissue injury, vascular endothelium becomes activated, losing its antithrombotic properties whereas inflammatory mediators build up a prothrombotic environment. Platelets are the first elements to be activated following endothelial damage; they participate in physiological haemostasis, but also in inflammatory and thrombotic events occurring in an injured tissue. While physiological haemostasis develops rapidly to prevent excessive blood loss in the endothelium activated by inflammation, hypoxia or by altered blood flow, thrombosis develops slowly. Activated platelets release the content of their granules, including ATP and ADP released from their dense granules. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 dephosphorylates ATP to ADP and to AMP, which in turn, is hydrolysed to adenosine by ecto-5'-nucleotidase (CD73). NTPDase1/CD39 has emerged has an important molecule in the vasculature and on platelet surfaces; it limits thrombotic events and contributes to maintain the antithrombotic properties of endothelium. The aim of the present review is to provide an overview of platelets as cellular elements interfacing haemostasis and inflammation, with a particular focus on the emerging role of NTPDase1/CD39 in controlling both processes.

Topics: Animals; Antigens, CD; Apyrase; Humans; Inflammation; Nucleotides; Platelet Activation; Signal Transduction; Thrombosis

ATP and its ultimate degradation product adenosine are potent extracellular signalling molecules that elicit a variety of pathophysiological functions in the kidney through the activation of P2 and P1 purinergic receptors, respectively. Extracellular purines can modulate immune responses, balancing inflammatory processes and immunosuppression; indeed, alterations in extracellular nucleotide and adenosine signalling determine outcomes of inflammation and healing processes. The functional activities of ectonucleotidases such as CD39 and CD73, which hydrolyse pro-inflammatory ATP to generate immunosuppressive adenosine, are therefore pivotal in acute inflammation. Protracted inflammation may result in aberrant adenosinergic signalling, which serves to sustain inflammasome activation and worsen fibrotic reactions. Alterations in the expression of ectonucleotidases on various immune cells, such as regulatory T cells and macrophages, as well as components of the renal vasculature, control purinergic receptor-mediated effects on target tissues within the kidney. The role of CD39 as a rheostat that can have an impact on purinergic signalling in both acute and chronic inflammation is increasingly supported by the literature, as detailed in this Review. Better understanding of these purinergic processes and development of novel drugs targeting these pathways could lead to effective therapies for the management of acute and chronic kidney disease.

Topics: 5'-Nucleotidase; Acute Kidney Injury; Adenosine; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Carcinoma, Renal Cell; Diabetic Nephropathies; Graft Rejection; Humans; Immune Tolerance; Inflammation; Kidney Diseases; Kidney Neoplasms; Kidney Transplantation; Macrophages; Polycystic Kidney Diseases; Receptors, Purinergic P1; Receptors, Purinergic P2; Renal Insufficiency, Chronic; Reperfusion Injury; Signal Transduction; T-Lymphocytes, Regulatory

Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease with periods of exacerbation and remission. SLE is characterized by the irreversible breakdown of immunological self-tolerance, where there is deregulation of multiple aspects of the immune system. SLE immune dysfunction is characterized by activation of autoreactive T lymphocytes, and hyperactivity of B lymphocytes with consequent production of several autoantibodies. ATP is a purinergic mediator released into the extracellular space in response to cell and tissue damage which operates as a danger signal to modulate immune and inflammatory responses. ATP binds to P2 receptors and its levels are regulated by NTPDase (CD39). SLE patients exhibit increased levels of ATP which binds to P2X receptors resulting in activation of the inflammasome and consequent release of IL-1β and IL-18, cytokines associated with disease pathogenesis. CD39 is upregulated in SLE representing an important immunoregulatory mechanism by controlling inflammation and favoring the production of adenosine. The aim of this review is to clarify the effects of ATP on the modulation of the inflammatory process and immune responses via P2 receptors as well as the role of NTPDase in the immunopathogenesis of SLE.

Topics: Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; CD4-Positive T-Lymphocytes; Humans; Inflammation; Lupus Erythematosus, Systemic; Receptors, Purinergic P2X; Self Tolerance; Signal Transduction

Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphate; Antigens, CD; Apyrase; Endothelial Cells; Humans; Immunity, Cellular; Inflammation; Receptors, Purinergic P1; Receptors, Purinergic P2X; Receptors, Purinergic P2Y

Migration and positioning of immune cells is fundamental for their differentiation and recruitment at sites of infection. Besides the fundamental role played by chemokines and their receptors, recent studies demonstrate that a complex network of purinergic signaling events plays a key role in these trafficking events. This process includes the release of nucleotides (such as ATP and ADP) and subsequent autocrine and paracrine signaling events through nucleotide receptors. At the same time, surface-expressed ectoapyrases and nucleotidases convert extracellular nucleotides to adenosine, and adenosine signaling events play additional functional roles in leucocyte trafficking. In this review we revisit classical paradigms of inflammatory cell trafficking in the context of recent studies implicating purinergic signaling events in this process.

Topics: Adenosine; Adenosine Triphosphate; Animals; Apyrase; Cell Communication; Cell Movement; Chemokines; Humans; Inflammation; Leukocytes; Lymphocyte Activation; Nucleotidases; Receptors, Purinergic; Signal Transduction

The ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, or CD39) catalyzes the phosphohydrolysis of extracellular ATP (eATP) and ADP (eADP) released under conditions of inflammatory stress and cell injury. CD39 generates AMP, which is in turn used by the ecto-5'-nucleotidase CD73 to synthesize adenosine. These ectonucleotidases have a major impact on the dynamic equilibrium of proinflammatory eATP and ADP nucleotides versus immunosuppressive adenosine nucleosides. Indeed, CD39 plays a dominant role in the purinergic regulation of inflammation and the immune response because its expression is influenced by genetic and environmental factors. We review the specific role of CD39 in the kinetic regulation of cellular immune responses in the evolution of disease. We focus on the effects of CD39 on T cells and explore potential clinical applications in autoimmunity, chronic infections, and cancer.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Monophosphate; Animals; Antigens, CD; Apyrase; Autoimmune Diseases; Autoimmunity; Gene-Environment Interaction; Humans; Immune Tolerance; Infections; Inflammation; Lymphocyte Activation; Neoplasms; T-Lymphocytes

Adenosine, an immunomodulatory biomolecule, is produced by the ecto-enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) by dephosphorylation of extracellular ATP. CD73 is expressed by many cell types during injury, infection and during steady-state conditions. Besides host cells, many bacteria also have CD39-CD73-like machinery, which helps the pathogen subvert the host inflammatory response. The major function for adenosine is anti-inflammatory, and most recent research has focused on adenosine's control of inflammatory mechanisms underlying various autoimmune diseases (e.g., colitis, arthritis). Although adenosine generated through CD73 provides a feedback to control tissue damage mediated by a host immune response, it can also contribute to immunosuppression. Thus, inflammation can be a double-edged sword: it may harm the host but eventually helps by killing the invading pathogen. The role of adenosine in dampening inflammation has been an area of active research, but the relevance of the CD39/CD73-axis and adenosine receptor signaling in host defense against infection has received less attention. Here, we review our recent knowledge regarding CD73 expression during murine Salmonellosis and Helicobacter-induced gastric infection and its role in disease pathogenesis and bacterial persistence. We also explored a possible role for the CD73/adenosine pathway in regulating innate host defense function during infection.

Topics: 5'-Nucleotidase; Adenosine; Animals; Antigens, CD; Apyrase; Helicobacter Infections; Immunity, Innate; Inflammation; Mice; Salmonella Infections

Extracellular nucleotides play a critical role in vascular thrombosis and inflammation. Alterations in purinergic extracellular nucleotide concentrations activate pathways that result in platelet degranulation and aggregation, and endothelial and leukocyte activation and recruitment. CD39, the dominant vascular nucleotidase, hydrolyzes ATP and ADP to provide the substrate for generation of the anti-inflammatory and antithrombotic mediator adenosine. The purinergic signaling system, with CD39 at its center, plays an important role in modulating vascular homeostasis and the response to vascular injury, as seen in clinically relevant diseases such as stroke, ischemia-reperfusion injury, and pulmonary hypertension. A growing body of knowledge of the purinergic signaling pathway implicates CD39 as a critical modulator of vascular thrombosis and inflammation. Therapeutic strategies targeting CD39 offer promising opportunities in the management of vascular thromboinflammatory diseases.

Topics: Antigens, CD; Apyrase; Atherosclerosis; Endothelium, Vascular; Humans; Hypertension, Pulmonary; Inflammation; Myocardial Ischemia; Myocardial Reperfusion Injury; Signal Transduction; Stroke; Thrombosis; Vasculitis

The enzymatic activities of CD39 and CD73 play strategic roles in calibrating the duration, magnitude, and chemical nature of purinergic signals delivered to immune cells through the conversion of ADP/ATP to AMP and AMP to adenosine, respectively. This drives a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine. The CD39/CD73 pathway changes dynamically with the pathophysiological context in which it is embedded. It is becoming increasingly appreciated that altering this catabolic machinery can change the course or dictate the outcome of several pathophysiological events, such as AIDS, autoimmune diseases, infections, atherosclerosis, ischemia-reperfusion injury, and cancer, suggesting these ectoenzymes are novel therapeutic targets for managing a variety of disorders.

