Lung Neutrophil Sequestration Research Articles

A 2A ADENOSINE RECEPTORS REGULATE MULTIPLE ORGAN FAILURE AFTER HEMORRHAGIC SHOCK IN MICE.

Trauma hemorrhagic shock (T/HS) is a clinical condition that causes multiple organ failure that needs rapid intervention. Restricted oxygen at the cellular level causes inflammation and subsequent cell death. Adenosine triphosphate is the universal intracellular energy currency and an important extracellular inflammatory signaling molecule. Adenosine, an endogenous nucleotide formed as a result of the breakdown of adenosine triphosphate, is also released during T/HS. Adenosine binds to four G protein-coupled receptors (A 1R , A 2a , A 2b , A 3R ) called adenosine receptors or P1 receptors. In the present study, we evaluated the effect of activation, inactivation, and genetic absence of A2aR (A2aR -/- mice) on T/HS-induced multiple organ failure. Wild-type mice were pretreated (30 min before shock induction) with an agonist or antagonist and then subjected to T/HS by withdrawing arterial blood and maintaining the blood pressure between 28 and 32 mm Hg. A2aR -/- mice were subjected to T/HS in the absence of pharmacologic treatment. Neutrophil sequestration was assessed by detecting myeloperoxidase, and Evans blue dye (EBD) method was used to analyze lung permeability. Blood and lung inflammatory cytokine levels were determined by sandwich enzyme-linked immunosorbent assay. The liver enzymes aspartate aminotransferase and alanine aminotransferase were determined spectrophotometrically from plasma. Activation of the apoptotic cascade was evaluated using a mouse apoptosis array. Our results demonstrate that the selective A2aR agonist CGS21680 decreases lung neutrophil sequestration, lung proinflammatory cytokines IL-6 and TNF-α, and bronchoalveolar lavage EBD. Pretreatment with the selective antagonist ZM241385 and genetic blockade in A2aR -/- mice increased neutrophil sequestration, proinflammatory cytokine levels, and bronchoalveolar lavage fluid EBD. The myeloperoxidase level in the lung was also increased in A2aR -/- mice. We observed that antiapoptotic markers decreased significantly with the absence of A2aR in the lung and spleen after T/HS. In conclusion, our data demonstrate that activation of A2aR regulates organ injury and apoptosis in the setting of T/HS.

Rabeprazole Promotes Vascular Repair and Resolution of Sepsis-Induced Inflammatory Lung Injury through HIF-1α.

There are currently no effective treatments for sepsis and acute respiratory distress syndrome (ARDS). The repositioning of existing drugs is one possible effective strategy for the treatment of sepsis and ARDS. We previously showed that vascular repair and the resolution of sepsis-induced inflammatory lung injury is dependent upon endothelial HIF-1α/FoxM1 signaling. The aim of this study was to identify a candidate inducer of HIF-1α/FoxM1 signaling for the treatment of sepsis and ARDS. Employing high throughput screening of a library of 1200 FDA-approved drugs by using hypoxia response element (HRE)-driven luciferase reporter assays, we identified Rabeprazole (also known as Aciphex) as a top HIF-α activator. In cultured human lung microvascular endothelial cells, Rabeprazole induced HIF1A mRNA expression in a dose-dependent manner. A dose-response study of Rabeprazole in a mouse model of endotoxemia-induced inflammatory lung injury identified a dose that was well tolerated and enhanced vascular repair and the resolution of inflammatory lung injury. Rabeprazole treatment resulted in reductions in lung vascular leakage, edema, and neutrophil sequestration and proinflammatory cytokine expression during the repair phrase. We next used Hif1a/Tie2Cre knockout mice and Foxm1/Tie2Cre knockout mice to show that Rabeprazole promoted vascular repair through HIF-1α/FoxM1 signaling. In conclusion, Rabeprazole is a potent inducer of HIF-1α that promotes vascular repair and the resolution of sepsis-induced inflammatory lung injury via endothelial HIF-1α/FoxM1 signaling. This drug therefore represents a promising candidate for repurposing to effectively treat severe sepsis and ARDS.

