Tobias Woehrle

Autocrine regulation of T-cell activation by ATP release and P2X7 receptors

T‐cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X7 receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real‐time PCR and immu‐nohistochemistry, we find that Jurkat T cells and human CD4+ T cells express abundant P2X7 receptors. We show, using a novel fluorescent microscopy technique, that T‐cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 μM). This release of ATP is required for TCR‐mediated calcium influx, NFAT activation, and interleukin‐2 (IL‐2) production. TCR activation up‐regulates P2X7 receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi‐anion channel blocker gadolinium chloride, or siRNA silencing of P2X7 receptors blocks calcium entry and inhibits T‐cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X7 receptors, compared to control BALB/c mice, which express fully functional P2X7receptors. We conclude that ATP release and autocrine, positive feedback through P2X7 receptors is required for the effective activation of T cells.—Yip, L., Woe–hrle, T.,Corriden, R., Hirsh, M., Chen, Y., Inoue, Y., Ferrari, V., Insel, P.A., Junger, W.G. Autocrine regulation of T‐cell activation by ATP release and P2X7 receptors. FASEBJ. 23, 1685–1693 (2009)

Purinergic Signaling: A Fundamental Mechanism in Neutrophil Activation

Neutrophil activation by infectious and inflammatory signals requires ATP release and its feedback through purinergic receptors. Feedback for Function Neutrophils migrate to sites of infection, where they kill pathogens by processes such as phagocytosis and the release of reactive oxygen species. However, activated neutrophils can also result in tissue damage and inflammatory diseases in the host; thus, a better understanding of the mechanisms that regulate neutrophil activation could help in the development of therapies that could curb their destructive side effects. Chen et al. found that neutrophils responded to a range of infectious and inflammatory signals by releasing adenosine triphosphate (ATP). In addition to its role as a cellular energy source, ATP and its metabolites function as intercellular signaling molecules by stimulating purinergic receptors. The authors found that stimulation of formyl peptide receptors (FPRs) on neutrophils triggered the release, through pannexin-1 hemichannels, of ATP that signaled in an autocrine fashion through P2Y2 receptors. Moreover, this autocrine signal was required for neutrophil activation. In addition, mice deficient in P2Y2 receptors were less capable of clearing bacteria than were their wild-type counterparts. Together, these data suggest that feedback signaling by ATP released by neutrophils contributes to their activation. Efficient activation of neutrophils is a key requirement for effective immune responses. We found that neutrophils released cellular adenosine triphosphate (ATP) in response to exogenous stimuli such as formylated bacterial peptides and inflammatory mediators that activated Fcγ, interleukin-8, C5a complement, and leukotriene B4 receptors. Stimulation of the formyl peptide receptor (FPR) led to ATP release through pannexin-1 (panx1) hemichannels, and FPRs colocalized with P2Y2 nucleotide receptors on the cell surface to form a purinergic signaling system that facilitated neutrophil activation. Disruption of this purinergic signaling system by inhibiting or silencing panx1 hemichannels or P2Y2 receptors blocked neutrophil activation and impaired innate host responses to bacterial infection. Thus, purinergic signaling is a fundamental mechanism required for neutrophil activation and immune defense.

Hypertonic stress regulates T cell function via pannexin-1 hemichannels and P2X receptors

Hypertonic saline (HS) resuscitation increases T cell function and inhibits posttraumatic T cell anergy, which can reduce immunosuppression and sepsis in trauma patients. We have previously shown that HS induces the release of cellular ATP and enhances T cell function. However, the mechanism by which HS induces ATP release and the subsequent regulation of T cell function by ATP remain poorly understood. In the present study, we show that inhibition of the gap junction hemichannel pannexin‐1 (Panx1) blocks ATP release in response to HS, and HS exposure triggers significant changes in the expression of all P2X‐type ATP receptors in Jurkat T cells. Blocking or silencing of Panx1 or of P2X1, P2X4, or P2X7 receptors blunts HS‐induced p38 MAPK activation and the stimulatory effects of HS on TCR/CD28‐induced IL‐2 gene transcription. Moreover, treatment with HS or agonists of P2X receptors overcomes T cell suppression induced by the anti‐inflammatory cytokine IL‐10. These findings indicate that Panx1 hemichannels facilitate ATP release in response to hypertonic stress and that P2X1, P2X4, and P2X7 receptor activation enhances T cell function. We conclude that HS and P2 receptor agonists promote T cell function and thus, could be used to improve T cell function in trauma patients.