Topics: 5'-Nucleotidase; Animals; Antigens, CD; Apyrase; Humans; Immunity; Inflammation

Interactions between the vascular endothelium and polymorphonuclear leukocytes (PMNs) are central to PMN emigration into inflamed tissues, and to neutrophil-endothelial crosstalk pathways that modulate inflammatory responses and vascular barrier function. For example, during episodes of inflammation, the transendothelial migration (TEM) of PMNs potentially disturbs vascular barrier and gives rise to intravascular fluid extravasation and edema. However, because of the close special relationship between PMNs and the vascular endothelium, TEM creates an ideal situation for neutrophil-endothelial crosstalk. While investigating innate mechanisms to dampen intravascular fluid loss and edema occurring during TEM, we observed that PMNs release adenine nucleotides after activation (adenosine triphosphate [ATP] and adenosine monophosphate [AMP]). ATP and AMP are metabolized by endothelial cell-surface enzymes, the ecto-apyrase (CD39, metabolizes ATP to AMP) and the 5'-ecto-nucleotidase (CD73, metabolizes AMP to adenosine). Adenosine generated in this fashion can activate endothelial adenosine receptors, leading to increases in intracellular cyclic AMP and resealing of the endothelial junctions, thereby promoting vascular barrier function. This crosstalk pathway provides an endogenous mechanism to dampen vascular leak syndrome during neutrophil-endothelial interaction. In other words, during TEM, neutrophils close the door behind them.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Antigens, CD; Apyrase; Capillary Permeability; Cell Movement; Cells, Cultured; Coculture Techniques; Edema; Endothelial Cells; Endothelium, Vascular; Humans; Inflammation; Neutrophils; Signal Transduction

The interaction between platelets and endothelium has been implicated in many disease processes. It is now known that platelets may be involved in the initiation of atheroma, modulate various inflammatory responses, and contribute to endothelial dysfunction, in addition to their classical role in thombosis. Indeed, various drugs targeting platelets and endothelium have been shown to be of benefit in disease states. The last few years has seen an exponential increase in our knowledge of platelet and endothelial biology. As a result of this, many new drugs, for example the ADP-antagonists and the GpIIbIIIa antagonists, have been developed for use in the clinical setting. There is also renewed interest in the use of older drugs (for example, the statins) in modulating platelet-endothelial interactions. This review focuses on the recent advances in our understanding of the interaction(s) between platelets and endothelium and how this knowledge could be manipulated for therapeutic gain.

Topics: Adenosine Triphosphatases; Antigens, CD; Apyrase; Blood Platelets; Cardiovascular Diseases; CD40 Ligand; Cell Adhesion; Chemokine CCL5; Cytokines; Endothelium, Vascular; Humans; Inflammation; Nitric Oxide; P-Selectin; Prostaglandins

Platelet activation as a result of vascular injury provokes endothelial cells to respond in a manner which limits or reverses the occlusive consequences of platelet accumulation. If the agonistic forces are strong, platelet accumulation is irreversible. In vitro data from our laboratory have repeatedly demonstrated that platelets become unresponsive to all agonists when in proximity to endothelial cells. This unresponsiveness is due to at least three separate endothelial "thromboregulatory" systems: eicosanoids, endothelium-derived relaxing factor (EDRF/NO), and most importantly an endothelial cell ecto-nucleotidase which metabolizes released platelet adenosine diphosphate (ADP) with consequent restoration of platelets to the resting state. This nucleotidase is operative in the complete absence of EDRF/NO and eicosanoids, indicating that the latter two are dispensable thromboregulators. We have solubilized the human endothelial cell ectoADPase, as well as that from placental tissue. Candidate proteins from a purified ADPase fraction are now being studied in further detail. An understanding of the molecular biology of the ADPase gene may lead to development of therapeutic agents such as soluble forms of the enzyme as well as approaches toward up-regulation of ectoADPase activity. This could result in "early thromboregulation", i.e. prevention and/or reversal of platelet accumulation at sites of vascular damage via immediate metabolic removal of the prime platelet agonist-ADP.

Topics: Adenosine Diphosphate; Apyrase; Aspirin; Blood Platelets; Cyclooxygenase Inhibitors; Eicosanoids; Endothelium, Vascular; HSP70 Heat-Shock Proteins; Humans; Inflammation; Nitric Oxide; Platelet Activation; Thrombosis

Induced regulatory T-cells (iT-reg) and T helper type 17 (Th17) in the mouse share common CD4 progenitor cells and exhibit overlapping phenotypic and functional features. Here, we show that human Th17 cells endowed with suppressor activity (supTh17) can be derived following exposure of iT-reg populations to Th17 polarizing conditions. In contrast to "pathogenic" Th17, supTh17 display immune suppressive function and express high levels of CD39, an ectonucleotidase that catalyzes the conversion of pro-inflammatory extracellular nucleotides ultimately generating nucleosides. Accordingly, supTh17 exhibit nucleoside triphosphate diphosphohydrolase activity, as demonstrated by the efficient generation of extracellular AMP, adenosine and other purine derivatives. In addition supTh17 cells are resistant to the effects of adenosine as result of the low expression of the A2A receptor and accelerated adenosine catalysis by adenosine deaminase (ADA). These supTh17 can be detected in the blood and in the lamina propria of healthy subjects. However, these supTh17 cells are diminished in patients with Crohn's disease. In summary, we describe a human Th17 subpopulation with suppressor activity, which expresses high levels of CD39 and consequently produces extracellular adenosine. As these uniquely suppressive CD39+ Th17 cells are decreased in patients with inflammatory bowel disease, our findings might have implications for the development of novel anti-inflammatory therapeutic approaches in these and potentially other immune disorders.

Topics: Animals; Antigens, CD; Apyrase; Crohn Disease; Female; Humans; Immune Tolerance; Inflammation; Male; Mice; Th17 Cells

CD39 (ENTPD1) is expressed by subsets of pathogenic human CD4(+) T cells, such as Th17 cells. These Th17 cells are considered important in intestinal inflammation, such as seen in Crohn's disease (CD). Recently, CD161 (NKR-P1A) was shown to be a phenotypic marker of human Th17 cells. In this study, we report that coexpression of CD161 and CD39 not only identifies these cells but also promotes Th17 generation. We note that human CD4(+)CD39(+)CD161(+) T cells can be induced under stimulatory conditions that promote Th17 in vitro. Furthermore, CD4(+)CD39(+)CD161(+) cells purified from blood and intestinal tissues, from both healthy controls and patients with CD, are of the Th17 phenotype and exhibit proinflammatory functions. CD39 is coexpressed with CD161, and this association augments acid sphingomyelinase (ASM) activity upon stimulation of CD4(+) T cells. These pathways regulate mammalian target of rapamycin and STAT3 signaling to drive the Th17 phenotype. Inhibition of ASM activity by pharmacological blockers or knockdown of ASM abrogates STAT3 signaling, thereby limiting IL-17 production in CD4(+) T cells obtained from both controls and patients with active CD. Increased levels of CD39(+)CD161(+) CD4(+) T cells in blood or lamina propria are noted in patients with CD, and levels directly correlate with clinical disease activity. Hence, coexpression of CD39 and CD161 by CD4(+) T cells might serve as a biomarker to monitor Th17 responsiveness. Collectively, CD39 and CD161 modulate human Th17 responses in CD through alterations in purinergic nucleotide-mediated responses and ASM catalytic bioactivity, respectively.

Topics: Adult; Aged; Antigens, CD; Apyrase; Biomarkers; Crohn Disease; Female; Humans; Inflammation; Interleukin-17; Male; Middle Aged; Mucous Membrane; NK Cell Lectin-Like Receptor Subfamily B; Signal Transduction; Sphingomyelin Phosphodiesterase; STAT3 Transcription Factor; Th17 Cells

Alcoholic liver disease caused by chronic excessive drinking has become one of the most common types of liver disease. Alcohol-induced inflammatory immune responses play a central role in the development of alcohol-associated steatohepatitis. The content and expression of ATP and P2X4 in the livers of alcoholic steatohepatitis mice are significantly increased. The content of ATP increased by 20 percent and the expression of P2X4 receptor protein was 1.3 times higher than that in the livers of normal mice. Treatment with 5-BDBD, a P2X4 receptor-specific inhibitor, significantly reduced alcohol-induced liver inflammation and lipid deposition. In RAW264.7 cell experiments, 5-BDBD inhibited the expression of P2X4 and alleviated alcohol-induced inflammation, while the CD39-specific inhibitor POM-1 reduced extracellular ATP degradation and promoted the expression of P2X4, thereby exacerbating inflammation. After treatment with 5-BDBD, P2X4 receptor protein expression decreased by 0.2 times and after treatment with POM-1, P2X4 receptor protein expression increased by 0.1 times compared to the alcohol-stimulated group. In addition, inhibition of P2X4 expression in RAW264.7 cells reduced calcium influx in RAW264.7 cells. P2X4 may induce the activation of NLRP3 inflammasomes by mediating calcium influx, thus exacerbating the inflammatory response, and inhibition of P2X4 expression can effectively block this process. Conclusion: These results suggest that the ATP-P2X4 signaling pathway promotes the inflammatory response in alcoholic steatohepatitis and that CD39 may play a protective role in regulating P2X4 expression by hydrolyzing ATP. In conclusion, the CD39 and ATP-P2X4 signaling pathways may be potential therapeutic targets for alcoholic steatohepatitis.