Prevention of Pulmonary Injury in Isolated Perfused Rat Lungs by Activated Human Neutrophils Preincubated With Anti-Mo1 Monoclonal Antibody

Neutrophil activation results in neutrophil adherence and may subsequently cause lung injury through the generation of oxidants, release of granule proteases, and generation of a variety of mediator substances. We hypothesized that inhibition of neutrophil adherence and subsequent lung sequestration would attenuate the lung injury caused by activated neutrophils. Using isolated perfused rat lungs, we determined if anti-Mo1 monoclonal antibody (binds to the alpha subunit of a neutrophil glycoprotein [gp 155.94] that facilitates adherence) would attenuate lung neutrophil sequestration and lung injury caused by human neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated neutrophils but not PMA or neutrophils alone caused lung injury as assessed by accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid. Incubation of neutrophils with anti-Mo1 antibody prior to stimulation with PMA attenuated lung injury and neutrophil sequestration. Furthermore, a histological survey revealed that anti-Mo1 antibody inhibited neutrophils present in the lung from spreading following exposure to PMA. Anti-Mo1 antibody did not inhibit PMA-stimulated neutrophil release of granule constituents or toxic O2 metabolites as evidenced by lysozyme and lactoferrin release or the reduction of ferricytochrome c in the lung perfusate. The inhibition of lung injury caused by the anti-Mo1 antibody was not likely due to a nonspecific effect of the antibody, since another murine monoclonal antibody of the same class (anti-Mo5) did not inhibit lung neutrophil sequestration or lung injury. Thus, in this experimental model, interference with the close approximation of the neutrophil to its target site inhibited the ability of the activated human neutrophil to cause injury.

Hydrogen sulfide limits neutrophil transmigration, inflammation, and oxidative burst in lipopolysaccharide-induced acute lung injury

Transmigration and activation of neutrophils in the lung reflect key steps in the progression of acute lung injury (ALI). It is known that hydrogen sulfide (H2S) can limit neutrophil activation, but the respective mechanisms remain elusive. Here, we aimed to examine the underlying pathways in pulmonary inflammation. In vivo, C57BL/6N mice received the H2S slow releasing compound GYY4137 prior to lipopolysaccharide (LPS) inhalation. LPS challenge led to pulmonary injury, inflammation, and neutrophil transmigration that were inhibited in response to H2S pretreatment. Moreover, H2S reduced mRNA expression of macrophage inflammatory protein-2 (MIP-2) and its receptor in lung tissue, as well as the accumulation of MIP-2 and interleukin-1β in the alveolar space. In vitro, GYY4137 did not exert toxic effects on Hoxb8 neutrophils, but prevented their transmigration through an endothelial barrier in the presence and absence of MIP-2. In addition, the release of MIP-2 and reactive oxygen species from LPS-stimulated Hoxb8 neutrophils were directly inhibited by H2S. Taken together, we provide first evidence that H2S limits lung neutrophil sequestration upon LPS challenge. As proposed underlying mechanisms, H2S prevents neutrophil transmigration through the inflamed endothelium and directly inhibits pro-inflammatory as well as oxidative signalling in neutrophils. Subsequently, H2S pretreatment ameliorates LPS-induced ALI.

Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury.

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

TNFα-stimulated gene-6 (TSG6) activates macrophage phenotype transition to prevent inflammatory lung injury

