Neutrophil Sequestration Research Articles

Inhaled thrombolytics reduce lung microclot and leukocyte infiltration after acute blood loss.

We showed previously that a 30% blood loss in rats, without resuscitation, caused significant accumulation of microthrombi and leukocytes within the pulmonary circulation by 24 h. We hypothesized that the microthrombi formed spontaneously as a consequence of hemorrhage-induced stasis within the low-pressure pulmonary circuit and that the leukocytes were attracted to them. This suggested that elimination of the microthrombi, using an inhaled thrombolytic agent, could prevent the neutrophil sequestration after blood loss. To test this hypothesis, we removed 30% of the calculated blood volume from isoflurane-anesthetized, male Sprague-Dawley rats (350-500 g) over 5 min and allowed them to recover. Six hours later, we re-anesthetized the rats and nebulized tissue plasminogen activator (80 or 320 µg/kg), lactated Ringer's solution (LRS), or ipratropium bromide (i-bromide) into their lungs. We used i-bromide as a control after we discovered that nebulized LRS had thrombolytic properties. At 24 h, we removed and fixed the lungs and prepared sections for immunohistochemistry using antibodies against fibrinogen (microthrombi) and CD16 (leukocytes). Digital images of each section were obtained using a confocal microscope. Pixel counts of the images showed significantly less accumulation of microthrombi and leukocytes in lungs nebulized with tissue plasminogen activator or LRS than in non-nebulized lungs or in lungs nebulized with i-bromide (P ≤ 0.05). Lactated Ringer's solution becomes positively charged when nebulized (unlike i-bromide), suggesting that it eliminated microthrombi by fibrin depolymerization. We confirmed this using an in vitro assay. Our results demonstrate that lyses of microthrombi that accumulate in the lung after acute blood loss prevent subsequent leukocyte sequestration.

Expression of Human CD46 Modulates Inflammation Associated With GalTKO Lung Xenograft Injury

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120' 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120' 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60' 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Role of endothelial sialic acid expression on xenogenic neutrophil adhesion (1003.4)

Objective: Transplantation of organs across species, xenotransplantation, is limited by the sequestration of platelets and neutrophils in injured organs; we hypothesized that sialic acids on porcine endothelial cells affects adhesion of human neutrophils.Methods: An adhesion assay was developed with freshly isolated human neutrophils and porcine aortic endothelial cells (pAECs). Neutrophils labeled with 0.5 uM of Calcein were quantified using a Spectramax fluorescent reader. Neutrophil adhesion was expressed as arbitrary fluorescence units. 25 mU of Neuraminidase (Clostridium Perfringens) was added to remove sialic acids from pAECs 30 minutes before neutrophil addition. Sialic acid expression was assessed by flow cytometry using lectins (Elderberry lectin, (SNA), Peanut agglutinin (PNA) and Maackia amurensis hemoagglutinin (MAC)). Some pAECs wells were incubated with human TNF (25ng/ml) for 3 hrs at 37 °C before addition of neuraminidase and neutrophils.Results: Human neutrophil adhesion was detectable on resting porcine endothelial cells (52 +/‐15) and was increased by TNF activation (72 +/‐ 4.7). Neuraminidase treatment increased neutrophil adhesion on resting pAECs to a level similar to TNF activation (71 +/‐ 8.8).Conclusion: Removal of sialic acid with neuraminidase increased the adhesion of human neutrophils to swine endothelial cells. Thus, targeting levels of sialic acid expression may be a novel therapy to limit neutrophil adhesion during xenotransplantation. Future studies will examine the role of endothelial sialic acid on platelet adhesion as well as the role of neutrophils and platelet sialic acid expression.Grant Funding Source: Supported by: NIH 5T32HL007698‐19 &amp; U19AI090959; United Therapeutics unrestricted gift

Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks 'outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

Human CD34+ Progenitor Cells Freshly Isolated from Umbilical Cord Blood Attenuate Inflammatory Lung Injury following LPS Challenge

