Emmanuel Lorne

Activation of AMPK attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury

AMP-activated protein kinase (AMPK) is activated by increases in the intracellular AMP-to-ATP ratio and plays a central role in cellular responses to metabolic stress. Although activation of AMPK has been shown to have anti-inflammatory effects, there is little information concerning the role that AMPK may play in modulating neutrophil function and neutrophil-dependent inflammatory events, such as acute lung injury. To examine these issues, we determined the effects of pharmacological activators of AMPK, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) and barberine, on Toll-like receptor 4 (TLR4)-induced neutrophil activation. AICAR and barberine dose-dependently activated AMPK in murine bone marrow neutrophils. Exposure of LPS-stimulated neutrophils to AICAR or barberine inhibited release of TNF-alpha and IL-6, as well as degradation of IkappaBalpha and nuclear translocation of NF-kappaB, compared with findings in neutrophil cultures that contained LPS without AICAR or barberine. Administration of AICAR to mice resulted in activation of AMPK in the lungs and was associated with decreased severity of LPS-induced lung injury, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-alpha and IL-6 in bronchoalveolar lavage fluid. These results suggest that AMPK activation reduces TLR4-induced neutrophil activation and diminishes the severity of neutrophil-driven proinflammatory processes, including acute lung injury.

The passive leg-raising maneuver cannot accurately predict fluid responsiveness in patients with intra-abdominal hypertension.

Objectives:The passive leg-raising maneuver is a reversible fluid-loading procedure used to predict fluid responsiveness in mechanically ventilated patients. The aim of the present study was to determine whether intra-abdominal hypertension (which impairs venous return) reduces the ability of passive leg raising to detect fluid responsiveness in critically ill ventilated patients. Design:A prospective study. Setting:The medical and surgical intensive care unit of a university medical center. Patients:Forty-one mechanically ventilated patients with a pulse pressure variation of >12%. Interventions:Stroke volume was continuously monitored by esophageal Doppler. Intra-abdominal pressure was measured via bladder pressure. After a passive leg-raising maneuver and a return to baseline, fluid loading with 500 mL of saline was performed. Hemodynamic parameters were recorded at each step. Nonresponders to volume loading were not analyzed (10 patients). Thirty-one patients were classified into two groups according to their response to passive leg raising: responders to passive leg raising (at least a 12% increase in stroke volume) and nonresponders to passive leg raising. Measurements and Main Results:Sixteen patients (52%) were responders to passive leg raising, and 15 (48%) were nonresponders to passive leg raising (i.e., false negatives). At baseline, the median intra-abdominal pressure was significantly higher in the nonresponders to passive leg raising than in the responders to passive leg raising (20 [6.5] vs. 11.5 [5.5], respectively; p < .0001). The area under the receiver-operating characteristic curve was 0.969 ± 0.033. An intra-abdominal pressure cutoff value of 16 mm Hg discriminated between responders to passive leg raising and nonresponders to passive leg raising with a sensitivity of 100% (confidence interval, 78–100) and a specificity of 87.5% (confidence interval, 61.6–98.1). An intra-abdominal pressure of ≥16 mm Hg was the only independent predictor of nonresponse to passive leg raising in a multivariate analysis (odds ratio, 2.6 [confidence interval, 1.1–6.6]; p = .04). Conclusions:An intra-abdominal pressure of ≥16 mm Hg seems to be responsible for false negatives to passive leg raising. Hence, the intra-abdominal pressure should be measured in critically ill ventilated patients, especially before performing passive leg raising.

High Mobility Group Protein-1 Inhibits Phagocytosis of Apoptotic Neutrophils through Binding to Phosphatidylserine

Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with an unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. High mobility group protein-1 (HMGB1), a nuclear nonhistone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and fluorescence resonance energy transfer demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage fluid from Scnn+ mice, a murine model of cystic fibrosis lung disease which contains elevated concentrations of HMGB1, inhibits neutrophil efferocytosis. Anti-HMGB1 Abs reverse the inhibitory effect of Scnn+ bronchoalveolar lavage on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses.

Postoperative Ketamine Administration Decreases Morphine Consumption in Major Abdominal Surgery : A Prospective, Randomized, Double-Blind, Controlled Study