TNFα-stimulated gene-6 (TSG6), a 30-kDa protein generated by activated macrophages, modulates inflammation; however, its mechanism of action and role in the activation of macrophages are not fully understood. Here we observed markedly augmented LPS-induced inflammatory lung injury and mortality in TSG6-/- mice compared with WT (TSG6+/+) mice. Treatment of mice with intratracheal instillation of TSG6 prevented LPS-induced lung injury and neutrophil sequestration, and increased survival in mice. We found that TSG6 inhibited the association of TLR4 with MyD88, thereby suppressing NF-κB activation. TSG6 also prevented the expression of proinflammatory proteins (iNOS, IL-6, TNFα, IL-1β, and CXCL1) while increasing the expression of anti-inflammatory proteins (CD206, Chi3l3, IL-4, and IL-10) in macrophages. This shift was associated with suppressed activation of proinflammatory transcription factors STAT1 and STAT3. In addition, we observed that LPS itself up-regulated the expression of TSG6 in TSG6+/+ mice, suggesting an autocrine role for TSG6 in transitioning macrophages. Thus, TSG6 functions by converting macrophages from a proinflammatory to an anti-inflammatory phenotype secondary to suppression of TLR4/NF-κB signaling and STAT1 and STAT3 activation.

Inhibition of Na-K-Cl cotransporter isoform 1 reduces lung injury induced by ischemia–reperfusion

ObjectivesIschemia–reperfusion acute lung injury is characterized by increased vascular permeability, lung edema, and neutrophil sequestration. Ischemia–reperfusion acute lung injury occurs in lung transplantation and other major surgical procedures. Effective regulation of alveolar fluid balance is critical for pulmonary edema. Sodium-potassium-chloride co-transporter regulates alveolar fluid and is associated with inflammation. We hypothesized that sodium-potassium-chloride co-transporter is important in ischemia–reperfusion acute lung injury. Bumetanide, a sodium-potassium-chloride co-transporter inhibitor, is used to treat pulmonary edema clinically. We studied the effect of bumetanide in ischemia–reperfusion acute lung injury. MethodsIsolated perfusion of mouse lungs in situ was performed. The main pulmonary artery and left atrium were catheterized for lung perfusion and effluent collection for recirculation, respectively, with perfusate consisting of 1 mL blood and 9 mL physiologic solution. Ischemia–reperfusion was induced by 120 minutes of ischemia (no ventilation or perfusion) and reperfused for 60 minutes. Wild-type, SPAK knockout (SPAK−/−), and WNK4 knockin (WNK4D561A/+) mice were divided into control, ischemia–reperfusion, and ischemia–reperfusion + bumetanide groups (n = 6 per group). Bumetanide was administered via perfusate during reperfusion. Measurements were taken of lung wet/dry weight, microvascular permeability, histopathology, cytokine concentrations, and activity of the nuclear factor-κB pathway. ResultsIn wild-type mice, ischemia–reperfusion caused lung edema (wet/dry weight 6.30 ± 0.36) and hyperpermeability (microvascular permeability, 0.29 ± 0.04), neutrophil sequestration (255.0 ± 55.8 cells/high-power field), increased proinflammatory cytokines, and nuclear factor-κB activation (1.33 ± 0.13). Acute lung injury was more severe in WNK4 mice with more lung edema, permeability, neutrophil sequestration, and nuclear factor-κB activation. Severity of acute lung injury was attenuated in SPAK−/−mice. Bumetanide decreased pulmonary edema (wild-type: wet/dry weight 5.05 ± 0.44, WNK4: wet/dry weight 5.13 ± 0.70), neutrophil sequestration (wild-type: 151.7 ± 27.8 cells/high-power field, WNK4: 135.3 ± 19.1 cells/high-power field), permeability (wild-type: 0.19 ± 0.01, WNK4: 0.21 ± 0.03), cytokines, and nuclear factor-κB activation after ischemia–reperfusion. ConclusionsFunctional reduction of sodium-potassium-chloride co-transporter by genetic or pharmacologic treatment to inhibit sodium-potassium-chloride co-transporter resulted in lower severity of acute lung injury induced by ischemia–reperfusion. Sodium-potassium-chloride co-transporter may present a promising target for therapeutic interventions in a clinical setting.