Adult stem cell-based therapy is a promising novel approach for treatment of acute lung injury. Here we investigated the therapeutic potential of freshly isolated human umbilical cord blood CD34+ progenitor cells (fCB-CD34+ cells) in a mouse model of acute lung injury. At 3 h post-lipopolysaccharide (LPS) challenge, fCB-CD34+ cells were transplanted i.v. to mice while CD34− cells or PBS were administered as controls in separate cohorts of mice. We observed that fCB-CD34+ cell treatment inhibited lung vascular injury evident by decreased lung vascular permeability. In contrast, CD34− cells had no effects on lung vascular injury. Lung inflammation determined by myeloperoxidase activity, neutrophil sequestration and expression of pro-inflammatory mediators was attenuated in fCB-CD34+ cell-treated mice at 26 h post-LPS challenge compared to PBS or CD34− cell-treated controls. Importantly, lung inflammation in fCB-CD34+ cell-treated mice was returned to normal levels as seen in basal mice at 52 h post-LPS challenge whereas PBS or CD34− cell-treated control mice exhibited persistent lung inflammation. Accordingly, fCB-CD34+ cell-treated mice exhibited a marked increase of survival rate. Employing in vivo 5-bromo-2′-deoxyuridine incorporation assay, we found a drastic induction of lung endothelial proliferation in fCB-CD34+ cell-treated mice at 52 h post-LPS compared to PBS or CD34− cell-treated controls, which contributed to restoration of vascular integrity and thereby inhibition of lung inflammation. Taken together, these data have demonstrated the protective effects of fCB-CD34+ cell on acute lung injury induced by LPS challenge, suggesting fCB-CD34+ cells are an important source of stem cells for the treatment of acute lung injury.

Improving effect of Sivelestat on lipopolysaccharide‐induced lung injury in rats

Sepsis causes neutrophil sequestration in the lung, which leads to acute lung injury (ALI). Neutrophil elastase (NE) is thought to play an important role in the pathogenesis of ALI. This study investigated whether Sivelestat, a specific NE inhibitor, can attenuate ALI induced by lipopolysaccharide (LPS). In vivo, 30 male Wistar rats were divided into three groups (n=10 each groups) on the basis of the reagent used, which were subjected to LPS injection with or without Sivelestat treatments to induce ALI model. Lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (W/D) ratio, immunohistochemical analysis of intercellular adhesion molecule-1 (ICAM-1), the number of myeloperoxidase (MPO)-positive cells, and gene expression of ICAM-1. In vitro, pulmonary microvascular endothelial cells (PMVECs) were stimulated with LPS in the presence and absence of Sivelestat; nuclear factor-κB (NF-κB) p65 was measured by immunocytochemistry staining and Western blotting. Infusion of LPS induced lung injury, in vivo, as demonstrated by pulmonary edema with infiltration of neutrophils, the increase in lung W/D ratio, the number of MPO-positive cells and enhanced expression of ICAM-1 and ICAM-1 gene. In vitro, the significant increased release of NF-κB p65 and its subsequent translocation into the nucleus in PMVECs. In contrast, Sivelestat treatment significantly ameliorated the LPS-induced lung injury, as judged by the marked improvement in all these indices. These results indicated that inhibition of NE attenuated LPS-induced lung injury through an inhibition of the inflammatory signaling pathway, besides the direct inhibitory effect on NE.