Topics: Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Fatty Liver, Alcoholic; Inflammasomes; Inflammation; Liver Diseases, Alcoholic; Mice; Receptors, Purinergic P2X4

Emerging evidences have linked the air pollution particulate matters, especially the fine particulate matter PM2.5, to the disease development of chronic obstructive pulmonary disease (COPD). Our previous studies reported that biofuel PM2.5 can induce devastated damage of human bronchial epithelial cells, this study aims to further investigate the underlying molecular mechanisms how biofuel PM2.5 induces bronchial epithelial cell death and dysfunction. In this study, biofuel PM2.5 extracted from wood smoke (WSPM2.5) was used according to our previous publication. A 16-HBE cell line was used as the cell model. Results showed that: Firstly, WSPM2.5 induced significant pyroptosis in 16-HBE cells, reflected by the typical changes including elevated release of lactate dehydrogenase release (LDH) and activated activity and expression of Caspase-1/IL-1β/IL-18 signaling pathway. Then, specific inhibitors for both Caspases (Z-VAD-FMK) and Caspase-1 (VX-765), as well as specific siRNA knockdown of IL-1β all effectively attenuated the WSPM2.5-induced upregulation of downstream inflammatory cytokines and chemokines (IL-6, IL-8, CXCL-1, CXCL-2, etc), respectively. Notably, WSPM2.5 caused a novel increase of intracellular-to-extracellular ATP secretion, which could also contribute to the WSPM2.5-induced pyroptosis and inflammation by activating the Caspase-1/IL-1β/IL-18 signaling pathway through possible autocrine and/or paracrine mechanisms. Antagonism of ATP (Apyrase) or specific siRNA knockdown against ATP receptors (P2Y2 and P2Y7) both significantly inhibited the WSPM2.5-induced pyroptosis and inflammation. These results add up to the current knowledge and bring up novel insights that WSPM2.5 could induce significant pyroptosis and inflammation of human bronchial epithelial cells, through both a classic NLRP3/Caspase-1/IL-1β-dependent and a novel ATP/P2Y-dependent mechanisms.

Topics: Adenosine Triphosphate; Apyrase; Biofuels; Caspase 1; Epithelial Cells; Humans; Inflammation; Interleukin-18; Interleukin-1beta; Interleukin-6; Interleukin-8; Lactate Dehydrogenases; Nicotiana; NLR Family, Pyrin Domain-Containing 3 Protein; Particulate Matter; Pyroptosis; RNA, Small Interfering; Smoke; Wood

Pro-inflammatory stimuli such as hyperglycemia and cytokines have been shown to negatively affect endothelial cell functions. The aim of this study is to assess the potential of quercetin and its human metabolites to overcome the deleterious effects of hyperglycemic or inflammatory conditions on the vascular endothelium by modulating endothelial cell metabolism.. A metabolomics approach enabled identification and quantification of 27 human umbilical vein endothelial cell (HUVEC) metabolites. Treatment of HUVECs with high-glucose concentrations causes significant increases in lactate and glutamate concentrations. Quercetin inhibits glucose-induced increases in lactate and adenosine 5'-triphosphate (ATP) and also increased inosine concentrations. Tumor necrosis factor α-treatment (TNFα) of HUVECs causes increases in asparagine and decreases in aspartate concentrations. Co-treatment with quercetin reduces pyruvate concentrations compared to TNFα-only treated controls. Subsequently, it was shown that quercetin and its HUVEC phase-2 conjugates inhibit adenosine deaminase, xanthine oxidase and 5'nucleotidase (CD73) but not ectonucleoside triphosphate diphosphohydrolase-1 (CD39) or purine nucleoside phosphorylase activities.. Quercetin was shown to alter the balance of HUVEC metabolites towards a less inflamed phenotype, both alone and in the presence of pro-inflammatory stimuli. These changes are consistent with the inhibition of particular enzymes involved in purine metabolism by quercetin and its HUVEC metabolites.

Topics: 5'-Nucleotidase; Adenosine Deaminase; Apyrase; Cytokines; Endothelium, Vascular; Energy Metabolism; Glucose; GPI-Linked Proteins; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Purine-Nucleoside Phosphorylase; Purines; Quercetin; Tumor Necrosis Factor-alpha; Xanthine Oxidase

The epithelial cell-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) initiate type 2 inflammation in allergic diseases, including asthma. However, the signaling pathway regulating these cytokines expression remains elusive. Since microRNAs are pivotal regulators of gene expression, we profiled microRNA expression in bronchial epithelial brushings from type 2-low and type 2-high asthma patients. miR-206 was the most highly expressed epithelial microRNA in type 2-high asthma relative to type 2-low asthma but was downregulated in both subsets compared with healthy controls. CD39, an ectonucleotidase degrading ATP, was a target of miR-206 and upregulated in asthma. Allergen-induced acute extracellular ATP accumulation led to miR-206 downregulation and CD39 upregulation in human bronchial epithelial cells, forming a feedback loop to eliminate excessive ATP. Airway ATP levels were markedly elevated and strongly correlated with IL-25 and TSLP expression in asthma patients. Intriguingly, airway miR-206 antagonism increased Cd39 expression; reduced ATP accumulation; suppressed IL-25, IL-33, and Tslp expression and group 2 innate lymphoid cell expansion; and alleviated type 2 inflammation in a mouse model of allergic airway inflammation. In contrast, airway miR-206 overexpression had opposite effects. Overall, epithelial miR-206 upregulates airway IL-25 and TSLP expression by targeting the CD39-extracellular ATP axis, which represents a potentially novel therapeutic target in type 2-high asthma.

Topics: Adenosine Triphosphate; Adult; Allergens; Animals; Antigens, CD; Apyrase; Asthma; Bronchi; Bronchoscopy; Cells, Cultured; Cytokines; Epithelial Cells; Female; Humans; Inflammation; Interleukin-17; Interleukin-33; Interleukins; Lymphocytes; Male; Mice; MicroRNAs; Middle Aged; Signal Transduction; Thymic Stromal Lymphopoietin

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Adult; Aminopyridines; Animals; Apyrase; Cells, Cultured; Cytokines; GPI-Linked Proteins; Humans; Infertility, Male; Inflammation; Male; Mice; Mice, Inbred C57BL; Middle Aged; Receptor, Adenosine A2B; Receptors, Purinergic P1; RNA Interference; RNA, Small Interfering; Testis

Topics: 5'-Nucleotidase; Age Factors; Aging; Animals; Anti-Inflammatory Agents; Antigens, CD; Apyrase; Cytokines; Female; Inflammation; Interleukin-17; Listeriosis; Mice; Mice, Inbred C57BL; T-Lymphocytes, Regulatory; Th17 Cells

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on

Topics: 5'-Nucleotidase; Activins; Animals; Antigens, CD; Apyrase; Cell Differentiation; Encephalomyelitis, Autoimmune, Experimental; GPI-Linked Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Th17 Cells

In the current study, we explored the role of extracellular ATP (eATP) in promoting systemic inflammation during development of acute pancreatitis (AP). Release of extracellular (e)ATP was evaluated in plasma and bronchoalveolar lavage fluid (BALF) of mice with experimental acute pancreatitis (AP). Prophylactic intervention using apyrase or suramin was used to understand the role and contribution of eATP in pancreatitis-associated systemic injury. AP of varying severity was induced in C57BL/6 mice using 1-day or 2-day caerulein, caerulein + LPS and l-arginine models. eATP was measured in plasma and BALF. Mice were treated with suramin or apyrase in the caerulein and l-arginine models of AP. Plasma cytokines, lung, and pancreatic myeloperoxidase, and morphometric analysis of pancreatic and lung histology, were used to assess the severity of pancreatitis. Plasma eATP and purinergic 2 (P2) receptors in the pancreas and lungs were significantly elevated in the experimental models of AP. Blocking the effect of eATP by suramin led to reduced levels of plasma IL-6 and TNFα as well as reduced lung, and pancreatic injury. Neutralizing eATP with apyrase reduced systemic injury but did not ameliorate local injury. The results of this study support the role of eATP and P2 receptors in promoting systemic inflammation during AP. Modulating purinergic signaling during AP can be an important therapeutic strategy in controlling systemic inflammation and, thus, systemic inflammatory response syndrome during AP.