TSG6 as a central mediator of macrophage M2 polarization (IRC5P.626)

Abstract TNF-α stimulated gene-6 (TSG6) is an anti-inflammatory protein, secreted by activated monocytes and endothelial cells. TSG6 binds to CD44 receptor expressed on multiple cell types including monocytes/macrophages. We tested the concept that TSG6 via binding to CD44 induces a phenotypic shift in macrophages from pro-inflammatory M1 to anti-inflammatory M2 type that protects the mice against lung inflammation and injury induced by endotoxin LPS. We show that TSG6 suppressed the induction of M1 markers (IL-6, IL-1β, CXCL1, iNOS) and activated expression of M2 marker genes (such as IL-10, Mannose receptor (CD206) and Chitinase-3-like-3). The shift was abrogated in macrophages isolated from CD44-/- mice. TSG6 also suppressed the activation of NF-ΚB signaling by inhibiting interaction of MyD88 with TLR4. In addition, TSG6 induced activation of anti-inflammatory transcription factor STAT6 in LPS exposed macrophages. Intra-tracheal instillation of TSG6 protected the mice against LPS induced mortality. Moreover, mice treated with TSG6 showed significantly suppressed lung vascular injury and neutrophil sequestration on challenge with LPS. That TSG6 binding to CD44 activates the macrophage anti-inflammatory program suggests that TSG6 is a specialized regulator of inflammation and potential therapeutic target.

P47phox contributes to albuminuria and kidney fibrosis in mice

Reactive oxygen species (ROS) play an important pathogenic role in the development of many diseases, including kidney disease. Major ROS generators in the glomerulus of the kidney are the p47phox-containing NAPDH oxidases NOX1 and NOX2. The cytosolic p47phox subunit is a key regulator of the assembly and function of NOX1 and NOX2 and its expression and phosphorylation are up-regulated in the course of renal injury, and have been shown to exacerbate diabetic nephropathy. However, its role in non-diabetic-mediated glomerular injury is unclear. To address this, we subjected p47phox-null mice to either adriamycin-mediated or partial renal ablation-mediated glomerular injury. Deletion of p47phox protected the mice from albuminuria and glomerulosclerosis in both injury models. Integrin α1-null mice develop more severe glomerulosclerosis than wild type mice in response to glomerular injury mainly due to increased production of ROS. Interestingly, the protective effects of p47phox knockout were more profound in p47phox/integrin α1 double knockout mice. In vitro analysis of primary mesangial cells showed that deletion of p47phox led to reduced basal levels of superoxide and collagen IV production. Thus, p47phox-dependent NADPH oxidases are a major glomerular source of ROS, contribute to kidney injury, and are potential targets for antioxidant therapy in fibrotic disease.

Blocking TNF-α enhances Pseudomonas aeruginosa-induced mortality in burn mice through induction of IL-1β

PurposeTumor necrosis factor (TNFα) is a proinflammatory cytokine and has been a target for intervention in human sepsis. However, inhibition of TNF-α with a high dose of a TNF-receptor fusion protein in patients with septic shock worsened patient survival. This study was designed to investigate whether blocking TNF-α enhances mortality in infected burn mice through the induction of IL-1β. MethodsWT or Tnfrsf1a−/− mice received Pseudomonas aeruginosa injection in the back at 8h after burn injury. The animals were sacrificed at 24h after burn and lung tissues were harvested and examined for determining myeloperoxidase (MPO) activity, pulmonary microvascular dysfunction, NF-κB DNA binding activity, and IL-1β expression. Also, the lung and blood were harvested for bacterial count assay. ResultThermal injury alone induced NF-κB DNA binding activity and neutrophil infiltration in the lung in WT but not in Tnfrsf1a−/− mice. A 50% total body surface area (TBSA) burn induced a significant increase of mortality in WT compared with Tnfrsf1a−/− mice. In contrast, P. aeruginosa injection with a 30% TBSA burn pretreatment enhanced IL-1β expression, bacterial counts in lung and blood, pulmonary microvascular dysfunction, and mortality in Tnfrsf1a−/− mice compared with WT mice. Injection of the IL-1 receptor antagonist, Anakinra, reduced P. aeruginosa infection with burn pretreatment-induced blood bacterial counts, IL-1β levels as well as permeability of lung, and mortality in Tnfrsf1a−/− mice. ConclusionsOur findings suggest that thermal injury induces lung NF-κB activation and neutrophil sequestration through TNFα signaling. However, blocking TNF-α enhances P. aeruginosa infection-induced lung damage in burn mice via induction of IL-1β. Using an IL-1 receptor antagonist combined with the neutralization of TNF-α could be a useful strategy for decreasing P. aeruginosa infection-induced mortality in burn patients.

