Intratracheal Instillation Of LPS Research Articles

Thrombin-Activatable Fibrinolysis Inhibitor Protects against Acute Lung Injury by Inhibiting the Complement System

Acute lung injury (ALI) is a devastating disease with an overall mortality rate of 30 to 40%. The coagulation/fibrinolysis system is implicated in the pathogenesis of ALI. Thrombin-activatable fibronolysis inhibitor (TAFI) is an important component of the fibrinolysis system. Recent studies have shown that the active form of TAFI can also regulate inflammatory responses by its ability to inhibit complement C3a, C5a, and osteopontin. We hypothesized that TAFI might have a protective role in ALI. To demonstrate this hypothesis, the development of ALI was compared between wild-type (WT) and TAFI-deficient mice. ALI was induced by intratracheal instillation of LPS. Control mice were treated with saline. Animals were killed 24 hours after LPS. The number of inflammatory cells and the concentration of total protein and inflammatory cytokines were significantly increased in bronchoalveolar lavage fluid from LPS-treated, TAFI-deficient mice compared with their WT counterparts. Significantly higher concentrations of C5a were found in bronchoalveolar lavage fluid and plasma in LPS-treated TAFI knockout mice compared with WT mice. Pretreatment with inhaled C5a receptor antagonist blocked the detrimental effects of TAFI deficiency to levels found in WT mice. Our results show that TAFI protects against ALI, at least in part, by inhibiting the complement system.

NADPH Oxidase Limits Lipopolysaccharide-Induced Lung Inflammation and Injury in Mice through Reduction-Oxidation Regulation of NF-κB Activity

Although reactive oxygen species (ROS) produced by NADPH oxidase are known to regulate inflammatory responses, the impact of ROS on intracellular signaling pathways is incompletely understood. In these studies, we treated wild-type (WT) and p47(phox)-deficient mice with LPS to investigate mechanisms by which NADPH oxidase regulates signaling through the NF-κB pathway. After intratracheal instillation of LPS, ROS generation was impaired in p47(phox)(-/-) mice, whereas these mice had increased neutrophilic alveolitis and greater lung injury compared with WT controls. In mice interbred with transgenic NF-κB reporters (HIV-long terminal repeat/luciferase [HLL]), we found exaggerated LPS-induced NF-κB activation and increased expression of proinflammatory cytokines in lungs of p47(phox)(-/-)/HLL mice compared with controls. Both lung macrophages and bone marrow-derived macrophages (BMDMs) isolated from p47(phox)(-/-)/HLL mice showed enhanced LPS-stimulated NF-κB activity compared with controls. Although nuclear translocation of NF-κB proteins was similar between genotypes, EMSAs under nonreducing conditions showed increased DNA binding in p47(phox)(-/-)/HLL BMDMs, suggesting that ROS production reduces NF-κB binding to DNA without affecting nuclear translocation. Increased intracellular reduced glutathione/glutathione disulfide ratio and greater nuclear redox factor 1 (Ref-1) levels were present in p47(phox)(-/-)/HLL compared with WT BMDMs, pointing to NADPH oxidase modulating intracellular redox status in macrophages. Treatment with the Ref-1-specific inhibitor E3330 or hydrogen peroxide inhibited LPS-induced NF-κB activation in p47(phox)(-/-)/HLL BMDMs but not in WT/HLL cells. Consistent with these findings, small interfering RNA against Ref-1 selectively reduced NF-κB activity in LPS-treated p47(phox)(-/-)/HLL BMDMs. Together, these results indicate that NADPH oxidase limits LPS-induced NF-κB transcriptional activity through regulation of intracellular redox state.

Effect of low level laser therapy on lung mechanics and inflammatory response

Low level laser therapy (LLLT) is currently prescribed as an adjuvant therapy for inflammatory diseases, however its use in internal viscera is controversial. Therefore, we assessed the effect of LLLT on acute lung injury (ALI) induced by intratracheal LPS instillation. C57 black mice (n=10) were exposed to intratracheal LPS (5mg/kg) or PBS. Six hours after instillation, both groups (PBS and LPS) were irradiated with laser at 660 nm, power output 30mW, fluency 10J/cm2 Twenty‐four hours later, bronchoalveolar lavage was collected; lungs were submitted to functional analysis and quantitation of tumor necrosis factor (TNF‐α), interleukin (IL) 1 β and intercellular adhesion molecule 1 (ICAM ‐1).LPS instillation induced infiltration of large number of inflammatory cells to distal airways, which was reduced by LLLT (2.92±0.34 vs 1.63±051 mononuclear cells; 2.25± 0.84 vs 0.43±0.19 neutrophils; p<0.05). We also observed that LLLT was efficient in reducing secretion of TNF‐α, IL‐1β and ICAM‐1. In spite of reducing inflammation, LLLT treatment did not affect lung mechanical properties, since no change was observed in tissue resistance or elastance.Therefore, we can conclude that LLLT is able to reduce inflammatory reaction in lungs exposed to LPS, without affecting the pulmonary function and recovery.