Treatment of Low Molecular Weight Heparin Inhibits Systemic Inflammation and Prevents Endotoxin-Induced Acute Lung Injury in Rats

To determine whether low molecular weight heparin (LMWH) is able to reduce pulmonary inflammation and improve the survival in rats with endotoxin-induced acute lung injury (ALI). Rat ALI model was reproduced by injection of lipopolysaccharide (LPS) into tail vein. Rats were divided randomly into three groups: control group, ALI group, LMWH-treated group. Blood was collected and lung tissue was harvested at the designated time points for analysis. The lung specimens were harvested for morphological studies, streptavidin-peroxidase immunohistochemistry examination. Lung tissue edema was evaluated by tissue water content. The levels of lung tissue myeloperoxidase (MPO) were determined. Meanwhile, the nuclear factor-kappa B (NF-κB) activation, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) levels and high mobility group box 1 (HMGB1) and intercellular adhesion molecule-1 (ICAM-1) protein levels in the lung were studied. In survival studies, a separate group of rats were treated with LMWH or sterile saline after LPS administration. Then, the mortality was recorded. Treatment with LMWH after ALI was associated with a reduction in the severity of LPS-induced lung injury. Treatment with LMWH significantly decreased the expression of TNF-α, IL-1β, HMGB1 and ICAM-1 in the lung of ALI rats. Similarly, treatment with LMWH dramatically diminished LPS-induced neutrophil sequestration and markedly reduced the enhanced lung permeability. In the present study, LMWH administration inhibited the nuclear translocation of NF-κB in the lung. Survival was significantly higher among the LMWH-treated group compared with the ALI group. These data suggest that LMWH attenuates inflammation and prevents lethality in endotoxemic rats.

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: >800U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91phox−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury.

Translational toxicological research: investigating and preventing acute lung injury in organophosphorus insecticide poisoning

Poisoning through ingestion of organophosphorus (OP) insecticide is a leading cause of suicide globally. Severe poisoning with OP compounds creates an unconscious, paralysed patient with respiratory failure. These symptoms make...

Leukocyte Depletion During CPB: Effects on Inflammation and Lung Function

Cardiopulmonary bypass (CPB) is related to inflammatory response and pulmonary dysfunction. The aim of this study was to evaluate the effects of CPB leukocyte filtration on inflammation and lung function after coronary artery bypass grafting (CABG). A prospective randomized study was performed to compare CABG patients undergoing CPB leukocyte filtration (n = 9) or standard CPB (n = 11). Computed tomography, oxygenation, leukocyte count, hemodynamic data, PaO2/FiO2, shunt fraction, interleukins, elastase, and myeloperoxidase were evaluated. Data were analyzed using two-factor ANOVA for repeated measurements. The filtered group showed lower neutrophil counts up to 50 min of CPB, lower shunt fraction up to 6 h after surgery, and lower levels of IL-10 at the end of surgery (p < 0.05). There was no statistically significant difference between groups related to other parameters. Leukodepletion during CPB results in neutrophil sequestration by a short time, decreased IL-10 serum levels, and lower worsening of lung function only temporarily.

Targeting Aspiration Pneumonitis

Targeting Aspiration Pneumonitis

Carbon monoxide inhalation increases microparticles causing vascular and CNS dysfunction

We hypothesized that circulating microparticles (MPs) play a role in pro-inflammatory effects associated with carbon monoxide (CO) inhalation. Mice exposed for 1h to 100ppm CO or more exhibit increases in circulating MPs derived from a variety of vascular cells as well as neutrophil activation. Tissue injury was quantified as 2000kDa dextran leakage from vessels and as neutrophil sequestration in the brain and skeletal muscle; and central nervous system nerve dysfunction was documented as broadening of the neurohypophysial action potential (AP). Indices of injury occurred following exposures to 1000ppm for 1h or to 1000ppm for 40min followed by 3000ppm for 20min. MPs were implicated in causing injuries because infusing the surfactant MP lytic agent, polyethylene glycol telomere B (PEGtB) abrogated elevations in MPs, vascular leak, neutrophil sequestration and AP prolongation. These manifestations of tissue injury also did not occur in mice lacking myeloperoxidase. Vascular leakage and AP prolongation were produced in naïve mice infused with MPs that had been obtained from CO poisoned mice, but this did not occur with MPs obtained from control mice. We conclude that CO poisoning triggers elevations of MPs that activate neutrophils which subsequently cause tissue injuries.

