Yuka Sumi

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.

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.

Early activation of γδ T lymphocytes in patients with severe systemic inflammatory response syndrome

Innate immunity plays an important role in host defense after severe insult. γδ T lymphocytes are recognized as the first line of defense against microbial invasion. In this study, we evaluated γδ T lymphocytes in the peripheral blood of patients with severe systemic inflammatory response syndrome (SIRS), and examined on role of these cells. Thirty-seven patients with severe SIRS (SIRS criteria and serum C-reactive protein ≥ 10 mg/dL) and 27 healthy volunteers were studied. Severe SIRS was caused by trauma in 14 patients (Injury Severity Score of 30.1 ± 10.8) and by sepsis in 23 patients. The counts of γδ and αβ T lymphocytes were determined by flow cytometry of cells stained with monoclonal antibodies to γδ and αβ T lymphocyte receptors. The activation of these cells was evaluated by flow cytometry of cells stained with monoclonal antibodies to CD69 and HLA-DR. Serial counts and activation of γδ and αβ T lymphocytes were also determined in eight trauma patients (Injury Severity Score of 31.0 ± 13.5) during a 2-week observation period. The count of γδ T lymphocytes in the peripheral blood of SIRS patients (30.1 ± 6.0/μL) was significantly lower (P < 0.05) than that of the healthy volunteers (104.3 ± 10.9/μL). The expression of CD69, an index of early activation of T lymphocytes, was significantly greater on γδ T lymphocytes from SIRS patients (patients 23.9% ± 3.4%, healthy controls 4.8% ± 0.6%, P < 0.05). In trauma patients, the expression of CD69 on γδ T lymphocytes increased rapidly within 48 h after injuries. In conclusion, γδ T lymphocytes are activated and decreased in the peripheral blood of severe SIRS patients. In trauma patients, the activation of γδ T lymphocytes occurs in the fairly acute phase after injuries. These results suggest a significant role for γδ T lymphocytes as early responders after severe insult.

The analysis of efficacy for AutoPulse™ system in flying helicopter.

AIM OF THE STUDY The helicopter emergency medical service (HEMS) was introduced in Japan in 2001, and some cardiopulmonary arrest (CPA) patients are transported using this service. However, it is difficult to maintain continuous and effective manual cardiopulmonary resuscitation (CPR) in flying helicopters. To overcome this problem, the AutoPulse™ system, automated mechanical CPR devices, was induced. We conducted a retrospective study to clarify the efficacy of AutoPulse™ on CPA patients in flying helicopters. METHODS In total, 92 CPA patients were enrolled in this study. Of these, 43 CPA patients received manual CPR (between April 2004 and June 2008), and 49 patients received AutoPulse™ CPR (between July 2008 and March 2011). We compared the manual CPR group with the AutoPulse™ group using logistic regression analysis and examined the efficacy of AutoPulse™ in flying helicopters. RESULTS Rates for return of spontaneous circulation (ROSC) and survival to hospital discharge were increased in the AutoPulse™ group compared to the manual CPR group (ROSC, 30.6% [15 patients] vs. 7.0% [3 patients]; survival to hospital discharge, 6.1% [3 patients] vs. 2.3% [1 patient]). In multivariate analysis, the factors associated with ROSC were the use of AutoPulse™ (odds ratio [OR], 7.22; P=0.005) and patients aged ≤65 years (OR, 0.31; P=0.042). CONCLUSION The present study demonstrates that the use of AutoPulse™ in flying helicopters was significantly effective for the ROSC in CPA patients. The use of automated chest compression devices such as AutoPulse™ might be recommended at least for CPA patients transported by helicopters.

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.

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.

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.

Hepatocyte growth factor in polymorphonuclear leukocytes is increased in patients with systemic inflammatory response syndrome

BACKGROUND Hepatocyte growth factor (HGF) has a significant effect on the regeneration of epithelial and endothelial cells. Studies have also shown an important role of HGF in wound healing and organ regeneration. Because recent studies indicate that polymorphonuclear leukocytes (PMNLs) store HGF in their specific granules and that HGF can be degranulated in the inflammatory tissue in which activated PMNLs migrate, we evaluated the storage and release of HGF in PMNLs from patients with systemic inflammatory response syndrome (SIRS) and attempted to examine the role of HGF from PMNLs in the systemic inflammatory process. METHODS Twenty-four patients with SIRS (serum C-reactive protein, 20.2 +/- 12.4 mg/dL [mean +/- SD]) and 18 healthy volunteers were studied. HGF in PMNLs was measured by flow cytometry by using a monoclonal antibody to HGF. The oxidative activity in PMNLs was also measured by flow cytometry. Serum HGF, interleukin (IL)-6, and IL-8 levels in each patient were measured by enzyme-linked immunosorbent assay. HGF degranulation from PMNLs was evaluated in 10 patients. RESULTS Immunocytochemistry under fluorescence microscopy revealed enhanced expression of HGF in the granules of PMNLs. HGF in PMNLs significantly increased in patients with SIRS compared with PMNLs from healthy volunteers (SIRS, 171.0 +/- 6.6 fluorescence/cell; control, 130.7 +/- 3.8 fluorescence/cell). N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharide stimulation induced further increase of HGF fluorescence in PMNLs from patients. HGF degranulation from PMNLs was also significantly enhanced in patients. Moreover, oxidative activity in PMNLs was significantly enhanced in patients with SIRS. Plasma HGF (pHGF) correlated positively with IL-6 and IL-8 levels in patients (pHGF and IL-6, gamma = 0.635, p < 0.05; pHGF and IL-8, gamma = 0.827, p < 0.01), but these values did not correlate with HGF in PMNLs. CONCLUSION Activated PMNLs in SIRS patients increased HGF in their granules and demonstrate enhanced degranulation of HGF. The release of HGF from migrated PMNLs in the inflammatory tissue may play an important role in wound healing and organ regeneration under those conditions.

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.