Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction.

BACKGROUND: The function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals. RESULTS: Bacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging. CONCLUSIONS: Our findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer.

Bladder cancer is amongst the most common causes of cancer death worldwide. Muscle-invasive bladder cancer (MIBC) bears a particularly poor prognosis. Overexpression of purinergic P2X receptors (P2XRs) has been associated with worse outcome in several malignant tumors. Here, we investigated the role of P2XRs in bladder cancer cell proliferation in vitro and the prognostic value of P2XR expression in MIBC patients. Cell culture experiments with T24, RT4, and non-transformed TRT-HU-1 cells revealed a link between high ATP concentrations in the cell culture supernatants of bladder cell lines and a higher grade of malignancy. Furthermore, proliferation of highly malignant T24 bladder cancer cells depended on autocrine signaling through P2X receptors. P2X1R, P2X4R, and P2X7R expression was immunohistochemically analyzed in tumor specimens from 173 patients with MIBC. High P2X1R expression was associated with pathological parameters of disease progression and reduced survival time. High combined expression of P2X1R and P2X7R increased the risk of distant metastasis and was an independent negative predictor of overall and tumor-specific survival in multivariate analyses. Our results suggest that P2X1R/P2X7R expression scores are powerful negative prognostic markers in MIBC patients and that P2XR-mediated pathways are potential targets for novel therapeutic strategies in bladder cancer.

ATP breakdown in plasma of children limits the antimicrobial effectiveness of their neutrophils.

Neutrophils (PMNs) require extracellular ATP and adenosine (ADO) to fight bacterial infections, which often have life-threatening consequences in pediatric patients. We wondered whether the ATP and ADO levels in the plasma of children change with age and if these changes influence the antimicrobial efficacy of the PMNs of these children. We measured plasma concentrations of ATP and ADO and the activities of the enzymes responsible for the breakdown of these mediators in plasma samples from healthy children and adolescents (n = 45) ranging in age from 0.2 to 15 years. In addition, using blood samples of these individuals, we compared how effective their PMNs were in the phagocytosis of bacteria. In an experimental sepsis model with young (10 days) and adolescent mice (10 weeks), we studied how age influenced the resilience of these animals to bacterial infections and whether addition of ATP could improve the antimicrobial capacity of their PMNs. We found that plasma ATP levels correlated with age and were significantly lower in infants (< 1 year) than in adolescents (12-15 years). In addition, we observed significantly higher plasma ATPase and adenosine deaminase activities in children (< 12 years) when compared to the adolescent population. The activities of these ATP and ADO breakdown processes correlated inversely with age and with the ability of PMNs to phagocytize bacteria. Similar to their human counterparts, young mice also had significantly lower plasma ATP levels when compared to adolescent animals. In addition, we found that mortality of young mice after bacterial infection was significantly higher than that of adolescent mice. Moreover, bacterial phagocytosis by PMNs of young mice was weaker when compared to that of older mice. Finally, we found that ATP supplementation could recover bacterial phagocytosis of young mice to levels similar to those of adolescent mice. Our findings suggest that rapid ATP hydrolysis in the plasma of young children lowers the antimicrobial functions of their PMNs and that this may contribute to the vulnerability of pediatric patients to bacterial infections.

Optimized HPLC method to elucidate the complex purinergic signaling dynamics that regulate ATP, ADP, AMP, and adenosine levels in human blood.

ATP released into the bloodstream regulates immune responses and other physiological functions. Excessive accumulation of extracellular ATP interferes with these functions, and elevated plasma ATP levels could indicate infections and other pathological disorders. However, there is considerable disagreement about what constitutes normal plasma ATP levels. Therefore, we optimized a method to accurately assess ATP concentrations in blood. We found that rapid chilling of heparinized blood samples is essential to preserve in vivo ATP levels and that differential centrifugation minimizes inadvertent ATP release due to cell damage and mechanical stress. Plasma samples were stabilized with perchloric acid, etheno-derivatized, and delipidated for sensitive analysis of ATP and related compounds using high-performance liquid chromatography (HPLC) and fluorescence detection. We measured 33 ± 20 nM ATP, 90 ± 45 nM ADP, 100 ± 55 nM AMP, and 81 ± 51 nM adenosine in the blood of healthy human adults (n = 10). In critically ill patients, ATP levels were 6 times higher than in healthy subjects. The anticoagulant greatly affected results. ATP levels were nearly 8 times higher in EDTA plasma than in heparin plasma, while AMP levels were 3 times lower and adenosine was entirely absent in EDTA plasma. If EDTA blood was not immediately chilled, ATP, ADP, and AMP levels continued to rise, which indicates that EDTA interferes with the endogenous mechanisms that regulate plasma adenylate levels. Our optimized method eliminates artifacts that prevent accurate determination of plasma adenylates and will be useful for studying mechanisms that regulate adenylate levels and for monitoring of pathological processes in patients with infections and other diseases.