BACKGROUND:Ketamine decreases postoperative morphine consumption, but its optimal dosing and duration of administration remain unclear. In this study, we compared the effects of ketamine administration on morphine consumption limited to the intraoperative period, or continued for 48 h postoperatively. METHODS:Eighty-one patients scheduled for abdominal surgery were prospectively randomized under double-blind conditions to three groups: (1) PERI group receiving intraoperative and postoperative ketamine for the first 48 h after surgery (2 &mgr;g · kg−1 · min−1 after a 0.5 mg/kg bolus); (2) INTRA group receiving intraoperative ketamine administration only (2 &mgr;g · kg−1 · min−1 after a 0.5 mg/kg bolus); and (3) CTRL group receiving placebo. Morphine consumption, visual analog scale scores and side effects (sedation score, nausea-vomiting score, nightmares, psychiatric disorders, or delusions) were recorded for the first 48 h. RESULTS:Cumulative morphine consumption 24 h after surgery was significantly lower in the PERI group (median = 27 mg, interquartile range = [19]) than in the INTRA group (48 mg [41.5]) and CTRL group (50 mg [21]) (P < 0.005). Postoperative visual analog scale scores were significantly lower in the PERI group and INTRA group than in the CTRL group (P < 0.001). A higher rate of nausea was observed in the CTRL group compared with the PERI group (27% vs 4%, P = 0.005). No difference in sedation scores or psychiatric disorders was observed among groups. CONCLUSIONS:Low-dose ketamine improved postoperative analgesia with a significant decrease of morphine consumption when its administration was continued for 48 h postoperatively, with a lower incidence of nausea and with no side effects of ketamine.

Mitochondrial Respiratory Complex I Regulates Neutrophil Activation and Severity of Lung Injury

RATIONALE Mitochondria have important roles in intracellular energy generation, modulation of apoptosis, and redox-dependent intracellular signaling. Although reactive oxygen species (ROS) participate in the regulation of intracellular signaling pathways, including activation of nuclear factor (NF)-kappaB, there is only limited information concerning the role of mitochondrially derived ROS in modulating cellular activation and tissue injury associated with acute inflammatory processes. OBJECTIVES To examine involvement of the mitochondrial electron transport chain complex I on LPS-mediated NF-kappaB activation in neutrophils and neutrophil-dependent acute lung injury. METHODS Neutrophils incubated with rotenone or metformin were treated with bacterial lipopolysaccharide (LPS) to determine the effects of mitochondrial complex I inhibition on intracellular concentrations of reactive oxygen species, NF-kappaB activation, and proinflammatory cytokine expression. Acute lung injury was produced by intratracheal injection of LPS into control, metformin, or rotenone-treated mice. MEASUREMENTS AND MAIN RESULTS Inhibition of complex I with either rotenone or the antihyperglycemic agent metformin was associated with increased intracellular levels of both superoxide and hydrogen peroxide, as well as inhibition of LPS-induced I kappaB-alpha degradation, NF-kappaB nuclear accumulation, and proinflammatory cytokine production. Treatment of LPS-exposed mice with rotenone or metformin resulted in inhibition of complex I in the lungs, as well as diminished severity of lung injury. CONCLUSIONS These results demonstrate that mitochondrial complex I plays an important role in modulating Toll-like receptor 4-mediated neutrophil activation and suggest that metformin, as well as other agents that inhibit mitochondrial complex I, may be useful in the prevention or treatment of acute inflammatory processes in which activated neutrophils play a major role, such as acute lung injury.

p53 Attenuates Lipopolysaccharide-Induced NF-κB Activation and Acute Lung Injury

The transcriptional factor p53 has primarily been characterized for its central role in the regulation of oncogenesis. A reciprocal relationship between the activities of p53 and NF-κB has been demonstrated in cancer cells, but there is little information concerning interactions between p53 and NF-κB in inflammatory processes. In this study, we found that neutrophils and macrophages lacking p53, i.e., p53−/−, have elevated responses to LPS stimulation compared with p53+/+ cells, producing greater amounts of proinflammatory cytokines, including TNF-α, IL-6, and MIP-2, and demonstrating enhanced NF-κB DNA-binding activity. p53−/− mice are more susceptible than are p53+/+ mice to LPS-induced acute lung injury (ALI). The enhanced response of p53−/− cells to LPS does not involve alterations in intracellular signaling events associated with TLR4 engagement, such as activation of MAPKs, phosphorylation of IκB-α or the p65 subunit of NF-κB, or IκB-α degradation. Culture of LPS-stimulated neutrophils and macrophages with nutlin-3a, a specific inducer of p53 stabilization, attenuated NF-κB DNA-binding activity and production of proinflammatory cytokines. Treatment of mice with nutlin-3a reduced the severity of LPS-induced ALI. These data demonstrate that p53 regulates NF-κB activity in inflammatory cells and suggest that modulation of p53 may have potential therapeutic benefits in acute inflammatory conditions, such as ALI.