NF-κB activation in myeloid cells mediates ventilator-induced lung injury

BackgroundAlthough use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.MethodsTo study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ△mye), IL-6-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.ResultsOur results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ△mye mice as well as in IL6-/- to WT chimeric mice.ConclusionGiven that IKKβ△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

Gene Dosage-Dependent Negative Regulatory Role of β-Arrestin-2 in Polymicrobial Infection-Induced Inflammation

β-arrestin-2 (β-arr2) is a scaffolding protein of the arrestin family with a wide variety of cellular functions. Recent studies have demonstrated differential roles for β-arr2 in inflammation following endotoxemia and cecal ligation and puncture (CLP) models of sepsis. Because CLP-induced inflammation involves response to fecal contents and necrotic cecum in addition to microbial challenge, in this study, we examined the role of β-arr2 in an exclusively polymicrobial infection (PMI) model. In addition, we examined the role of gene dosage of β-arr2 in polymicrobial sepsis. Our studies demonstrate that β-arr2 is a negative regulator of systemic inflammation in response to polymicrobial infection and that one allele is sufficient for this process. Our results further reveal that loss of β-arr2 leads to increased neutrophil sequestration and overt inflammation specifically in the lungs following polymicrobial infection. Consistent with this, specific NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways were differentially activated in the β-arr2 knockout (KO) mice lungs compared to the wild type (WT) following PMI. Associated with enhanced inflammation in the KO mice, PMI-induced mortality was also significantly higher in KO mice than in WT mice. To understand the differential role of β-arr2 in different sepsis models, we used cell culture systems to evaluate inflammatory cytokine production following endotoxin and polymicrobial stimulation. Our results demonstrate cell-type- as well as stimulus-specific roles for β-arr2 in inflammation. Taken together, our results reveal a negative regulatory role for β-arr2 in polymicrobial infection-induced inflammation and further demonstrate that one allele of β-arr2 is sufficient to mediate most of these effects.

Lung Re-Expansion Following One-Lung Ventilation Induces Neutrophil Cytoskeletal Rearrangements in Rats

To investigate the morphological and functional behavior of neutrophils during and after one-lung ventilation (OLV). We utilized an OLV rat model system and performed 3 hours of OLV followed by either re-expansion (RE) and 30 minutes of two-lung ventilation (TLV) (RE group), only two-lung ventilation (TLV group), or only OLV (OLV group). Cytoskeletal rearrangements of circulating neutrophils were assessed by determining the localization of filamentous actin (F-actin). In addition, the number of sequestered neutrophils in the lung capillary and the cytokine-induced neutrophil chemoattractant 1 (CINC-1) levels in the plasma were determined. The F-actin rimmed neutrophils in the RE group increased after RE, but did not increase in the other groups. In the RE group, the sequestered neutrophils in the ventilated lung were significantly more numerous, and the plasma CINC-1 levels were significantly higher than in the other groups. Lung RE following OLV induces cytoskeletal rearrangements in circulating neutrophils and would thereby promote their sequestration in the lung capillaries. The plasma CINC-1 elevation after RE can be involved in neutrophil recruitment.

Transfusion-related acute lung injury: advances in understanding the role of proinflammatory mediators in its genesis

Transfusion-related acute lung injury (TRALI) is the most common cause of serious morbidity and mortality due to hemotherapy. The pathogenesis is the result of two events: the first related to the recipient’s clinical condition, predisposing to acute lung injury (ALI) through neutrophil or polymorphonuclear leukocyte sequestration, and the second being the infusion of antibodies or mediators that activate these adherent polymorphonuclear neutrophils, resulting in endothelial damage, capillary leak and ALI. TRALI is most prevalent in the critically ill, although many of these cases are termed ALI. Although mitigation strategies, such as the use of male-only plasma, have decreased the number of TRALI cases and deaths, TRALI still occurs. This review will detail the pathophysiology of TRALI, provide insight into newer areas of research and critically assess current practices to mitigate TRALI and improve transfusion safety.