Frontline Science: P2Y11 receptors support T cell activation by directing mitochondrial trafficking to the immune synapse.

T cells form an immune synapse (IS) with antigen-presenting cells (APCs) to detect antigens that match their TCR. Mitochondria, pannexin-1 (panx1) channels, and P2X4 receptors congregate at the IS where mitochondria produce the ATP that panx1 channels release in order to stimulate P2X4 receptors. P2X4 receptor stimulation causes cellular Ca2+ influx that up-regulates mitochondrial metabolism and localized ATP production at the IS. Here we show that P2Y11 receptors are essential players that sustain these T cell activation mechanisms. We found that P2Y11 receptors retract from the IS toward the back of cells where their stimulation by extracellular ATP induces cAMP/PKA signaling that redirects mitochondrial trafficking to the IS. P2Y11 receptors thus reinforce IS signaling by promoting the aggregation of mitochondria with panx1 ATP release channels and P2X4 receptors at the IS. This dual purinergic signaling mechanism involving P2X4 and P2Y11 receptors focuses mitochondrial metabolism to the IS where localized ATP production sustains synaptic activity in order to allow successful completion of T cell activation responses. Our findings have practical implications because rodents lack P2Y11 receptors, raising concerns as to the validity of rodent models to study treatment of infections and inflammatory conditions.

Mitochondria Synergize With P2 Receptors to Regulate Human T Cell Function.

Intracellular ATP is the universal energy carrier that fuels many cellular processes. However, immune cells can also release a portion of their ATP into the extracellular space. There, ATP activates purinergic receptors that mediate autocrine and paracrine signaling events needed for the initiation, modulation, and termination of cell functions. Mitochondria contribute to these processes by producing ATP that is released. Here, we summarize the synergistic interplay between mitochondria and purinergic signaling that regulates T cell functions. Specifically, we discuss how mitochondria interact with P2X1, P2X4, and P2Y11 receptors to regulate T cell metabolism, cell migration, and antigen recognition. These mitochondrial and purinergic signaling mechanisms are indispensable for host immune defense. However, they also represent an Achilles heel that can render the host susceptible to infections and inflammatory disorders. Hypoxia and mitochondrial dysfunction deflate the purinergic signaling mechanisms that regulate T cells, while inflammation and tissue damage generate excessive systemic ATP levels that distort autocrine purinergic signaling and impair T cell function. An improved understanding of the metabolic and purinergic signaling mechanisms that regulate T cells may lead to novel strategies for the diagnosis and treatment of infectious and inflammatory diseases.

Negative feedback control of neuronal activity by microglia.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

The purinergic receptor P2Y11 choreographs the polarization, mitochondrial metabolism, and migration of T lymphocytes.

T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP-such as in response to inflammation, sepsis, and cancer-disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense.

Author Correction: NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RIG-I and TLR4 responses and adverse outcomes in pediatric influenza-related critical illness.

BACKGROUND: Decreased TNF-α production in whole blood after ex vivo LPS stimulation indicates suppression of the Toll-like receptor (TLR)4 pathway. This is associated with increased mortality in pediatric influenza critical illness. Whether antiviral immune signaling pathways are also suppressed in these patients is unclear. OBJECTIVES: We sought to evaluate suppression of the TLR4 and the antiviral retinoic acid-inducible gene-I (RIG-I) pathways with clinical outcomes in children with severe influenza infection. METHODS: In this 24-center, prospective, observational cohort study of children with confirmed influenza infection, blood was collected within 72 hours of intensive care unit admission. Ex vivo whole blood stimulations were performed with matched controls using the viral ligand polyinosinic-polycytidylic acid-low-molecular-weight/LyoVec and LPS to evaluate IFN-α and TNF-α production capacities (RIG-I and TLR4 pathways, respectively). RESULTS: Suppression of either IFN-α or TNF-α production capacity was associated with longer duration of mechanical ventilation and hospitalization, and increased organ dysfunction. Children with suppression of both RIG-I and TLR4 pathways (n = 33 of 103 [32%]) were more likely to have prolonged (≥7 days) multiple-organ dysfunction syndrome (30.3% vs 8.6%; P = .004) or prolonged hypoxemic respiratory failure (39.4% vs 11.4%; P = .001) compared with those with single- or no pathway suppression. CONCLUSIONS: Suppression of both RIG-I and TLR4 signaling pathways, essential for respective antiviral and antibacterial responses, is common in previously immunocompetent children with influenza-related critical illness and is associated with bacterial coinfection and adverse outcomes. Prospective testing of both pathways may aid in risk-stratification and in immune monitoring.