Topics: Acute Disease; Adenosine Triphosphate; Animals; Apyrase; Arginine; Bronchoalveolar Lavage Fluid; Ceruletide; Cytokines; Inflammation; Lung; Mice; Mice, Inbred C57BL; Pancreas; Pancreatitis; Peroxidase; Receptors, Purinergic; Signal Transduction; Suramin

Unfettered inflammation is a leading cause of multiple organ failures in sepsis. The anti‑inflammatory role of cluster of differentiation (CD)39 has been previously reported. The present study aimed to investigate the role of unfettered inflammation in sepsis‑induced acute kidney injury (AKI). Lipopolysaccharide (LPS) was introduced to construct a sepsis mouse model. Kidney function and pathological changes in mice were measured at 12, 24 and 48 h. CD39 overexpression and inhibition vectors were transfected into renal tubular epithelial (HK‑2) cells, followed by LPS treatment (10 µg/ml), and the cell viability changes at 24 h after treatment were assessed and the expression of NLR family pyrin domain containing 3 (NLRP3), cleaved caspase‑1 and CD39 were determined by performing ELISAs. Cell apoptosis and reactive oxygen species (ROS) levels were determined by flow cytometry. It was found that after LPS administration, kidney injury was the most serious at 24 h in mice. CD39 overexpression could suppress the upregulation of pro‑inflammatory cytokines induced by LPS treatment. In addition, the cell apoptosis and ROS level exhibited an obvious decrease, while cell viability increased. The NLRP3 expression and activity also showed a great inhibition in CD39‑overexpressed cells. By contrast to CD39 overexpression, CD39 inhibition promoted the activation of the NLRP3 inflammasome. These data indicate the protective role of CD39 in LPS‑induced renal tubular epithelial cell damage through inhibiting NLRP3 inflammasome activation and that CD39 might be a potential therapeutic target in sepsis‑induced AKI.

Topics: Acute Kidney Injury; Animals; Antigens, CD; Apoptosis; Apyrase; Cell Survival; Cytokines; Disease Models, Animal; Epithelial Cells; Inflammasomes; Inflammation; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; Sepsis; Signal Transduction

Oxidative stress is one of the primary factors leading to endothelial dysfunction, a major underlying cause of vascular disorders. This study aims to understand the key signalling pathways regulated by sorghum (Shawaya short black 1 variety; characterised to be very high in its antioxidant activity) under oxidative stress in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were pre-treated with non-cytotoxic concentrations of phenolic-rich black sorghum extract (BSE) prior to induction of oxidative stress using hydrogen peroxide (H

Topics: Antioxidants; Apyrase; Gene Expression Regulation; Heme Oxygenase-1; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Inflammation Mediators; Intercellular Adhesion Molecule-1; NADPH Oxidase 4; Nitric Oxide Synthase Type III; Oxidative Stress; Phenols; Plant Extracts; Sorghum

Inflammation plays a central role in the progression of alcoholic liver disease. ATP-P2Y2R signaling and CD39 play an important role in various diseases, but little is known about their role in alcoholic liver steatosis and inflammation. As a transmembrane hydrolase, CD39 hydrolyzes ATP, while the mutual regulation of CD39 and ATP-P2Y2R in alcoholic steatohepatitis is poorly understood. Here, we found that the expression of ATP, P2Y2R, and CD39 is increased significantly both in the liver of alcohol-fed mice and alcohol-induced RAW264.7 cell lines. In this study, C57BL/6 mice were intrapretationally injected with P2Y2R inhibitor suramin from day 4 until day 10 during the induction of a chronic/binge drinking model. Pharmacological blockade of P2Y2R largely prevents liver damage, lipid accumulation, and inflammation, with concomitant down-expression of CD39 in liver. We found that the inhibition of P2Y2R in vitro reduces inflammation via down-expression of interleukin 6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α), and the expression of CD39 was reduced, whereas the activation of P2Y2R showed an opposite effect. Silencing of CD39 promoted the expression of ATP and P2Y2R. These results indicate that CD39 attenuates alcohol-induced steatohepatitis by scavenging extracellular ATP to indirectly regulate the expression of P2Y2R. Interestingly, P2Y2R paradoxically boosts CD39 activity. Thus, blockade of the extracellular ATP-P2Y2R signalling represents a potential therapeutic approach against alcoholic liver disease, and CD39 is a potential therapeutic target.

Topics: Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Cytokines; Fatty Liver, Alcoholic; Inflammation; Male; Mice; Mice, Inbred C57BL; RAW 264.7 Cells; Receptors, Purinergic P2Y2

Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.

Topics: Antigens, CD; Apyrase; B-Lymphocytes; Cell Death; Cellular Microenvironment; Child; Cohort Studies; Colon; Dendritic Cells; Dipyridamole; Endothelial Cells; Epithelial Cells; Fibroblasts; Gene Expression Regulation; Genetic Predisposition to Disease; Homeostasis; Humans; Immunoglobulin G; Immunologic Memory; Inflammation; Inflammatory Bowel Diseases; Interferon Type I; Intestinal Mucosa; Macrophages; Methylprednisolone; Myeloid Cells

Thymol is a phytochemical component present in many plants used as food additive in order to promote animal growth due to its several biological properties. However, possible side effects of thymol remain poorly known limited to few reports. In this sense, we evaluated the enzymes of the purinergic signaling such as, ectonucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA), that play an important role on toxicity induced by excessive adenosine triphosphate (ATP) content in the extracellular environment. Thus, the aim of this study was to evaluate whether purinergic signaling could be considered a potential target of thymol-induced inflammation, and the toxicity in tissues and immune cells of mice after thymol administration. NTPDase activity (ATP as substrate) in serum, spleen, and splenic lymphocytes was lower after 30 days of oral treatment at doses of 10, 20, and 40 mg/kg of thymol, while ADA activity was stimulated at 20 and 40 mg/kg. No differences were observed between groups regarding NTPDase (ADP as substrate) and 5'-nucleotidase activities in all evaluated tissues. Based on these evidences, adenine nucleotide hydrolysis is modified in serum, spleen, and splenic lymphocytes of mice treated with thymol, contributing to inflammation and toxicity by a reduction on ATP hydrolyses and its possible accumulation in the extracellular medium and increased Ado desamination and its possible reduction in the extracellular environment, leading to a self-sustained pro-inflammatory deleterious cycle. In summary, all tested thymol concentrations induced inflammation and toxicity in tissues and immune cells of treated mice.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Animals; Anti-Infective Agents; Antigens, CD; Apyrase; Inflammation; Lymphocytes; Male; Mice; Purines; Spleen; Thymol

CD8. We compared the cytokine production of CD8. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in the vessel wall induces an immune regulatory CD8

Topics: Animals; Antigens, CD; Apyrase; Atherosclerosis; CD8-Positive T-Lymphocytes; Cells, Cultured; Cellular Microenvironment; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Receptors, Antigen, T-Cell; Signal Transduction

Extracellular adenosine triphosphate (eATP), released following inflammatory stimulation or infection, is a potent signaling molecule in activating innate immune responses in fish. However, the regulation of eATP-mediated innate immunity in fish remains unknown. Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a critical molecular switch for controlling the ATP levels in the extracellular space. CD39 plays a key role in regulating eATP-activated innate immune responses through the phosphohydrolysis of pro-inflammatory eATP to inactive AMP. Here, we identified and characterized a CD39 homolog (namely, poCD39) in the Japanese flounder Paralichthys olivaceus and analyzed its regulatory role in eATP-mediated innate immunity. Real-time quantitative PCR analysis revealed that poCD39 is ubiquitously present in all tested normal tissues with dominant expression in enriched Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments demonstrated that poCD39 expression was upregulated by inflammatory stimulation and Edwardsiella tarda infection. Biochemical and immunofluorescence analysis revealed that poCD39 is a functional glycosylated membrane protein for the hydrolysis of eATP. Inhibition of poCD939 activity with the ecto-NTPDase inhibitor ARL 67156 resulted in increased IL-1beta gene expression and ROS production in Japanese flounder HKMs. In contrast, overexpression of poCD39 in Japanese flounder FG-9307 cells reduced eATP-induced pro-inflammatory cytokine IL-1beta gene expression. Finally, poCD39 expression was significantly induced by eATP stimulation in the HKMs, suggesting that eATP may provide a feedback mechanism for transcriptional regulation of fish CD39. Taken together, we identified and characterized a functional fish CD39 protein involved in regulating eATP-mediated innate immune responses in fish.

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Proteins; Flounder; Gene Expression Regulation; Head Kidney; Immunity, Innate; Inflammation; Japan; Macrophages; Reactive Oxygen Species; Transcription, Genetic; Up-Regulation

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS.. We studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity.. aPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression.. We demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.

Topics: 5'-Nucleotidase; Abortion, Spontaneous; Adult; Animals; Antibodies, Antiphospholipid; Antigens, CD; Antiphospholipid Syndrome; Apyrase; Complement C3d; Disease Models, Animal; Female; Humans; Immunization, Passive; Inflammation; Inflammation Mediators; Lipid Peroxidation; Mice; Mice, Knockout; Mice, Transgenic; Pregnancy; Pregnancy Complications; Thromboplastin; Tumor Necrosis Factor-alpha

The ectoenzymes CD39 and CD73 degrade extracellular ATP to adenosine. ATP is released by stressed or damaged cells and provides pro-inflammatory signals to immune cells through P2 receptors. Adenosine, on the other hand, suppresses immune cells by stimulating P1 receptors. Thus, CD39 and CD73 can shape the quality of immune responses. Here we demonstrate that upregulation of CD39 is a consistent feature of activated conventional CD4+ and CD8+ T cells. Following stimulation in vitro, CD4+ and CD8+ T cells from human blood gained surface expression of CD39 but displayed only low levels of CD73. Activated human T cells from inflamed joints largely presented with a CD39+CD73- phenotype. In line, in spleens of mice with acute Listeria monocytogenes, listeria-specific CD4+ and CD8+ T cells acquired a CD39+CD73- phenotype. To test the function of CD39 in control of bacterial infection, CD39-deficient (CD39-/-) mice were infected with L. monocytogenes. CD39-/- mice showed better initial control of L. monocytogenes, which was associated with enhanced production of inflammatory cytokines. In the late stage of infection, CD39-/- mice accumulated more listeria-specific CD8+ T cells in the spleen than wildtype animals suggesting that CD39 attenuates the CD8+ T-cell response to infection. In conclusion, our results demonstrate that CD39 is upregulated on conventional CD4+ and CD8+ T cells at sites of acute infection and inflammation, and that CD39 dampens responses to bacterial infection.