Vagal nerve stimulation modulates gut injury and lung permeability in trauma-hemorrhagic shock

Hemorrhagic shock is known to disrupt the gut barrier leading to end-organ dysfunction. The vagus nerve can inhibit detrimental immune responses that contribute to organ damage in hemorrhagic shock. Therefore, we explored whether stimulation of the vagus nerve can protect the gut and recover lung permeability in trauma-hemorrhagic shock (THS). Male Sprague-Dawley rats were subjected to left cervical vagus nerve stimulation at 5 V for 10 minutes. The right internal jugular and femoral artery were cannulated for blood withdrawal and blood pressure monitoring, respectively. Animals were then subjected to hemorrhagic shock to a mean arterial pressure between 30 mm Hg and 35 mm Hg for 90 minutes then reperfused with their own whole blood. After observation for 3 hours, gut permeability was assessed with fluorescein dextran 4 in vivo injections in a ligated portion of distal ileum followed by Evans blue dye injection to assess lung permeability. Pulmonary myeloperoxidase levels were measured and compared. Vagal nerve stimulation abrogated THS-induced lung injury (mean [SD], 8.46 [0.36] vs. 4.87 [0.78]; p < 0.05) and neutrophil sequestration (19.39 [1.01] vs. 12.83 [1.16]; p < 0.05). Likewise, THS gut permeability was reduced to sham levels. Neuromodulation decreases injury in the THS model as evidenced by decreased gut permeability as well as decreased lung permeability and pulmonary neutrophil sequestration in a rat model.

Increased Survival and Reduced Neutrophil Infiltration of the Liver in Rab27a- but Not Munc13-4-Deficient Mice in Lipopolysaccharide-Induced Systemic Inflammation

Genetic defects in the Rab27a or Munc13-4 gene lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the role of Rab27a and Munc13-4 in the regulation of systemic inflammation initiated by Gram-negative bacterium-derived pathogenic molecules is currently unknown. Using a model of lipopolysaccharide-induced systemic inflammation, we show that Rab27a-deficient (Rab27a(ash/ash)) mice are resistant to lipopolysaccharide (LPS)-induced death, while Munc13-4-deficient (Munc13-4(jinx/jinx)) mice show only moderate protection. Rab27a(ash/ash) but not Munc13-4(jinx/jinx) mice showed significantly decreased tumor necrosis factor alpha (TNF-α) plasma levels after LPS administration. Neutrophil sequestration in lungs from Rab27a(ash/ash) and Munc13-4(jinx/jinx) LPS-treated mice was similar to that observed for wild-type mice. In contrast, Rab27a- but not Munc13-4-deficient mice showed decreased neutrophil infiltration in liver and failed to undergo LPS-induced neutropenia. Decreased liver infiltration in Rab27a(ash/ash) mice was accompanied by lower CD44 but normal CD11a and CD11b expression in neutrophils. Both Rab27a- and Munc13-4-deficient mice showed decreased azurophilic granule secretion in vivo, suggesting that impaired liver infiltration and improved survival in Rab27a(ash/ash) mice is not fully explained by deficient exocytosis of this granule subset. Altogether, our data indicate that Rab27a but not Munc13-4 plays an important role in neutrophil recruitment to liver and LPS-induced death during endotoxemia, thus highlighting a previously unrecognized role for Rab27a in LPS-mediated systemic inflammation.

A Novel Function of Sphingosine Kinase 1 Suppression of JNK Activity in Preventing Inflammation and Injury

The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappaB activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.