Airway brush cells generate cysteinyl leukotrienes through the ATP sensor P2Y2.

Chemosensory epithelial cells (EpCs) are specialized cells that promote innate type 2 immunity and protective neurally mediated reflexes in the airway. Their effector programs and modes of activation are not fully understood. Here, we define the transcriptional signature of two choline acetyltransferase-expressing nasal EpC populations. They are found in the respiratory and olfactory mucosa and express key chemosensory cell genes including the transcription factor Pou2f3, the cation channel Trpm5, and the cytokine Il25 Moreover, these cells share a core transcriptional signature with chemosensory cells from intestine, trachea and thymus, and cluster with tracheal brush cells (BrCs) independently from other respiratory EpCs, indicating that they are part of the brush/tuft cell family. Both nasal BrC subsets express high levels of transcripts encoding cysteinyl leukotriene (CysLT) biosynthetic enzymes. In response to ionophore, unfractionated nasal BrCs generate CysLTs at levels exceeding that of the adjacent hematopoietic cells isolated from naïve mucosa. Among activating receptors, BrCs express the purinergic receptor P2Y2. Accordingly, the epithelial stress signal ATP and aeroallergens that elicit ATP release trigger BrC CysLT generation, which is mediated by the P2Y2 receptor. ATP- and aeroallergen-elicited CysLT generation in the nasal lavage is reduced in mice lacking Pou2f3, a requisite transcription factor for BrC development. Last, aeroallergen-induced airway eosinophilia is reduced in BrC-deficient mice. These results identify a previously undescribed BrC sensor and effector pathway leading to generation of lipid mediators in response to luminal signals. Further, they suggest that BrC sensing of local damage may provide an important sentinel immune function.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Adenosine 5'-Monophosphate Protects from Hypoxia by Lowering Mitochondrial Metabolism and Oxygen Demand.

Ischemia and reperfusion injury following severe trauma or cardiac arrest are major causes of organ damage in intensive care patients. The brain is particularly vulnerable because hypoxia rapidly damages neurons due to their heavy reliance on oxidative phosphorylation. Therapeutic hypothermia can reduce ischemia-induced brain damage, but cooling procedures are slow and technically difficult to perform in critical care settings. It has been previously reported that injection of naturally occurring adenosine 5'-monophosphate (AMP) can rapidly induce hypothermia in mice. We studied the underlying mechanisms and found that AMP transiently reduces the heart rate, respiratory rate, body temperature, and the consciousness of adult male and female C57BL/6J mice. Adding AMP to mouse or human neuronal cell cultures dose-dependently reduced the membrane potential (ΔΨm) and Ca signaling of mitochondria in these cells. AMP treatment increased intracellular AMP levels and activated AMP-activated protein kinase, which resulted in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and of mitochondrial and cytosolic Ca signaling in resting and stimulated neurons. Pretreatment with an intraperitoneal injection of AMP almost doubled the survival time of mice under hypoxic (6% O2) or anoxic (<1% O2) conditions when compared to untreated mice. These findings suggest that AMP induces a hypometabolic state that slows mitochondrial respiration, reduces oxygen demand, and delays the processes that damage mitochondria in the brain and other organs following hypoxia and reperfusion. Further examination of these mechanisms may lead to new treatments that preserve organ function in critical care patients.

Frontline Science: Escherichia coli use LPS as decoy to impair neutrophil chemotaxis and defeat antimicrobial host defense.