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.

Human EPCR Expression in GalTKO.hCD46 Lungs Extends Survival Time and Lowers PVR in a Xenogenic Lung Perfusion Model

Purpose Analyses of xenogenic GalTKO.hCD46 organ perfusions have revealed that activation of complement cascade and platelets play a substantial role in rejection of the perfused organ. With the aim of better controlling these mechanisms, human endothelial protein C receptor (hEPCR), a receptor facilitating protein C activation by the thrombin-thrombomodulin complex, has been added on the genetic GalTKO.hCD46 background. Here we evaluate effects of this additional genetic modification in a xenogenic lung perfusion model. Methods and Materials 6 GalTKO.hCD46.hEPCR and 37 GalTKO.hCD46 pig lungs were perfused with heparinized fresh human blood until failure (by oxygenation or PA flow and pressure criteria) or elective termination at 4 h. Functional parameters (oxygenation, PVR) were recorded, and blood samples were collected at pre-specified intervals throughout the perfusion for analysis of cell counts and cell activation. Results Median survival time in GalTKO.hCD46 organs was 171’ whereas GalTKO.hCD46.hEPCR lungs all “survived” to an elective termination (MST 240’, p=0.006). Platelet activation was significantly lower in the GalTKO.hCD46.hEPCR group (ΔBTG at 2h: 119±163 vs. 770±377, p=0.008). However, platelet and neutrophil sequestration was not prevented by the additional hEPCR expression. Thrombin generation was lower throughout the perfusion in hEPCR lungs without reaching statistical significance. PVR was lowered in hEPCR organs when compared to the GalTKO.hCD46 controls (PVR at 90’: 76±81 vs. 171±110, p=0.0536). Conclusions Results of this study demonstrate that genetic modifications to the pig, targeting anticoagulant mechanisms, do suppress platelet activation and lower activation of the complement cascade. As a consequence, PVR was lowered and survival was significantly improved. Expression of hEPCR, and perhaps other anticoagulant molecules, may facilitate successful lung xenotransplantation.

Outer Membrane Vesicles Derived from Escherichia coli Up-Regulate Expression of Endothelial Cell Adhesion Molecules In Vitro and In Vivo

Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1-/- and TLR4-/- mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide.

Arginase 1: An Unexpected Mediator of Pulmonary Capillary Barrier Dysfunction in Models of Acute Lung Injury

The integrity of epithelial and endothelial barriers in the lower airspaces of the lungs has to be tightly regulated, in order to prevent leakage and to assure efficient gas exchange between the alveoli and capillaries. Both G− and G+ bacterial toxins, such as lipopolysaccharide and pneumolysin, respectively, can be released in high concentrations within the pulmonary compartments upon antibiotic treatment of patients suffering from acute respiratory distress syndrome (ARDS) or severe pneumonia. These toxins are able to impair endothelial barrier function, either directly, or indirectly, by induction of pro-inflammatory mediators and neutrophil sequestration. Toxin-induced endothelial hyperpermeability can involve myosin light chain phosphorylation and/or microtubule rearrangement. Endothelial nitric oxide synthase (eNOS) was proposed to be a guardian of basal barrier function, since eNOS knock-out mice display an impaired expression of inter-endothelial junction proteins and as such an increased vascular permeability, as compared to wild type mice. The enzyme arginase, the activity of which can be regulated by the redox status of the cell, exists in two isoforms – arginase 1 (cytosolic) and arginase 2 (mitochondrial) – both of which can be expressed in lung microvascular endothelial cells. Upon activation, arginase competes with eNOS for the substrate l-arginine, as such impairing eNOS-dependent NO generation and promoting reactive oxygen species generation by the enzyme. This mini-review will discuss recent findings regarding the interaction between bacterial toxins and arginase during acute lung injury and will as such address the role of arginase in bacterial toxin-induced pulmonary endothelial barrier dysfunction.