Toll-like receptor 4 mediates neutrophil sequestration and lung injury induced by endotoxin and hyperinflation*

To address the role of Toll-like receptor 4 signaling in mediating neutrophil recruitment and lung injury induced by lipopolysaccharide challenge coupled to lung hyperinflation, using Toll-like receptor 4 knockout (tlr4) mice. Infiltration of polymorphonuclear neutrophils into the lung is an important feature of ventilator-induced lung injury associated with pneumonia, but the mechanisms involved in neutrophil recruitment are poorly understood. Experimental animal model. University laboratory. tlr4 and wild-type C57BL/6 mice. Wild-type or tlr4 mice were challenged by intratracheal instillation of lipopolysaccharide (0.3 mg/kg) for 2 hrs and then subjected to normal (7 mL/kg) or high (28 mL/kg) tidal volume ventilation for another 2 hrs. In other studies, neutrophils from wild-type or tlr4 mice were pretreated with lipopolysaccharide for 30 mins and then infused into the isolated lung preparation for 30 mins as the lungs were ventilated with 25 cm H2O peak inspiratory pressure. Lipopolysaccharide-challenged wild-type mice ventilated with a 28 mL/kg tidal volume exhibited 12-fold increase in neutrophil sequestration, 6-fold increase in bronchoalveolar lavage albumin concentration, and 1.6-fold increase in lung water content compared with unchallenged mice exposed to normal tidal volume ventilation. However, tlr4 mice showed negligible neutrophil sequestration, microvascular barrier breakdown, or edema formation. Mechanical ventilation alone or combined with lipopolysaccharide caused activation of circulating neutrophils and pulmonary endothelium in wild-type mice, whereas this was prevented in tlr4 mice. High tidal volume ventilation during pneumonia/sepsis induces lung neutrophil sequestration and injury via the Toll-like receptor 4-dependent signaling pathway. The results suggest an important role of Toll-like receptor 4 in the mechanism of lung neutrophil sequestration and acute lung injury when pneumonia/sepsis is coupled to lung hyperinflation.

Oxidant stress mediates inflammation and apoptosis in ventilator‐induced lung injury

Ventilator-induced lung injury (VILI) leads to airway epithelial cell apoptosis and lung inflammation. High tidal volume ventilation in vivo has been shown to induce MIP-2 production, lung neutrophil sequestration and apoptotic airway cell death. This study aimed to determine the effect of N-acetylcysteine (NAC), a scavenger of oxygen radicals, on lung inflammation and apoptosis in an in vivo model of VILI. Sprague-Dawley rats (n = 5 per group) were ventilated at low tidal volume (V(T) 7 mL/kg) or high tidal volume (V(T) 20 mL/kg) with or without administration of 140 mg/kg of intravenous NAC. Animals were ventilated for 30 min, 1 or 2 h, then allowed to recover for 2 h, at which time neutrophil infiltration, MIP-2, TNF-alpha and IL-6 in BAL fluid, as well as the percentage of apoptotic airway epithelial cells, were measured. Ventilation at V(T) 20 mL/kg increased oxidant release, as measured by serum isoprostane, and decreased lung glutathione, the major antioxidant in the lung. NAC treatment during ventilation at V(T) 20 mL/kg prevented the decrease in lung glutathione and significantly lowered serum isoprostane levels, neutrophil infiltration, cytokines in the BAL and apoptosis in the airways as compared with animals ventilated at V(T) 20 mL/kg without NAC (P < 0.05). These data point to an early role of oxidant-induced inflammation and apoptosis in VILI.