Topics: Animals; Antigens, CD; Apyrase; CD8-Positive T-Lymphocytes; Flow Cytometry; Gene Expression Regulation; Humans; Immunity, Innate; Infections; Inflammation; Listeria monocytogenes; Listeriosis; Lymphocyte Activation; Mice

Ferulic acid (FA), derived from fruits and vegetables, is well-known as a potent antioxidant of scavenging free radicals. However, the role and underlying mechanism of FA on kidney ischemia reperfusion (I/R) injury are limited. Here, we explored the effects of FA on kidney I/R injury. The kidney I/R injury models were carried out by clamping bilateral pedicles for 35 min followed by reperfusion for 24 h. Mice were orally pretreated with different doses of FA for three times 24 h before I/R. The renal function was assessed by serum creatine (Scr) and blood urea nitrogen (BUN). Kidney histology was examined by hematoxylin and eosin (HE) staining and terminal deoxynucleotidly transferased UTP nick-end labeling (TUNEL) assay. Proinflammatory cytokines, caspase-3 activity, adenosine generation, adenosine signaling molecules, and hypoxia inducible factor-1 alpha (HIF-1α) were also detected, respectively. The siHIF-1α adenovirus vectors were in vivo used to inhibit the expression of HIF-1α. The results showed that FA significantly attenuated kidney damage in renal I/R-operated mice as indicated by reducing levels of Scr and BUN, ameliorating renal pathological structural changes, and tubular cells apoptosis. Moreover, FA pretreatment inhibited I/R-induced renal proinflammatory cytokines and neutrophils recruitment. Interestingly, the levels of HIF-α, CD39, and CD73 mRNA and protein as well as adenosine production were all significantly increased after FA pretreatment in the kidney of I/R-performed mice, and inhibiting HIF-α expression using siRNA abolished this protection of FA on I/R-induced acute kidney injury as evidenced by more severe renal damage and reduced adenosine production. Our findings indicated that FA protected against kidney I/R injury by reducing apoptosis, alleviating inflammation, increasing adenosine generation, and upregulating CD39 and CD73 expression, which might be mediated by HIF-1α.

Topics: 5'-Nucleotidase; Adenosine; Animals; Antigens, CD; Apoptosis; Apyrase; Coumaric Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Kidney; Mice; Protective Agents; Reperfusion Injury

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

Topics: Animals; Antigens, CD; Apoptosis; Apyrase; Cell Proliferation; Cell-Derived Microparticles; Endothelial Cells; Inflammation; Mice, Inbred C57BL; MicroRNAs

Autoimmune diseases are characterized by an imbalance between regulatory T cells and effector T-cell subsets, such as Th1 and Th17 cells. Studies have confirmed that natural CD4

Topics: Animals; Antigens, CD; Apyrase; Autoimmunity; Cells, Cultured; DNA Methylation; Forkhead Transcription Factors; Graft vs Host Disease; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Mice; Mice, SCID; STAT1 Transcription Factor; STAT3 Transcription Factor; Stem Cell Transplantation; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Transplantation, Heterologous

Sepsis results in unfettered inflammation, tissue damage, and multiple organ failure. Diffuse brain dysfunction and neurological manifestations secondary to sepsis are termed sepsis-associated encephalopathy (SAE). Extracellular nucleotides, proinflammatory cytokines, and oxidative stress reactions are associated with delirium and brain injury, and might be linked to the pathophysiology of SAE. P2X7 receptor activation by extracellular ATP leads to maturation and release of IL-1β by immune cells, which stimulates the production of oxygen reactive species. Hence, we sought to investigate the role of purinergic signaling by P2X7 in a model of sepsis. We also determined how this process is regulated by the ectonucleotidase CD39, a scavenger of extracellular nucleotides. Wild type (WT), P2X7 receptor (P2X7

Topics: Animals; Antigens, CD; Apyrase; Brain; Catalase; Cytokines; Inflammation; Male; Mice; Mice, Knockout; Oxidative Stress; Receptors, Purinergic P2X7; Sepsis; Signal Transduction; Superoxide Dismutase

Naringenin (NGEN) is a natural flavonoid aglycone of naringin that has been reported to have a wide range of pharmacological properties, such as antioxidant activity and free radical scavenging capacity. The aim of this study was to investigate the protective effect of NGEN on oxidative and inflammatory parameters, as well as to evaluate the hydrolysis of adenine nucleotides in kidney and platelet membranes of rats exposed to a hypercholesterolemic diet (HCD) for 90 days. Kidney oxidative stress and mRNA expression of the ectonucleoside triphosphate diphosphohydrolases (NTPDases), ecto-5'-nucleotidase (CD73), inducible NO synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and the nuclear factor kappa B (NF-κB) genes were evaluated by real time RT-PCR. The co-administration of NGEN (50 mg kg(-1)) for 90 days significantly prevented renal failure in HCD rats as indicated by an improvement of renal markers. Histopathological observation findings are also consistent with these effects. Moreover, NGEN (50 mg kg(-1)) significantly decreased the lipid profile and inhibited pro-oxidant and inflammation marker levels in the kidney of HCD rats. Furthermore, the NTPDase activities were significantly decreased in platelets and kidney membranes of HCD-treated rats and these alterations were improved by NGEN. In conclusion, this study suggests that naringenin can potentially improve the renal failure and platelet alterations observed in rats fed a hypercholesterolemic diet probably through its antioxidant effects.

Topics: Adenine Nucleotides; Animals; Antioxidants; Apyrase; Blood Platelets; Cholesterol, Dietary; Flavanones; Free Radical Scavengers; Gene Expression; Hydrolysis; Hypercholesterolemia; Inflammation; Kidney; Male; Mitochondria; NF-kappa B; Nucleotidases; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Renal Insufficiency; Signal Transduction

Obesity-induced inflammation is conducted by a metabolic pathway, which eventually causes activation of specialized immune cells and leads to an unresolved inflammatory response within the tissue. For this reason, it is critically important to determine how hypertrophic fat tissue alters T cell balance to drive inflammation. In this study, we identify the purinergic signaling as a novel mechanism driving the adaptive Th17 response in human visceral adipose tissue (VAT) of metabolically unhealthy obese patients. We demonstrate that ATP acting via the P2X7 receptor pathway promotes a Th17 polarizing microenvironment with high levels of IL-1β, IL-6, and IL-17 in VAT explants from lean donors. Moreover, in vitro blockade of the P2X7 receptor abrogates the levels of these cytokines. These findings are consistent with a greater frequency of Th17 cells in tissue from metabolically unhealthy obese donors, revealed not only by the presence of a baseline Th17-promoting milieu, but also by the higher expression of steadily recognized Th17 markers, such as RORC, IL-17 cytokine, and IL-23R, in comparison with metabolically healthy obese and lean donors. In addition, we demonstrate that CD39 expression on CD4(+)effector T cells represents a novel Th17 marker in the inflamed VAT, which also confers protection against ATP-induced cell death. The manipulation of the purinergic signaling might represent a new therapeutic target to shift the CD4(+)T cell balance under inflammatory conditions.

Topics: Adenosine Triphosphate; Adult; Antigens, CD; Apoptosis; Apyrase; Cellular Microenvironment; Female; Humans; Inflammation; Interleukin-17; Interleukin-1beta; Interleukin-6; Intra-Abdominal Fat; Male; Nuclear Receptor Subfamily 1, Group F, Member 3; Obesity; Receptors, Interleukin; Receptors, Purinergic P2X7; Th17 Cells

T helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation.

Topics: 5'-Nucleotidase; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Cell Death; Cell Survival; Colitis; Hydrolysis; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-23; Intestines; Mice, Inbred C57BL; Phenotype; Receptors, Purinergic; Signal Transduction; Th17 Cells; Transforming Growth Factor beta1

The aim of this study was to evaluate seric NTPDase and 5'nucleotidase activities of cattle naturally infected by Eurytrema coelomanticum, as well as to correlate them to histopathological lesions in the pancreas and the degree of parasitism. Blood samples and pancreas of 51 bovines were collected on a slaughterhouse in Southern Brazil: 33 from cattle naturally infected by E. coelomanticum (the Group A), and 18 from uninfected animals (the Group B). Infected animals showed an average of 532 parasites per pancreas. In the pancreatic histology, ducts displayed hyperplasia, stenosis, proliferation of fibrous tissue, and interstitial inflammatory infiltration of lymphocytes. The serum from infected animals showed an increase in NTPDase activity when ATP was used as substrate (P<0.001). For the ADP substrate, there was no difference between groups regarding NTPDase activity (P=0.37), as well as 5'-nucleotidase activity (P=0.27). Correlating NTPDase activity (ATP substrate) with the degree of histopathological lesions (rho=0.66, P<0.001) and the parasitic load on the pancreas (rho=0.65, P<0.001), a positive correlation was observed. Similar results were found between the degree of histopathological lesions and NTPDase activity (ADP substrate; rho=0.29, P=0.03), and 5'nucleotidase activity (rho=0.35, P=0.01). Based on the results of NTPDase and 5'nucleotidase enzymes in cattle naturally infected by E. coleomanticum, it is possible to suggest that these enzymes are involved in the modulation of inflammation, and they can act as markers of inflammatory response.