Bacterial infections and sepsis are leading causes of morbidity and mortality in critically ill patients. Currently, there are no effective treatments available to improve clinical outcome in sepsis. Here, we elucidated a mechanism by which Escherichia coli (E. coli) bacteria impair neutrophil (PMN) chemotaxis and we studied whether this mechanism can be therapeutically targeted to improve chemotaxis and antimicrobial host defense. PMNs detect bacteria with formyl peptide receptors (FPR). FPR stimulation triggers mitochondrial ATP production and release. Autocrine stimulation of purinergic receptors exerts excitatory and inhibitory downstream signals that induce cell polarization and cell shape changes needed for chemotaxis. Here we show that the bacterial cell wall product LPS dose-dependently impairs PMN chemotaxis. Exposure of human PMNs to LPS triggered excessive mitochondrial ATP production and disorganized intracellular trafficking of mitochondria, resulting in global ATP release that disrupted purinergic signaling, cell polarization, and chemotaxis. In mice infected i.p. with E. coli, LPS treatment increased the spread of bacteria at the infection site and throughout the systemic circulation. Removal of excessive systemic ATP with apyrase improved chemotaxis of LPS-treated human PMNs in vitro and enhanced the clearance of E. coli in infected and LPS-treated mice. We conclude that systemic ATP accumulation in response to LPS is a potential therapeutic target to restore PMN chemotaxis and to boost the antimicrobial host immune defense in sepsis.

Lipopolysaccharide suppresses T cells by generating extracellular ATP that impairs their mitochondrial function via P2Y11 receptors.

T cell suppression contributes to immune dysfunction in sepsis. However, the underlying mechanisms are not well-defined. Here, we show that exposure of human peripheral blood mononuclear cells to bacterial lipopolysaccharide (LPS) can rapidly and dose-dependently suppress interleukin-2 (IL-2) production and T cell proliferation. We also report that these effects depend on monocytes. LPS did not prevent the interaction of monocytes with T cells, nor did it induce programmed cell death protein 1 (PD-1) signaling that causes T cell suppression. Instead, we found that LPS stimulation of monocytes led to the accumulation of extracellular ATP that impaired mitochondrial function, cell migration, IL-2 production, and T cell proliferation. Mechanistically, LPS-induced ATP accumulation exerted these suppressive effects on T cells by activating the purinergic receptor P2Y11 on the cell surface of T cells. T cell functions could be partially restored by enzymatic removal of extracellular ATP or pharmacological blocking of P2Y11 receptors. Plasma samples obtained from sepsis patients had similar suppressive effects on T cells from healthy subjects. Our findings suggest that LPS and ATP accumulation in the circulation of sepsis patients suppresses T cells by promoting inappropriate P2Y11 receptor stimulation that impairs T cell metabolism and functions. We conclude that inhibition of LPS-induced ATP release, removal of excessive extracellular ATP, or P2Y11 receptor antagonists may be potential therapeutic strategies to prevent T cell suppression and restore host immune function in sepsis.

Plasma Adenylate Levels are Elevated in Cardiopulmonary Arrest Patients and May Predict Mortality.

Cerebral and cardiac dysfunction cause morbidity and mortality in postcardiac arrest syndrome (PCAS) patients. Predicting clinical outcome is necessary to provide the optimal level of life support for these patients. In this pilot study, we examined whether plasma ATP and adenylate levels have value in predicting clinical outcome in PCAS patients. In total, 15 patients who experienced cardiac arrest outside the hospital setting and who could be reanimated were enrolled in this study. Healthy volunteers (n = 8) served as controls. Of the 15 PCAS patients, 8 died within 4 days after resuscitation. Of the 7 survivors, 2 lapsed into vegetative states, 1 survived with moderate disabilities, and 4 showed good recoveries. Arterial blood samples were drawn immediately after successful resuscitation and return of spontaneous circulation (ROSC). The concentrations of ATP and other adenylates in plasma were assessed with high-performance liquid chromatography. PCAS patients had significantly higher ATP levels than healthy controls. Plasma ATP levels correlated with lactate levels, Acute Physiology and Chronic Health Evaluation II scores, and the time it took to ROSC (time-to-ROSC). Plasma adenylate levels in patients who died after resuscitation were significantly higher than in survivors. Based on our results and receiver-operating characteristic curve analysis, we conclude that plasma adenylate levels may help predict outcome in PCAS patients.

Adenosine Triphosphate Release is Required for Toll-Like Receptor-Induced Monocyte/Macrophage Activation, Inflammasome Signaling, Interleukin-1ß Production, and the Host Immune Response to Infection.