Inhaled nitric oxide does not prevent postpneumonectomy pulmonary edema in pigs

Increase in lung permeability is an inevitable consequence of pneumonectomy in relation to inflammatory injury and increased perfusion flow. We tested whether inhaled nitric oxide, a potent vasodilatator and anti-inflammatory agent, prevents postpneumonectomy edema in the first 24 hours after pneumonectomy in pigs. We assessed hemodynamics, gas exchange, extravascular lung water estimated with the double-indicator dilution method, and lung neutrophil sequestration measured on the basis of lung myeloperoxidase activity at 1 and 24 hours after left pneumonectomy in 14 pigs randomly assigned to inhaled nitric oxide (10 ppm) or control groups. Extravascular lung water content markedly increased at 1 and 24 hours after pneumonectomy, with no difference between the 2 groups. Hemodynamics did not differ between the 2 groups. Myeloperoxidase activity was higher and PaO2 values were lower in the nitric oxide group compared with in the control group. Over the 24 hours after pneumonectomy, intraoperative inhaled nitric oxide levels neither improved gas exchange nor attenuated accumulation of lung water. On the contrary, they were associated with an increase in lung neutrophil sequestration and deterioration of arterial oxygenation, suggesting the occurrence of an early and toxic effect of nitric oxide.

Role of interleukin‐18 in the development of acute pulmonary injury induced by intestinal ischemia/reperfusion and its possible mechanism

Lung injury is an important target for the systemic inflammatory response associated with intestinal ischemia/reperfusion (I/R). In the present study, the role of interleukin (IL)-18 in the development of acute pulmonary injury induced by intestinal I/R and its possible mechanism in relation to the increased activity of inducible nitric oxide synthase and tumor necrosis factor (TNF)-alpha were investigated. Mice were randomly divided into three groups: normal control group without operation; sham group with sham operation; and I/R group in which mice underwent superior mesenteric artery occlusion for 30 min followed by reperfusion for 3 h. Each group received pretreatment with exogenous IL-18, anti-IL-18 neutralizing antibody or L-NIL, the selective inhibitor of inducible nitric oxide synthase, 30 min before ischemia. The expression of TNF-alpha was detected by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Lung injury was evaluated by means of Evans blue dye (EBD) concentration, myeloperoxidase (MPO) activity and morphological analysis. The experimental results showed that both in the sham-operated and I/R groups of animals, pretreatment with exogenous IL-18 clearly enhanced pulmonary MPO activity, microvascular leakage and the expression of TNF-alpha mRNA and protein. In contrast, IL-18 did not increase the TNF-alpha level and degree of lung injury, although it clearly enhanced the pulmonary MPO activity in normal animals. Meanwhile, IL-18 antibody given prior to ischemia led to a reduction in the sequestration of neutrophils, extravasation of EBD and downregulation of the serum level of TNF-alpha in the I/R group of animals. In addition, selective inhibition of inducible nitric oxide synthase (iNOS) that inhibited plasma extravasation and pulmonary injury without affecting the MPO activity could be demonstrated in all treated animals. These data suggested a role of IL-18 in the activation and sequestration of neutrophils in lungs. Our results were consistent with the hypothesis that increased sequestration of neutrophils and microvascular leakage might, respectively, relate to the increased IL-18 level and the elevation of TNF-alpha/iNOS activity, and these two aspects might synergically contribute to intestinal I/R-induced pulmonary dysfunction.

Activation of Toll‐like Receptors is a Critical Determinant of Lung Neutrophil Sequestration and Injury Induced by High Tidal Volume Mechanical Ventilation

The infiltration of polymorphonuclear neutrophils (PMNs) into lungs is an important feature of ventilator-induced lung injury (VILI) associated with pneumonia, but the mechanisms that recruit PMNs are poorly understood. Using Toll-like receptor 4 knockout mice (TLR4−/−), we addressed the role of TLR4 signaling in PMN recruitment. Wild type (WT) or TLR4−/− mice were challenged by intratracheal instillation of LPS (3.0 mg/kg) for 2 hr and then subjected to high tidal volume mechanical ventilation (28 ml/kg) for 2 hr. Ventilated WT mice exhibited significant increases in PMN sequestration (5-fold), bronchoalveolar lavage PMN count (3.6-fold), airway fluid albumin concentration (2.4-fold), and edema formation (1.6-fold). Quantitative lung histopathology demonstrated PMN infiltration, alveolar hemorrhage, edema, and alveolar wall thickening in lungs of ventilated, LPS-challenged WT mice. However, TLR4−/− mice showed negligible PMN sequestration, microvascular barrier breakdown, and edema formation. Thus, high tidal volume ventilation during pneumonia induces PMN sequestration and lung injury via TLR4-dependent signaling pathways. The results suggest the important role of lung innate immunity regulated by TLRs in the mechanism of PMN sequestration and injury resulting from mechanical ventilation at high tidal volumes. Supported by NIH R01 HL 071626, P01 HL 60678, and P01 HL 077806.

