Wolfgang G. Junger

Circulating Mitochondrial DAMPs Cause Inflammatory Responses to Injury

Injury causes a systemic inflammatory response syndrome (SIRS) that is clinically much like sepsis. Microbial pathogen-associated molecular patterns (PAMPs) activate innate immunocytes through pattern recognition receptors. Similarly, cellular injury can release endogenous ‘damage’-associated molecular patterns (DAMPs) that activate innate immunity. Mitochondria are evolutionary endosymbionts that were derived from bacteria and so might bear bacterial molecular motifs. Here we show that injury releases mitochondrial DAMPs (MTDs) into the circulation with functionally important immune consequences. MTDs include formyl peptides and mitochondrial DNA. These activate human polymorphonuclear neutrophils (PMNs) through formyl peptide receptor-1 and Toll-like receptor (TLR) 9, respectively. MTDs promote PMN Ca2+ flux and phosphorylation of mitogen-activated protein (MAP) kinases, thus leading to PMN migration and degranulation in vitro and in vivo. Circulating MTDs can elicit neutrophil-mediated organ injury. Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial ‘enemies within’ by cellular injury is a key link between trauma, inflammation and SIRS.

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.

Immune cell regulation by autocrine purinergic signalling

Stimulation of almost all mammalian cell types leads to the release of cellular ATP and autocrine feedback through a diverse array of purinergic receptors. Depending on the types of purinergic receptors that are involved, autocrine signalling can promote or inhibit cell activation and fine-tune functional responses. Recent work has shown that autocrine signalling is an important checkpoint in immune cell activation and allows immune cells to adjust their functional responses based on the extracellular cues provided by their environment. This Review focuses on the roles of autocrine purinergic signalling in the regulation of both innate and adaptive immune responses and discusses the potential of targeting purinergic receptors for treating immune-mediated disease.

Hypertonic Saline Resuscitation Decreases Susceptibility to Sepsis after Hemorrhagic Shock

BACKGROUND We hypothesized that improvements in cellular immune function after hypertonic saline (HTS) resuscitation will alter the outcome of sepsis after hemorrhage. METHODS To test this hypothesis, a two-hit model was used. Hemorrhage was induced in BALB/c mice by catheterizing the femoral artery and bleeding until a mean arterial pressure = 35 mm Hg was reached and maintained for 1 hour. Resuscitation was performed with HTS (NaCl 7.5%, 4 mL/kg) or lactated Ringers (LR, twice the shed blood volume), plus the shed blood. Cecal ligation and puncture (CLP) was performed 24 hours after hemorrhage. Mortality was assessed for 72 hours, comparing HTS (n = 14) and LR (n = 13) resuscitation. Another set of animals (n = 10 in each group at each time point) were killed at 2 and 24 hours after blood collection. Liver and blood were cultured for the presence of bacteria, and lung and liver samples were scored on a scale from 0 (normal) to 4 (most severe) in a blind fashion by a pathologist. RESULTS Mortality 72 hours after CLP was 14.3% in HTS and 76.9% in LR treated animals (p < 0.002). At 24 hours after CLP, 44% of HTS, but 77% of LR treated animals had > 1,000 colony forming units/mL of blood. Positive liver cultures (> 100,000 colony forming units/g) also showed the same trend (HTS = 30%, LR = 60%). Autopsies revealed a better containment of the infection (abscess formation) in the HTS group. At 2 hours, lung scores were 1.2 +/- 0.25 and 2.6 +/- 0.31 for HTS and LR, respectively (p < 0.002). At 24 hours, HTS treated animals showed marked improvement of lung injury, while the scores in the LR group remained high. A significant difference was also observed regarding liver injury. At 2 hours, scores were 0.4 +/- 0.22 and 2.3 +/- 0.16 for HTS and LR, respectively (p < 0.002). At 24 hours, HTS treated animals showed normal hepatic architecture, although mild injury was still observed in the LR group. CONCLUSION HTS resuscitation leads to increased survival after hemorrhage and CLP. Marked improvements were observed in lung and liver injury compared with isotonic resuscitation. The better containment of the infection observed with HTS resuscitation corresponds to a marked decreased in bacteremia. HTS resuscitation stands as an alternative resuscitation regimen with immunomodulatory potential.

Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation after hemorrhagic shock.