Adrenergic receptor activation involves ATP release and feedback through purinergic receptors

Formyl peptide receptor-induced chemotaxis of neutrophils depends on the release of ATP and autocrine feedback through purinergic receptors. Here, we show that adrenergic receptor signaling requires similar purinergic feedback mechanisms. Real-time RT-PCR analysis revealed that human embryonic kidney (HEK)-293 cells express several subtypes of adrenergic (α(1)-, α(2)-, and β-receptors), adenosine (P1), and nucleotide receptors (P2). Stimulation of G(q)-coupled α(1)-receptors caused release of cellular ATP and MAPK activation, which was blocked by inhibiting P2 receptors with suramin. Stimulation of G(i)-coupled α(2)-receptors induced weak ATP release, while G(s)-coupled β-receptors caused accumulation of extracellular ADP and adenosine. β-Receptors triggered intracellular cAMP signaling, which was blocked by scavenging extracellular adenosine with adenosine deaminase or by inhibiting A2a adenosine receptors with SCH58261. These findings suggest that adrenergic receptors require purinergic receptors to elicit downstream signaling responses in HEK-293 cells. We evaluated the physiological relevance of these findings using mouse aorta tissue rings. Stimulation of α(1)-receptors induced ATP release and tissue contraction, which was reduced by removing extracellular ATP with apyrase or in the absence of P2Y(2) receptors in aorta rings from P2Y(2) receptor knockout mice. We conclude that, like formyl peptide receptors, adrenergic receptors require purinergic feedback mechanisms to control complex physiological processes such as smooth muscle contraction and regulation of vascular tone.

Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

BackgroundDespite substantial progress, pathogenesis and therapy of chronic pain are still the focus of many investigations. The ATP-gated P2X7 receptor (P2X7R) has previously been shown to play a central role in animal models of nociceptive inflammatory and neuropathic pain. Recently, we found that the adaptive immune system is involved in the pathophysiology of chronic nociceptive and neuropathic pain in humans. So far, data regarding P2X7R expression patterns on cells of the adaptive immune system of pain patients are scarce. We therefore analyzed the P2X7R expression on peripheral blood lymphocytes and monocytes, as well as serum levels of IL-1β in patients suffering from chronic nociceptive and neuropathic pain in comparison to healthy volunteers in order to identify individuals who might benefit from a P2X7R modulating therapy.MethodsP2X7R messenger RNA (mRNA) and protein expression were determined in patients with either chronic nociceptive low back pain (CLBP) or neuropathic pain (NeP), and in healthy volunteers by quantitative real-time PCR (qPCR) and by fluorescence-assisted cell-sorting (FACS), respectively. IL-1β serum levels were measured with a multiplex cytokine assay.ResultsCompared to healthy volunteers, P2X7R mRNA (1.6-fold, p = 0.038) and protein levels were significantly increased on monocytes (NeP: 24.6 ± 6.2, healthy volunteers: 17.0 ± 5.4; p = 0.002) and lymphocytes (NeP: 21.8 ± 6.5, healthy volunteers: 15.6 ± 5.2; p = 0.009) of patients with NeP, but not in patients with CLBP. Similarly, IL-1β serum concentrations were significantly elevated only in NeP patients (1.4-fold, p = 0.04).ConclusionsA significant upregulation of P2X7R and increased IL-1β release seems to be a particular phenomenon in patients with NeP. P2X7R inhibitors may therefore represent a potential option for the treatment of this frequently intractable type of pain.German Clinical Trial Register (DRKS): Registration Trial DRKS00005954.

Plasma ATP is required for neutrophil activation in a mouse sepsis model.

ABSTRACT Our previous work has shown that polymorphonuclear neutrophils (PMNs) require cellular adenosine triphosphate (ATP) release and autocrine purinergic signaling for their activation. Here we studied in a mouse model of cecal ligation and puncture (CLP) whether sepsis affects this purinergic signaling process and thereby alters PMN responses after sepsis. Using high-performance liquid chromatography, we found that plasma ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) concentrations increased up to 6-fold during the first 8 h after CLP, reaching top levels that were significantly higher than those in sham control animals without CLP. Although leukocyte and PMN counts in sham animals increased significantly after 4 h, these blood cell counts decreased in sepsis animals. CD11b expression on the cell surface of PMNs of septic animals was significantly higher compared with sham and untreated control animals. These findings suggest increased PMN activation and sequestration of PMN from the circulation after sepsis. Plasma ATP levels correlated with CD11b expression, suggesting that increased ATP concentrations in plasma contribute to PMN activation. We found that treatment of septic mice with the ATP receptor antagonist suramin diminished CD11b expression, indicating that plasma ATP contributes to PMN activation by stimulating P2 receptors of PMNs. Increased PMN activation can protect the host from invading microorganisms. However, increased PMN activation can also be detrimental by promoting secondary organ damage. We conclude that pharmacological targeting of P2 receptors may allow modulation of PMN responses in sepsis.