Interleukin-6 Regulation of Direct Lung Ischemia Reperfusion Injury

Lung ischemia reperfusion injury continues to adversely affect patient and graft survival after transplantation. While the role of interleukin-6 has been studied in ischemia-reperfusion models of intestine, liver, and heart, its participation in lung reperfusion injury has not been characterized. We administered recombinant interleukin-6 to rat lungs through the intratracheal route before inducing left lung ischemia and reperfusion. Multiple in-vivo indicators of left lung injury were studied, as were transactivation patterns for nuclear factor kappa B and signal transduction and activators of transcription-3. Downstream effects on the elaboration of proinflammatory chemokines and cytokines were also studied. Recombinant interleukin-6 reduced endothelial disruption and neutrophil sequestration in left lung and alveolar spaces, resulting in improved oxygenation after ischemia and 4 hours of reperfusion. This protection was associated with decreased nuclear factor kappa B and signal transduction and activators of transcription-3 nuclear translocation early in reperfusion, and diminished proinflammatory mediator secretion late in reperfusion. Further studies focusing on the effects of recombinant interleukin-6 in large animal models are warranted, as this may be a novel strategy to improve outcomes after lung transplantation. Intratracheal administration may focus its efficacy on the lung while reducing effects on other organ systems during organ procurement.

Toll-like receptor 4 signaling leads to neutrophil migration impairment in polymicrobial sepsis*

We have documented an impaired neutrophil migration toward the infectious focus in severe sepsis. This phenomenon appears to be mediated by nitric oxide, the release of which is stimulated by circulating inflammatory cytokines released by immune cells after stimulation by bacteria and/or their products. Toll-like receptor 4 (TLR4) is the major recognition receptor for lipopolysaccharide, a component of Gram-negative bacterial cell walls. In the present study, we investigated whether TLR4 is involved in the failure of neutrophil migration in mice subjected to polymicrobial or Gram-negative sepsis. Controlled animal study. University research laboratory. Male C3H/HeJ (TLR4-deficient) and C3H/HePas (TLR4-normal) mice. Mice were subjected to sublethal or lethal polymicrobial sepsis, both induced by cecal ligation and puncture or intraperitoneal polymicrobial inoculation, and subjected to sublethal Gram-negative sepsis induced by intraperitoneal Salmonella typhimurium inoculation (GNI). Survival was monitored for 5 days. In separate experiments, mice were killed 6 hrs after sepsis induction, and intraperitoneal neutrophil migration, bacteremia, lung neutrophil sequestration, and levels of cytokines, chemokines, and nitrate were evaluated. TLR4-deficient (C3H/HeJ) mice presented incapacity to promote neutrophil recruitment to the infectious site after sublethal GNI, resulting in high mortality. However, TLR4 signaling is not essential to display neutrophil migration in sublethal polymicrobial sepsis induced by both cecal ligation and puncture and polymicrobial inoculation models, but surprisingly, it is crucial to establish the impairment of neutrophil migration in lethal polymicrobial sepsis, since TLR4-deficient mice that underwent lethal cecal ligation and puncture or polymicrobial inoculation did not present failure of neutrophil migration to infectious focus. As a consequence, these animals presented low bacteremia and a high survival rate and did not display systemic inflammation, determined by high levels of circulating cytokines and lung neutrophil sequestration and chemokine production. These results highlight the harmful role of TLR4 signaling in polymicrobial severe sepsis.