Topics: 5'-Nucleotidase; Abattoirs; Animals; Antigens, CD; Apyrase; Biomarkers; Brazil; Cattle; Cattle Diseases; Dicrocoeliidae; Inflammation; Lymphocytes; Pancreas; Parasite Load; Trematode Infections

Sepsis remains the leading cause of morbidity and mortality in critically ill patients. Excessive inflammation is a major cause of organ failure and mortality in sepsis. Ectonucleoside triphosphate diphosphohydrolase 1, ENTPDase1 (CD39) is a cell surface nucleotide-metabolizing enzyme, which degrades the extracellular purines ATP and ADP, thereby regulating purinergic receptor signaling. Although the role of purinergic receptor signaling in regulating inflammation and sepsis has been addressed previously, the role of CD39 in regulating the host's response to sepsis is unknown. We found that the CD39 mimic apyrase (250 U/kg) decreased and knockout or pharmacologic blockade with sodium polyoxotungstate (5 mg/kg; IC50 ≈ 10 μM) of CD39 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture. CD39 decreased inflammation, organ damage, immune cell apoptosis, and bacterial load. Use of bone marrow chimeric mice revealed that CD39 expression on myeloid cells decreases inflammation in septic mice. CD39 expression is upregulated during sepsis in mice, as well as in both murine and human macrophages stimulated with Escherichia coli. Moreover, E. coli increases CD39 promoter activity in macrophages. Altogether, these data indicate CD39 as an evolutionarily conserved inducible protective pathway during sepsis. We propose CD39 as a novel therapeutic target in the management of sepsis.

Topics: 5'-Nucleotidase; Animals; Antigens, CD; Apyrase; Chemokines; Cytokines; Escherichia coli; Humans; Inflammation; Interleukin-10; Interleukin-12; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Promoter Regions, Genetic; Sepsis; Transplantation Chimera

Regulatory T cells have various mechanisms to suppress the inflammatory response, among these, the modulation of the microenvironment through adenosine and with the participation of CD39, CD73 and A2A. The aim of this study was to assess the expression of CD73 and A2A in immune cells and the effect of activation of A2A by an adenosine analogue on apoptosis in patients with obesity and type 2 diabetes mellitus (T2D). CD73 and A2A expression were analyzed by flow cytometry in lymphocyte subpopulations from patients with obesity (n = 22), T2D (n = 22), and healthy subjects (n = 20). Lymphocytes were treated with the selective A2A antagonist (ZM241385) or the selective A2A agonist (CGS21680), and apoptotic cells were detected by Annexin V. We found an increased expression of CD39 coupled to a decrease in CD73 in the patient groups with obesity and T2D compared to the control group in the different studied lymphocyte subpopulations. A2A expression was found to be increased in different subpopulations of lymphocytes from T2D patients. We also detected positive correlations between CD39+ cells and age and BMI. Meanwhile, CD73+ cells showed negative correlations with age, WHR, BMI, FPG, HbAc1, triglycerides and cholesterol. Moreover, an increase in the percentage of apoptotic cells from T2D patients with regard to the groups with obesity and control was observed. In addition, the CD8+ T cells of patients with T2D exhibited decreased apoptosis when treated with the A2A agonist. In conclusion, our data suggest a possible role for CD73 and A2A in inflammation observed in patients with T2D and obesity mediated via apoptosis.

Topics: 5'-Nucleotidase; Adenosine; Adult; Antigens, CD; Apoptosis; Apyrase; Body Mass Index; CD8-Positive T-Lymphocytes; Diabetes Mellitus, Type 2; Gene Expression Regulation; GPI-Linked Proteins; Humans; Inflammation; Lymphocyte Subsets; Lymphocytes; Obesity; Phenethylamines; Receptor, Adenosine A2A; Triazines; Triazoles

Our understanding of the pathways that regulate lymphocyte metabolism, as well as the effects of metabolism and its products on the immune response, is still limited. We report that a metabolic program controlled by the transcription factors hypoxia inducible factor-1α (HIF1-α) and aryl hydrocarbon receptor (AHR) supports the differentiation of type 1 regulatory T cell (Tr1) cells. HIF1-α controls the early metabolic reprograming of Tr1 cells. At later time points, AHR promotes HIF1-α degradation and takes control of Tr1 cell metabolism. Extracellular ATP (eATP) and hypoxia, linked to inflammation, trigger AHR inactivation by HIF1-α and inhibit Tr1 cell differentiation. Conversely, CD39 promotes Tr1 cell differentiation by depleting eATP. CD39 also contributes to Tr1 suppressive activity by generating adenosine in cooperation with CD73 expressed by responder T cells and antigen-presenting cells. These results suggest that HIF1-α and AHR integrate immunological, metabolic and environmental signals to regulate the immune response.

Topics: Adult; Animals; Antigens, CD; Apyrase; Cell Differentiation; Female; Hematopoiesis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunity, Cellular; Inflammation; Lymphocyte Activation; Mice; Receptors, Aryl Hydrocarbon; T-Lymphocytes, Regulatory

Chronic inflammation is known to promote metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory response in obesity is unclear, it is known that overproduction of proinflammatory cytokines by innate immune cells affects metabolism. For example, TNF-α contributes to the inability of cells to respond to insulin and to the increase in levels of insulin. Whether this hyperinsulinemia itself is part of a feedback loop that affects the progression of chronic adipose inflammation is unknown. In this article, we show that regulatory T cells (Tregs) express the insulin receptor, and that high levels of insulin impair the ability of Tregs to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin activated AKT signaling in Tregs, leading to inhibition of both IL-10 production and the ability of Tregs to suppress the production of TNF-α by macrophages in a contact-independent manner. The effect of insulin on Treg suppression was limited to IL-10 production and it did not alter the expression of other proteins associated with Treg function, including CTLA-4, CD39, and TGF-β. In a model of diet-induced obesity, Tregs from the visceral adipose tissue of hyperinsulinemic, obese mice showed a similar specific decrease in IL-10 production, as well as a parallel increase in production of IFN-γ. These data suggest that hyperinsulinemia may contribute to the development of obesity-associated inflammation via a previously unknown effect of insulin on the IL-10-mediated function of Tregs.

Topics: Animals; Antigens, CD; Apyrase; Cells, Cultured; CTLA-4 Antigen; Epithelial Cells; Epithelium; Hyperinsulinism; Inflammation; Insulin; Interferon-gamma; Interleukin-10; Intra-Abdominal Fat; Macrophages; Mice; Mice, Inbred C57BL; Obesity; Proto-Oncogene Proteins c-akt; Receptor, Insulin; Signal Transduction; T-Lymphocytes, Regulatory; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The mammalian immune system constitutively senses vast quantities of commensal bacteria and their products through pattern recognition receptors, yet excessive immune reactivity is prevented under homeostasis. The intestinal microbiome can influence host susceptibility to extra-intestinal autoimmune disorders. Here we report that polysaccharide A (PSA), a symbiosis factor for the human intestinal commensal Bacteroides fragilis, protects against central nervous system demyelination and inflammation during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, through Toll-like receptor 2 (TLR2). TLR2 mediates tissue-specific expansion of a critical regulatory CD39(+) CD4 T-cell subset by PSA. Ablation of CD39 signalling abrogates PSA control of EAE manifestations and inflammatory cytokine responses. Further, CD39 confers immune-regulatory phenotypes to total CD4 T cells and Foxp3(+) CD4 Tregs. Importantly, CD39-deficient CD4 T cells show an enhanced capability to drive EAE progression. Our results demonstrate the therapeutic potential and underlying mechanism by which an intestinal symbiont product modulates CNS-targeted demyelination.