Post‐treatment with the heat shock protein 90 (hsp90) inhibitor, 17‐AAG, reduces pulmonary inflammation, hyper‐permeability and airway dysfunction associated with LPS‐induced acute lung injury (ALI) in mice.

Pre‐treatment with the heat shock protein 90 (hsp90) inhibitor, 17‐ AAG, reduces the magnitude of LPS‐induced ALI in mice. We now tested the hypothesis that post‐treatment with 17‐AAG would also reduce the magnitude of LPS‐induced ALI. 24h after the intratracheal instillation of vehicle or LPS (1.5mg/kg), mice received either vehicle or 17‐AAG (i.p. 10mg/kg). 48h later bronchoalveolar lavage fluid (BALF) was collected, and the number of cells and protein concentration were estimated as indicators of inflammation and permeability, respectively. In separate groups, lung capillary permeability was estimated by the Evans’ Blue Dye (EBD) method. LPS alone increased BALF cellularity (452±50 ×103cells/ml vs 33±2 ×103cells/ml in vehicle) and BALF protein concentration (204±5% from vehicle). 17‐AAG post‐treatment reduced the LPS‐induced BALF cellularity (140±32 ×103cells/ml) and protein concentration (119±18% from vehicle). Furthermore, LPS produced a 193±18% increase in EBD extravasation which was also significantly reduced to 135±27% in mice post‐treated with 17‐AAG. In additional mice, pulmonary function tests were performed. LPS caused a nearly 100% increase in airway resistance that was completely prevented by post‐treatment with 17‐AAG. These data strongly suggest important reparative actions of hsp90 inhibitors when administered after the LPS insult.

Pulmonary inflammation‐induced muscle atrophy involves a GSK‐3beta‐GR signaling axis

Skeletal muscle atrophy is induced by raised glucocorticoid (GC) levels and reduced insulin/IGF‐1‐Akt signaling. Glycogen synthase kinase‐3 beta (GSK‐3b) is a downstream kinase of Akt involved in regulation of muscle protein turnover.We hypothesized that muscle GC receptor (GR) and GSK‐3b are required for muscle atrophy in response to pulmonary inflammation.Control, muscle specific‐GR (mGRKO) or ‐GSK‐3b (mGSK‐3bKO) mice were subjected to intra‐tracheal LPS instillation to induce acute pulmonary inflammation.Increases in circulating corticosterone levels following IT‐LPS were similar in control, mGRKO and mGSK‐3KO mice, but accompanied by substantial muscle atrophy in control mice only. Accordingly, increases in mRNA transcripts encoding effectors of the Ub 26S‐proteasome (MuRF1 and Atrogin‐1) and autophagy‐lysosomal (Bnip3, LC3b) systems were strongly attenuated in muscle of mGRKO or mGSK‐3KO mice. Interestingly, increased expression of GC‐sensitive genes (Glul, KLF15 and REDD1) and FOXO1 and ‐3a following IT‐LPS, was suppressed in skeletal muscle of GRKO and GSK‐3KO mice. This dependency of GR signaling on GSK‐3b was confirmed in cultured muscle cells.We conclude that pulmonary inflammation‐induced muscle atrophy is dependent on an intact GSK‐3b‐GR signaling axis.Support: Dutch Astma Foundation (3.2.07.017) and Dutch Top Institute Pharma (T1–201)

Overexpression of CD39 in Mouse Airways Promotes Bacteria-Induced Inflammation

In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases. We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8(+) T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X(1) or P2X(7) receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation.

NF-κB Activation Is Required for the Transition of Pulmonary Inflammation to Muscle Atrophy

Disease exacerbations and muscle wasting comprise negative prognostic factors of chronic obstructive pulmonary disease (COPD). Transient systemic inflammation and malnutrition have been implicated in skeletal muscle wasting after acute exacerbations of COPD. However, the interactions between systemic inflammation and malnutrition in their contributions to muscle atrophy, as well as the molecular basis underlying the transition of systemic inflammation to muscle atrophy, remain unresolved. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS to model acute disease exacerbation. Systemic inflammation, nutritional intake, and body and muscle weights were determined. Muscle inflammatory signaling and atrophy signaling were examined, and the effect of the muscle-specific inactivation of NF-κB on muscle atrophy was assessed in genetically modified mice. The intratracheal LPS instillation was followed by markedly elevated circulating cytokine concentrations and NF-κB activation in extrapulmonary tissues, including skeletal muscle. The administration of intratracheal LPS increased the expression of muscle E3 ubiquitin ligases, which govern muscle proteolysis, in particular MuRF1, and caused a rapid loss of muscle mass. Reduced food intake only partly accounted for the observed muscle atrophy, and did not activate NF-κB in muscle. Rather, plasma transfer experiments revealed the presence of NF-κB-signaling and atrophy-signaling properties in the circulation of intratracheal LPS-treated mice. The genetic inhibition of muscle NF-κB activity suppressed intratracheal LPS-induced MuRF1 expression and resulted in a significant sparing of muscle tissue. Systemic inflammation and malnutrition contribute to the muscle wasting induced by acute pulmonary inflammation via distinct mechanisms, and muscle NF-κB activation is required for the transition from inflammatory to muscle atrophy signaling.