ATP release and autocrine signaling through P2X4 receptors regulate γδ T cell activation

Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional αβ T cells release ATP upon TCR cross‐linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the “unconventional” γδ T lymphocytes. We observed that γδ T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti‐CD3/CD28‐coated beads or IPP. Pretreatment of γδ T cells with 10panx‐1, CBX, or Bf A reversed the stimulation‐induced increase in extracellular ATP concentration, indicating that panx‐1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with 10panx‐1 inhibited Ca2+ signaling in response to TCR stimulation. qPCR revealed that γδ T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP‐ATP inhibited transcriptional up‐regulation of TNF‐α and IFN‐γ in γδ T cells stimulated with anti‐CD3/CD28‐coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human γδ T cells.

A3 adenosine receptor inhibition improves the efficacy of hypertonic saline resuscitation

We reported previously that hypertonic saline (HS) treatment can prevent or upregulate the function of polymorphonuclear neutrophils (PMNs) via A2a-type adenosine receptors or A3-type adenosine receptors (A3R), respectively. A3R translocate to the cell surface upon PMN stimulation, and thus, HS promotes PMN responses under conditions of delayed HS treatment. Here we investigated if inhibition of A3R improves the protective effects of HS resuscitation in a mouse sepsis model. We found that HS nearly triples extracellular adenosine concentrations in whole blood and that inhibition of A3R with the selective antagonist MRS-1191 dose dependently improves the inhibitory effect of HS. MRS-1191 at a concentration of 1 nM enhanced the inhibitory effect of HS and reduced stimulatory effects of delayed HS treatment. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that MRS-1191 reduces acute lung injury and PMN accumulation in lung tissue. Whereas delayed HS treatment (4 mL/kg of 7.5% NaCl) of mice 1 h after CLP aggravated PMN accumulation, lung tissue damage, and mortality 24 h after CLP, infusion of MRS-1191 (2 ng/kg body weight) combined with HS reduced these detrimental effects of delayed HS treatment. Our data thus show that A3 receptor antagonists can strengthen the beneficial effects of HS resuscitation by avoiding stimulatory adverse effects that result from delayed HS administration.

Mitochondrial Dysfunction, Depleted Purinergic Signaling, and Defective T Cell Vigilance and Immune Defense

T cell suppression in sepsis is a well-known phenomenon; however, the underlying mechanisms are not fully understood. Previous studies have shown that T cell stimulation up-regulates mitochondrial adenosine triphosphate (ATP) production to fuel purinergic signaling mechanisms necessary for adequate T cell responses. Here we show that basal mitochondrial ATP production, ATP release, and stimulation of P2X1 receptors represent a standby purinergic signaling mechanism that is necessary for antigen recognition. Inhibition of this process impairs T cell vigilance and the ability of T cells to trigger T cell activation, up-regulate mitochondrial ATP production, and stimulate P2X4 and P2X7 receptors that elicit interleukin 2 production and T cell proliferation. T cells of patients with sepsis lack this standby purinergic signaling system owing to defects in mitochondrial function, ATP release, and calcium signaling. These defects impair antigen recognition and T cell function and are correlated with sepsis severity. Pharmacological targeting of these defects may improve T cell function and reduce the risk of sepsis.

Inhibition of Neutrophils by Hypertonic Saline Involves Pannexin-1, CD39, CD73, and Other Ectonucleotidases.

ABSTRACT Hypertonic saline (HS) resuscitation has been studied as a possible strategy to reduce polymorphonuclear neutrophil (PMN) activation and tissue damage in trauma patients. Hypertonic saline blocks PMNs by adenosine triphosphate (ATP) release and stimulation of A2a adenosine receptors. Here, we studied the underlying mechanisms in search of possible reasons for the inconsistent results of recent clinical trials with HS resuscitation. Purified human PMNs or PMNs in whole blood were treated with HS to simulate hypertonicity levels found after HS resuscitation (40 mmol/L beyond isotonic levels). Adenosine triphosphate release was measured with a luciferase assay. Polymorphonuclear neutrophil activation was assessed by measuring oxidative burst. The pannexin-1 (panx1) inhibitor 10panx1 and the gap junction inhibitor carbenoxolone (CBX) blocked ATP release from PMNs in purified and whole blood preparations, indicating that HS releases ATP via panx1 and gap junction channels. Hypertonic saline blocked N-formyl-Met-Leu-Phe–induced PMN activation by 40% in purified PMN preparations and by 60% in whole blood. These inhibitory effects were abolished by 10panx1 but only partially reduced by CBX, which indicates that panx1 has a central role in the immunomodulatory effects of HS. Inhibition of the ectonucleotidases CD39 and CD73 abolished the suppressive effect of HS on purified PMN cultures but only partially reduced the effect of HS in whole blood. These findings suggest redundant mechanisms in whole blood that may strengthen the immunomodulatory effect of HS in vivo. We conclude that HS resuscitation exerts anti-inflammatory effects that involve panx1, CD39, CD73, and other ectonucleotidases, which produce the adenosine that blocks PMNs by stimulating their A2a receptors. Our findings shed new light on the immunomodulatory mechanisms of HS and suggest possible new strategies to improve the clinical efficacy of hypertonic resuscitation.