Topics: Animals; Antigens, CD; Apyrase; Bacteroides fragilis; CD4-Positive T-Lymphocytes; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Forkhead Transcription Factors; Humans; Inflammation; Intestinal Mucosa; Intestines; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Polysaccharides, Bacterial; Signal Transduction; Symbiosis; Toll-Like Receptor 2

Mast cells (MCs) are major contributors to an inflammatory milieu. One of the most potent drivers of inflammation is the cytokine IL-1β, which is produced in the cytoplasm in response to danger signals like LPS. Several controlling mechanisms have been reported which limit the release of IL-1β. Central to this regulation is the NLRP3 inflammasome, activation of which requires a second danger signal with the capacity to subvert the homeostasis of lysosomes and mitochondria. High concentrations of extracellular ATP have the capability to perturb the plasma membrane by activation of P2X7 channels and serve as such a danger signal. In this study we investigate the role of P2X7 channels and the ecto-5'-nucleotidase CD39 in ATP-triggered release of IL-1β from LPS-treated mast cells.. We report that in MCs CD39 sets an activation threshold for the P2X7-dependent inflammatory cell death and concomitant IL-1β release. Knock-out of CD39 or stimulation with non-hydrolysable ATP led to a lower activation threshold for P2X7-dependent responses. We found that stimulation of LPS-primed MCs with high doses of ATP readily induced inflammatory cell death. Yet, cell death-dependent release of IL-1β yielded only minute amounts of IL-1β. Intriguingly, stimulation with low ATP concentrations augmented the production of IL-1β in LPS-primed MCs in a P2X7-independent but caspase-1-dependent manner.. Our study demonstrates that the fine-tuned interplay between ATP and different surface molecules recognizing or modifying ATP can control inflammatory and cell death decisions.

Topics: Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Bone Marrow Cells; Caspase 1; Cell Death; Cells, Cultured; Inflammation; Interleukin-1beta; Lipopolysaccharides; Male; Mast Cells; Mice; Receptors, Purinergic P2X7

Sepsis is a highly fatal disease caused by an initial hyperinflammatory response followed by a state of profound immunosuppression. Although it is well appreciated that the initial production of proinflammatory cytokines by macrophages accompanies the onset of sepsis, it remains unclear what causes the transition to an immunosuppressive state. In this study, we reveal that macrophages themselves are key regulators of this transition and that the surface enzyme CD39 plays a critical role in self-limiting the activation process. We demonstrate that Toll-like receptor (TLR)-stimulated macrophages modulate their activation state by increasing the synthesis and secretion of adenosine triphosphate (ATP). This endogenous ATP is paradoxically immunosuppressive due to its rapid catabolism into adenosine by CD39. Macrophages lacking CD39 are unable to transition to a regulatory state and consequently continue to produce inflammatory cytokines. The importance of this transition is demonstrated in a mouse model of sepsis, where small numbers of CD39-deficient macrophages were sufficient to induce lethal endotoxic shock. Thus, these data implicate CD39 as a key "molecular switch" that allows macrophages to self-limit their activation state. We propose that therapeutics targeting the release and hydrolysis of ATP by macrophages may represent new ways to treat inflammatory diseases.

Topics: Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Cell Line; Cells, Cultured; Cytokines; Female; Flow Cytometry; Gene Expression; Homeostasis; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; Toll-Like Receptors

The ocular microenvironment uses a poorly defined mela5 receptor (MC5r)-dependent pathway to recover immune tolerance following intraocular inflammation. This dependency is seen in experimental autoimmune uveoretinitis (EAU), a mouse model of endogenous human autoimmune uveitis, with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. In this study, we found that the MC5r-dependent regulatory immunity increased CD11b(+)F4/80(+)Ly-6C(low)Ly-6G(+)CD39(+)CD73(+) APCs in the spleen of post-EAU mice. These MC5r-dependent APCs require adenosine 2A receptor expression on T cells to activate EAU-suppressing CD25(+)CD4(+)Foxp3(+) regulatory T cells. Therefore, in the recovery from autoimmune disease, the ocular microenvironment induces tolerance through a melanocortin-mediated expansion of Ly-6G(+) regulatory APCs in the spleen that use the adenosinergic pathway to promote activation of autoantigen-specific regulatory T cells.

Topics: 5'-Nucleotidase; Animals; Antigens, CD; Antigens, Ly; Apyrase; Autoantigens; Autoimmune Diseases; CD11b Antigen; CD4 Antigens; Disease Models, Animal; Eye; Eye Proteins; Forkhead Transcription Factors; Inflammation; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptor, Adenosine A2A; Receptors, Melanocortin; Retinol-Binding Proteins; Self Tolerance; Spleen; T-Lymphocytes, Regulatory; Uveitis

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.

Topics: Animals; Apyrase; Caco-2 Cells; Clostridioides difficile; Disease Models, Animal; Enterocolitis, Pseudomembranous; Enterotoxins; Humans; Inflammation; Interleukin-8; Intestinal Mucosa; Male; Mice; NF-kappa B; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Signal Transduction

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, including immune cells, comprise the tumor microenvironment where they create a milieu that appears to dictate cancer development. ATP and the phosphohydrolytic products ADP and adenosine by activating P2 and P1 receptors may participate in these interactions among malignant and immune cells. Purinergic receptor-mediated cell communication is closely regulated by ectonucleotidases, such as by members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, which hydrolyze extracellular nucleotides. We have shown that gliomas, unlike astrocytes, exhibit low NTPDase activity. Furthermore, ATP induces glioma cell proliferation and the co-administration of apyrase decreases progression of injected cells in vivo. We have previously shown that NTPDase2 reconstitution dramatically increases tumor growth in vivo. Here we evaluated whether NTPDase2 reconstitution to gliomas modulates systemic inflammatory responses. We observed that NTPDase2 overexpression modulated pro-inflammatory cytokine production and platelet reactivity. Additionally, pathological alterations in the lungs were observed in rats bearing these tumors. Our results suggest that disruption of purinergic signaling via ADP accumulation creates an inflammatory state that may promote tumor spread and dictate clinical progression.

Topics: Adenosine Triphosphatases; Animals; Apyrase; Brain Neoplasms; Cell Line, Tumor; Gene Expression Regulation, Enzymologic; Glioma; Inflammation; Inflammation Mediators; Lung Injury; Male; Rats; Rats, Wistar

The ectonucleotidase CD39 has recently been described as being highly expressed on regulatory Foxp3(+) CD4 T cells. Through hydrolysis of proinflammatory extracellular ATP, CD39 activity represents a newly described mechanism of regulatory T cell action. We report a novel population of human CD4 T cells that express CD39 yet are Foxp3 negative. These cells produce the proinflammatory cytokines IFN-gamma and IL-17 and fail to suppress proliferation; however, they still have high ATP hydrolysis activity. In the inflammatory site in human juvenile idiopathic arthritis, the CD39(+)Foxp3(-) population is greatly increased compared with peripheral blood of patients or healthy controls. We also show that cells expressing the AMPase CD73 are less frequent in the joint than in blood. To our knowledge, this is the first study to describe and characterize CD39 function on CD4 T cells from the target site in a human autoinflammatory condition. Our data suggest that in human CD4(+) T cells from the inflamed site, CD39 can be highly expressed on two populations, one regulatory and the other of a memory phenotype.

Topics: Adolescent; Antigens, CD; Apyrase; Arthritis, Juvenile; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Child; Female; Gene Expression Regulation, Enzymologic; Humans; Immunologic Memory; Immunophenotyping; Inflammation; Inflammation Mediators; Male; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Extracellular adenosine has been implicated as anti-inflammatory signaling molecule during acute lung injury (ALI). The main source of extracellular adenosine stems from a coordinated two-step enzymatic conversion of precursor nucleotides via the ecto-apyrase (CD39) and the ecto-5'-nucleotidase (CD73). In the present study, we hypothesized a critical role of CD39 and CD73 in mediating pulmonary neutrophil (PMN) transmigration during lipopolysaccharide (LPS) -induced lung injury. Initial studies revealed that pulmonary CD39 and CD73 transcript levels were elevated following LPS exposure in vivo. Moreover, LPS-induced accumulation of PMN into the lungs was enhanced in cd39(-/-) or cd73(-/-) mice, particularly into the interstitial and intra-alveolar compartment. Such increases in PMN trafficking were accompanied by corresponding changes in alveolar-capillary leakage. Similarly, inhibition of extracellular nucleotide phosphohydrolysis with the nonspecific ecto-nucleoside-triphosphate-diphosphohydrolases inhibitor POM-1 confirmed increased pulmonary PMN accumulation in wild-type, but not in gene-targeted mice for cd39 or cd73. Finally, treatment with apyrase or nucleotidase was associated with attenuated pulmonary neutrophil accumulation and pulmonary edema during LPS-induced lung injury. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in attenuating PMN trafficking into the lungs during LPS-induced lung injury and suggest treatment with their soluble compounds as a therapeutic strategy.

Topics: 5'-Nucleotidase; Adenosine; Animals; Antigens, CD; Apyrase; Cell Movement; Gene Expression Regulation; Inflammation; Lipopolysaccharides; Lung Diseases; Male; Mice; Mice, Knockout; Neutrophils; Nucleotidases; Phosphorylation; Solubility; Transcription, Genetic

In-stent thrombosis is mainly triggered by adenosine diphosphate (ADP)-dependent platelet aggregation after percutaneous coronary stent implantation. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) rapidly hydrolyzes ADP to adenosine monophosphate, inhibiting platelet aggregation. We tested the hypothesis that local delivery of human placental E-NTPDase (pE-NTPDase) gene into injured arteries via gene-eluting stent could prevent subacute in-stent thrombosis.. We generated gene-eluting stents by coating bare metal stents with cationic gelatin hydrogel containing pE-NTPDase cDNA (pE-NTPDase stent), and implanted the stents into rabbit femoral arteries (FA) prone to production of platelet-rich thrombi due to repeated balloon injury at 4-week intervals. After the second injury, E-NTPDase gene expression was severely decreased; however, the implantation of pE-NTPDase stent increased E-NTPDase mRNA levels and NTPDase activity to higher level than normal FA. The FAs with pE-NTPDase stents maintained patency in all rabbits (P<0.01), whereas the stent-implanted FAs without pE-NTPDase gene showed low patency rates (17% to 25%). The occlusive platelet-rich thrombi, excessive neointimal growth, and infiltration of macrophages were inhibited in stent implanted FA with pE-NTPDase gene, but not without pE-NTPDase gene.. Human pE-NTPDase gene transfer via cationic gelatin-coated stents inhibited subacute in-stent thrombosis and suppressed neointimal hyperplasia and inflammation without antiplatelet drugs.