Hypertonic saline (HS) resuscitation after hemorrhage and sepsis has been shown to markedly reduce the development of lung injury in animals, compared with traditional resuscitation with lactated Ringers (LR). These experiments examined the effect of HS on lung injury after hemorrhage without sepsis. The effects of HS and LR resuscitation on neutrophil trafficking, neutrophil adhesion, and neutrophil oxidative burst were studied. Methods: BALB/c mice were hemorrhaged to a mean arterial pressure of 40 torr for 1 h. Animals were resuscitated with shed blood and either 4 mL/kg of 7.5% HS or LR in twice the volume of the shed blood. Lung histology was examined 24 h after hemorrhage. Lung myeloperoxidase content and bronchoalveolar lavage fluid neutrophil counts were obtained. Peripheral blood smears were obtained to determine the neutrophil percentage. Peripheral blood neutrophil CD11b expression and neutrophil H2O2 production were assayed by flow cytometry. Results: HS animals had less lung injury than LR animals. The mean myeloperoxidase activity in HS versus LR animals was 1.79 ± 1.33 U/100 mg versus 3.0 ± 1.33 U/100 mg, respectively. The percentage of neutrophils in the bronchoalveolar lavage fluid of HS animals (3.8% ± .8) was significantly less than that of LR animals (10.8% ± 2.1). This corresponded to a significantly higher peripheral blood neutrophil count in HS animals compared with LR animals, 41% vs. 20%, respectively. There was no difference in neutrophil expression of the CD11b integrin between the HS and LR groups. The neutrophils of LR animals had basal H2O2 production that was 107% greater than that of controls; HS suppressed this hemorrhage-induced activation by > 60%. HS resuscitation after hemorrhagic shock protects against the development of lung injury. This protection is due, in part, to suppression of the hemorrhage-induced neutrophil oxidative burst. HS resuscitation offers immunomodulatory potential after hemorrhagic shock.

Hypertonic saline resuscitation: a tool to modulate immune function in trauma patients?

Hypertonic saline (HS) resuscitation has recently gained attention from trauma physicians because it may benefit the immune system of trauma patients. We have found that HS augments in vitro and in vivo immune function of healthy T-cells. In addition, HS restored the function of suppressed T-cells in vitro and in vivo and reduced immunosuppression after hemorrhage, protecting mice from subsequent sepsis. These effects of HS are based on its direct influence on cellular signaling events through specific signaling pathway(s) that include protein tyrosine kinase and mitogen-activated protein kinase p38 activation. HS provides a costimulatory signal that enhances the proliferation of activated T-cells. HS may be able to substitute signals lost through blockage as a result of trauma induced suppressive factors, thereby restoring the function of suppressed T-cells. Although further work is needed to determine the optimal conditions and possible risks of HS resuscitation, the data presented in this short review of our recent work shed a favorable light on HS as a simple but effective tool to modulate cellular immune function after trauma.

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)

Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.

Mice lacking P2Y2 receptors have salt-resistant hypertension and facilitated renal Na+ and water reabsorption

Extracellular nucleotides (e.g., ATP) regulate many physiological and pathophysiological pro‐cesses through activation of nucleotide (P2) receptors in the plasma membrane. Here we report that gene‐targeted (knockout) mice that lack P2Y2 receptors have salt‐resistant arterial hypertension in association with an inverse relationship between salt intake and heart rate, indicating intact baroreceptor function. Knockout mice have multiple alterations in their handling of salt and water: these include suppressed plasma renin and aldo‐sterone concentrations, lower renal expression of the aldosterone‐induced epithelial sodium channel α‐ENaC, greater medullary expression of the Na‐K‐2Cl‐cotrans‐porter NKCC2, and greater furosemide‐sensitive Na+ reabsorption in association with greater renal medullary expression of aquaporin‐2 and vasopressin‐depen‐dent renal cAMP formation and water reabsorption despite similar vasopressin levels compared with wild type. Of note, smaller increases in plasma aldosterone were required to adapt renal Na+ excretion to restricted intake in knockout mice, suggesting a facilitation in renal Na+ retention. The results thus identify a previously unrecognized role for P2Y2 receptors in blood pressure regulation that is linked to an inhibitory influence on renal Na+ and water reabsorption. Based on these findings in knockout mice, we propose that a blunting in P2Y2 receptor expression or activity is a new mechanism for salt‐resistant arterial hypertension.—Rieg, T., Bundey, R. A., Chen, Y., Deschenes, G., Junger, W., Insel, P. A., Vallon, V. Mice lacking P2Y2 receptors have salt‐resistant hypertension and facilitated renal Na+ and water reabsorption FASEB J. 21, 3717–3726 (2007)

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.