OBJECTIVES: Monocytes and macrophages produce interleukin-1ß by inflammasome activation which involves adenosine triphosphate release, pannexin-1 channels, and P2X7 receptors. However, interleukin-1ß can also be produced in an inflammasome-independent fashion. Here we studied if this mechanism also involves adenosine triphosphate signaling and how it contributes to inflammasome activation. DESIGN: In vitro studies with human cells and randomized animal experiments. SETTING: Preclinical academic research laboratory. SUBJECTS: Wild-type C57BL/6 and pannexin-1 knockout mice, healthy human subjects for cell isolation. INTERVENTIONS: Human monocytes and U937 macrophages were treated with different inhibitors to study how purinergic signaling contributes to toll-like receptor-induced cell activation and interleukin-1ß production. Wild-type and pannexin-1 knockout mice were subjected to cecal ligation and puncture to study the role of purinergic signaling in interleukin-1ß production and host immune defense. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor agonists triggered mitochondrial adenosine triphosphate production and adenosine triphosphate release within seconds. Inhibition of mitochondria, adenosine triphosphate release, or P2 receptors blocked p38 mitogen-activated protein kinase and caspase-1 activation and interleukin-1ß secretion. Mice lacking pannexin-1 failed to activate monocytes, to produce interleukin-1ß, and to effectively clear bacteria following cecal ligation and puncture. CONCLUSIONS: Purinergic signaling has two separate roles in monocyte/macrophage activation, namely to facilitate the initial detection of danger signals via toll-like receptors and subsequently to regulate nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 inflammasome activation. Further dissection of these mechanisms may reveal novel therapeutic targets for immunomodulation in critical care patients.

Purinergic P2X4 receptors and mitochondrial ATP production regulate T cell migration.

T cells must migrate in order to encounter antigen-presenting cells (APCs) and to execute their varied functions in immune defense and inflammation. ATP release and autocrine signaling through purinergic receptors contribute to T cell activation at the immune synapse that T cells form with APCs. Here, we show that T cells also require ATP release and purinergic signaling for their migration to APCs. We found that the chemokine stromal-derived factor-1α (SDF-1α) triggered mitochondrial ATP production, rapid bursts of ATP release, and increased migration of primary human CD4+ T cells. This process depended on pannexin-1 ATP release channels and autocrine stimulation of P2X4 receptors. SDF-1α stimulation caused localized accumulation of mitochondria with P2X4 receptors near the front of cells, resulting in a feed-forward signaling mechanism that promotes cellular Ca2+ influx and sustains mitochondrial ATP synthesis at levels needed for pseudopod protrusion, T cell polarization, and cell migration. Inhibition of P2X4 receptors blocked the activation and migration of T cells in vitro. In a mouse lung transplant model, P2X4 receptor antagonist treatment prevented the recruitment of T cells into allograft tissue and the rejection of lung transplants. Our findings suggest that P2X4 receptors are therapeutic targets for immunomodulation in transplantation and inflammatory diseases.

Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.

OBJECTIVE: Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis. DESIGN: Prospective randomized animal investigation and in vitro studies. SETTING: Preclinical academic research laboratory. SUBJECTS: Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies. INTERVENTIONS: Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis. MEASUREMENTS AND MAIN RESULTS: Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis. CONCLUSIONS: Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses, making this a promising new treatment strategy for sepsis.

Shock wave-induced ATP release from osteosarcoma U2OS cells promotes cellular uptake and cytotoxicity of methotrexate.

BACKGROUND: Osteosarcoma is the most prevalent primary malignant bone tumor, but treatment is difficult and prognosis remains poor. Recently, large-dose chemotherapy has been shown to improve outcome but this approach can cause many side effects. Minimizing the dose of chemotherapeutic drugs and optimizing their curative effects is a current goal in the management of osteosarcoma patients. METHODS: In our study, trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2OS cells. Cellular uptake of the fluorophores Lucifer Yellow CH dilithium salt and Calcein was measured by qualitative and quantitative methods. Human MTX ELISA Kit and MTT assay were used to assess the outcome for osteosarcoma U2OS cells in the present of shock wave and methotrexate. To explore the mechanism, P2X7 receptor in U2OS cells was detected by immunofluorescence and the extracellular ATP levels was detected by ATP assay kit. All data were analyzed using SPSS17.0 statistical software. Comparisons were made with t test between two groups. RESULTS: Treatment of human osteosarcoma U2OS cells with up to 450 shock wave pulses at 7 kV or up to 200 shock wave pulses at 14 kV had little effect on cell viability. However, this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2OS cells. Importantly, shock wave treatment also significantly enhanced the uptake of the chemotherapy drug methotrexate and increased the rate of methotrexate-induced apoptosis. We found that shock wave treatment increased the extracellular concentration of ATP and that KN62, an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis. CONCLUSIONS: Our results suggest that shock wave treatment promotes methotrexate-induced apoptosis by altering cell membrane permeability in a P2X7 receptor-dependent manner. Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.