Topics: Angioplasty, Balloon; Animals; Apyrase; Cell Proliferation; Coated Materials, Biocompatible; Disease Models, Animal; Female; Femoral Artery; Gelatin; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Hyperplasia; Inflammation; Male; Muscle, Smooth, Vascular; Nitric Oxide Synthase Type III; Peripheral Vascular Diseases; Placenta; Platelet Aggregation; Rabbits; Stents; Thrombosis; Time Factors; Tunica Intima; Vascular Patency

A clopidogrel loading dose administered during stenting attenuates inflammation marker release. However, less is known of the anti-inflammatory effect of clopidogrel maintenance therapy. Platelet reactivity to adenosine diphosphate and inflammation markers were measured in 110 consecutive patients (69 clopidogrel-naive patients and 41 patients receiving long-term clopidogrel therapy for >6 months) before nonemergent stenting by turbidimetric aggregometry and flow cytometry and multianalyte profiling, respectively. All patients were treated with aspirin. Prestenting adenosine diphosphate-induced platelet aggregation, P-selectin, and activated glycoprotein IIb/IIIa expression were lower in patients receiving long-term clopidogrel therapy compared with the clopidogrel-naive group (p <0.001), accompanied by lower levels of selected inflammation markers (p < or = 0.05). Additionally, there were strong correlations between platelet aggregation and flow cytometric measurements (p < or = 0.04) and between specific inflammation markers (p < or = 0.02). In conclusion, in addition to markedly lowering platelet reactivity to adenosine diphosphate, long-term clopidogrel therapy is associated with an anti-inflammatory effect.

Topics: Aged; Angioplasty, Balloon, Coronary; Apyrase; Blood Platelets; Clopidogrel; Female; Flow Cytometry; Humans; Inflammation; Male; Middle Aged; Myocardial Ischemia; P-Selectin; Pilot Projects; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Stents; Ticlopidine

Chronic inflammation, fibrosis, atrophy, malignant transformation, and thromboembolic events are hallmarks of chronic pancreatic disease. Extracellular nucleotides have been implicated as inflammatory mediators in many pathological situations. However, there are minimal data detailing expression of ectonucleotidases and type-2 purinergic receptors (P2R) in chronic pancreatitis and pancreatic cancer. We have therefore defined tissue distribution and localization of the CD39 family of ectonucleotidases and associated P2R in human disease. Transcripts of ectonucleotidases (CD39 and CD39L1) together with P2R (P2X7, P2Y2, and P2Y6) are significantly increased in both chronic pancreatitis and pancreatic cancer. CD39 and CD39L1 are preferentially associated with the vasculature and stromal elements in pathological tissues. P2X7 mRNA upregulation was associated with chronic pancreatitis, and heightened protein expression was found to be localized to infiltrating cells. P2Y2 was markedly upregulated in biopsies of pancreatic cancer tissues and expressed by fibroblasts adjacent to tumors. High-tissue mRNA levels of CD39 significantly correlated with better long-term survival after tumor resection in patients with pancreatic cancer. Heightened expression patterns and localization patterns of CD39, P2X7, and P2Y2 infer associations with chronic inflammation and neoplasia of the pancreas. Our data suggest distinct roles for CD39 and P2-purinergic signaling in both tissue remodeling and fibrogenesis with respect to human pancreatic diseases.

Topics: Adult; Alcoholism; Antigens, CD; Apyrase; Cell Transformation, Neoplastic; Chronic Disease; Female; Gene Expression Regulation; Humans; Inflammation; Male; Middle Aged; Neoplasm Staging; Pancreatic Diseases; Pancreatic Neoplasms; Pancreatitis; Receptors, Purinergic P2; Thromboembolism; Tissue Donors

The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in platelets from patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory markers, such as oxidized low-density lipoprotein, highly sensitive C-reactive protein and oxidized low-density lipoprotein autoantibodies, was also investigated. Lipid peroxidation was estimated by measurement of thiobarbituric acid reactive substances in serum. The following groups were studied: group I, < 150 mg.dL(-1) cholesterol; group II, 151-200 mg.dL(-1) cholesterol; group III, 201-250 mg.dL(-1) cholesterol; and group IV, > 251 mg.dL(-1) cholesterol. The results demonstrated that both ATP hydrolysis and ADP hydrolysis were enhanced as a function of cholesterol level. Low-density lipoprotein levels increased concomitantly with total cholesterol levels. Triglyceride levels were increased in the groups with total cholesterol above 251 mg.dL(-1). Oxidized low-density lipoprotein levels were elevated in groups II, III, and IV. Highly sensitive C-reactive protein was elevated in the group with cholesterol levels higher than 251 mg.dL(-1). Oxidized low-density lipoprotein autoantibodies were elevated in groups III and IV. Thiobarbituric acid reactive substance content was enhanced as a function of cholesterol level. In summary, hypercholesterolemia is associated with enhancement of inflammatory response, oxidative stress, and ATP and ADP hydrolysis. The increased ATP and ADP hydrolysis in group IV was confirmed by an increase in CD39 expression on its surface. The increase in CD39 activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia.

Topics: Adenine Nucleotides; Adult; Aged; Antigens, CD; Apyrase; Autoantibodies; Blood Glucose; Blood Platelets; C-Reactive Protein; Female; Humans; Hypercholesterolemia; Inflammation; Lipid Peroxidation; Lipoproteins, HDL; Lipoproteins, LDL; Male; Middle Aged; Triglycerides

Topics: Adenosine Triphosphatases; Animals; Antigens, CD; Apyrase; Humans; Immunity; In Vitro Techniques; Inflammation; Langerhans Cells; Mice; Mice, Knockout; Models, Immunological; Signal Transduction; Skin; T-Lymphocytes

CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39-/- mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39-/- mice. Paradoxically, T cell-mediated allergic contact hypersensitivity was severely attenuated in CD39-/- mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39-/- DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC-T cell communication in antigen presentation.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Female; Immunity; Inflammation; Irritants; Langerhans Cells; Male; Mice; Mice, Knockout; Receptors, Purinergic P2; Signal Transduction

Xeroderma pigmentosum group A (XPA) gene-deficient mice easily developed skin cancers by the application of topical chemical carcinogens as well as by UV irradiation. As certain chemical carcinogens have been shown to be immunosuppressive, we examined the inflammatory and immunosuppressive effects of dimethylbenz(a)anthracene (DMBA) on XPA mice. Compared with wild-type mice, XPA mice showed greater ear swelling and reduction of epidermal Langerhans cells after DMBA application. Topical application of DMBA impaired the induction of contact hypersensitivity, initiated either locally or at distant sites. These DMBA-induced local and systemic immunosuppressions were more greatly enhanced in XPA mice than in wild-type mice. DMBA application induced pronounced production of PGE(2), IL-10, and TNF-alpha in the skin of XPA mice. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited DMBA-induced inflammation and local immunosuppression. In XPA mice, increased serum IL-10 was detected after DMBA treatment. Excess production of PGE(2), TNF-alpha, and IL-10 after DMBA application may be involved in the enhanced local and systemic immunosuppression in DMBA-treated XPA mice. Susceptibility to DMBA-induced skin tumors in XPA mice may be due to easy impairment of the immune system by DMBA in addition to a defect in the repair of DMBA-DNA adduct. Enhanced immunosuppression by chemical carcinogens as well as the mutagenicity of these mutagens might be associated with the high incidence of internal malignancies seen in XP patients. Moreover, these results supported the hypothesis that persistent DNA damage is a trigger for the production of immunoregulatory cytokines.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adjuvants, Immunologic; Administration, Cutaneous; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apyrase; Carcinogens; Dermatitis, Contact; Dinitrofluorobenzene; Dinoprostone; Disease Models, Animal; DNA-Binding Proteins; Ear; Edema; Female; Immunosuppressive Agents; Indomethacin; Inflammation; Interleukin-10; Langerhans Cells; Mice; Mice, Hairless; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Skin; Tumor Necrosis Factor-alpha; Up-Regulation; Xeroderma Pigmentosum; Xeroderma Pigmentosum Group A Protein

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Antibodies; Aorta; Apyrase; Cells, Cultured; Endothelium, Vascular; Humans; Hydrogen Peroxide; Inflammation; Kinetics; Molecular Sequence Data; Oxidative Stress; Peptide Fragments; Platelet Aggregation; Reperfusion Injury; Swine; Thionucleotides; Tumor Necrosis Factor-alpha