Participation of Mammalian Target of Rapamycin Complex 1 in Toll-Like Receptor 2– and 4–Induced Neutrophil Activation and Acute Lung Injury

mTOR complex 1 (mTORC1) plays a central role in cell growth and cellular responses to metabolic stress. Although mTORC1 has been shown to be activated after Toll-like receptor (TLR)-4 engagement, there is little information concerning the role that mTORC1 may play in modulating neutrophil function and neutrophil-dependent inflammatory events, such as acute lung injury. To examine these issues, we determined the effects of rapamycin-induced inhibition of mTORC1 on TLR2- and TLR4-induced neutrophil activation. mTORC1 was dose- and time-dependently activated in murine bone marrow neutrophils cultured with the TLR4 ligand, LPS, or the TLR2 ligand, Pam(3) Cys-Ser-(Lys)(4) (PAM). Incubation of PAM- or LPS-stimulated neutrophils with rapamycin inhibited expression of TNF-alpha and IL-6, but not IkappaB-alpha degradation or nuclear translocation of NF-kappaB. Exposure of PAM or LPS-stimulated neutrophils to rapamycin inhibited phosphorylation of serine 276 in the NF-kappaB p65 subunit, a phosphorylation event required for optimal transcriptional activity of NF-kappaB. Rapamycin pretreatment inhibited PAM- or LPS-induced mTORC1 activation in the lungs. Administration of rapamycin also decreased the severity of lung injury after intratracheal LPS or PAM administration, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-alpha and IL-6 in bronchoalveolar lavage fluid. These results indicate that mTORC1 activation is essential in TLR2- and TLR4-induced neutrophil activation, as well as in the development and severity of acute lung injury.

Antiinflammatory Effects of Hydrogen Peroxide in Neutrophil Activation and Acute Lung Injury

RATIONALE Although reactive oxygen species (ROS) are generally considered to be proinflammatory and to contribute to cellular and organ dysfunction when present in excessive amounts, there is evidence that specific ROS, particularly hydrogen peroxide (H(2)O(2)), may have antiinflammatory properties. OBJECTIVES To address the role that increases in intracellular H(2)O(2) may play in acute inflammatory processes, we examined the effects of catalase inhibition or the absence of catalase on LPS-induced inflammatory responses. METHODS Neutrophils from control or acatalasemic mice, or control neutrophils incubated with the catalase inhibitor aminotriazole, were treated with LPS, and levels of reactive oxygen species, proteasomal activity, NF-kappaB activation, and proinflammatory cytokine expression were measured. Acute lung injury (ALI) was produced by intratracheal injection of LPS into control, acatalasemic-, or aminotriazole-treated mice. MEASUREMENTS AND MAIN RESULTS Intracellular levels of H(2)O(2) were increased in acatalasemic neutrophils and in neutrophils exposed to aminotriazole. Compared with LPS-stimulated neutrophils from control mice, neutrophils from acatalasemic mice or neutrophils treated with aminotriazole demonstrated reduced 20S and 26S proteasomal activity, IkappaB-alpha degradation, NF-kappaB nuclear accumulation, and production of the proinflammatory cytokines TNF-alpha and macrophage inhibitory protein (MIP)-2. The severity of LPS-induced ALI was less in acatalasemic mice and in mice treated with aminotriazole as compared with that found in control mice. CONCLUSIONS These results indicate that H(2)O(2) has antiinflammatory effects on neutrophil activation and inflammatory processes, such as ALI, in which activated neutrophils play a major role.

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production.

Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) and increased neutrophil production of the NF-kappaB-dependent cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-alpha or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-kappaB as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-kappaB and increases expression of NF-kappaB-dependent cytokines.

Participation of the urokinase receptor in neutrophil efferocytosis.

The urokinase receptor (uPAR) plays an important role in regulation of fibronolysis, cell migration, and adhesion. In this study, we examined whether uPAR plays a role in modulating efferocytosis of neutrophils. Macrophages from uPAR(-/-) mice demonstrated enhanced ability to engulf viable wild-type (WT) neutrophils in vitro and in vivo in the lungs. The increased phagocytic activity of uPAR(-/-) macrophages was abrogated by incubation with soluble uPAR (suPAR), arginine-glycine-aspartic acid (RGD)-containing peptides, or anti-integrin antibodies. There was increased uptake of viable uPAR(-/-) neutrophils by WT macrophages. Incubation of uPAR(-/-) neutrophils with suPAR or anti-integrin antibodies diminished uptake by WT macrophages to baseline. Uptake of uPAR(-/-) neutrophils by uPAR(-/-) macrophages was not enhanced. However, incubation of uPAR(-/-) neutrophils or uPAR(-/-) macrophages, but not both, with suPAR enhanced the uptake of viable uPAR(-/-) neutrophils by uPAR(-/-) macrophages. The adhesion of WT neutrophils to uPAR(-/-) macrophages was higher than to WT macrophages. uPAR(-/-) neutrophils demonstrated increased adhesion to suPAR, which was abrogated by blocking of low-density lipoprotein related protein and integrins. Expression of uPAR on the surface of apoptotic neutrophils was reduced compared with levels on viable neutrophils. These results demonstrate a novel role for uPAR in modulating recognition and clearance of neutrophils.