Effect of low level laser therapy on acute lung injury

Low level laser therapy (LLLT) is prescribed as adjuvant therapy for inflammatory diseases. Hence, we examined whether LLLT may ameliorate acute lung injury (ALI) induced by intratracheal LPS instillation. C57 black mice (n=10 per group) were treated with intratracheal LPS (5mg/kg) or PBS. Six hours after instillation, two groups (PBS and LPS) were irradiated with laser at 660 nm, power output 30mW, fluency 10J/cm2. We observed a marked decrease in the number of cells recovered by bronchoalveolar lavage in LPS + LLLT animals compared to LPS alone (2.0±0.8 × 4.4±1.3, respectively p<0.05). LLLT also decreased the number of inflammatory cells infiltrated in lung interstitium (49.6±3.15 × 71.8±3.92), p<0.05). There was also a decrease in the expression of F4/80 (macrophage surface marker) and MCP‐1 (monocyte chemoattractant protein‐1), detected by quantitative PCR, in animals submitted to LPS + LLLT, when compared to animals that received only LPS. A marked decrease in cytokines secretion (IL1β, TNFα, IL6, IL10) was also observed in LPS+LLLT group. No difference was observed in animals that received PBS, regardless of LLLT. Therefore, LLLT decreases pulmonary inflammatory cell infiltration, cytokines and chemokines secretion in an experimental model of ALI, supporting the notion that laser therapy attenuates inflammatory intensity, what can contribute to accelerate ALI resolution. Support CNPq, FAPESP.

Evaluation of lung injury and respiratory mechanics in a rat model of acute pancreatitis complicated with endotoxin

BackgroundAcute lung injury (ALI) is a common complication of acute pancreatitis (AP) and contributes to the majority of AP-associated deaths, particularly in the setting of secondary infection. This ‘two-hit’ model mimics clinical cases where the presentation of AP is associated with mild lung injury that, following a secondary direct lung infection, can result in respiratory dysfunction and death. We therefore aimed to characterize lung injury in a clinically-relevant ‘two-hit’ rat model of caerulein-induced AP combined with intratracheal endotoxin. MethodsRats received 7 hourly intraperitoneal injections of caerulein (50μg/kg). Twenty four hours following the first caerulein injection, rats were anaesthetised and LPS (15mg/kg) was instilled intratracheally. Following LPS instillation, rats were ventilated for a total of 2h. ResultsIn the present study, AP results in mild pulmonary injury indicated by increased lung myeloperoxidase (MPO) activity and edema, but with no alteration of respiratory function, while intratracheal instillation of LPS results in more substantial pulmonary injury. The induction of AP challenged with secondary intratracheal LPS results in an exacerbation of lung damage indicated by further increased lung edema, plasma and bronchoalveolar (BAL) CINC-1 concentration, lung damage histology score, and lung tissue resistance and elastance, compared with LPS alone. ConclusionsIn conclusion, the addition of instilled LPS acted as a “second-hit” and exacerbated caerulein-induced AP, compared with the induction of AP alone or the instillation of LPS alone. Given its clinical relevance, this model could prove useful for examination of therapeutic interventions for ALI following secondary infection.

LPS-Stimulated Cytokine Production in Type I Cells Is Modulated by the Renin–Angiotensin System

The alveolar epithelium serves as a barrier to the entry of potential respiratory pathogens. Alveolar Type II (TII) cells have immunomodulatory functions, but whether Type I (TI) cells, which comprise approximately 95% of the alveolar epithelium, also play a role in immunity is unknown. Because the renin-angiotensin system (RAS) is emerging as an important mediator of inflammation, and angiotensin-converting enzyme 2 (ACE2), an element of the RAS, has been implicated in lung injury, we hypothesize that TI cells can produce cytokines in response to LPS stimulation, and that this inflammation can be modulated by the RAS. Alveolar TI cells were isolated from adult Sprague-Dawley rat lungs that had been injured with an intratracheal instillation of LPS. PCR was performed to determine whether TI cells expressed transcripts for TNF-α, IL-6, or IL-1β at baseline and after lung injury. Immunocytochemical and protein analysis detected angiotensin II (Ang II) and ACE2, as well as angiotensin Type 1 receptor (AT1R) and Type 2 receptor (AT2R), in TI cells. To separate cell-specific responses, primary TI cells were isolated, cultured, and exposed to LPS, Ang II, or specific inhibitors of AT1R or AT2R. Cytokine production was assayed by ELISA. LPS stimulated the production of all cytokines, whereas ACE2 and losartan, an AT1R inhibitor, blocked elements of the LPS-induced cytokine response. Primary TI cells produce cytokines when treated with LPS, contain important components of the RAS, and can modulate LPS-induced cytokine production via the RAS, suggesting a role for TI cells in the innate immune response of the lung.

Activin-A Overexpression in the Murine Lung Causes Pathology That Simulates Acute Respiratory Distress Syndrome

Activin-A is up-regulated in various respiratory disorders. However, its precise role in pulmonary pathophysiology has not been adequately substantiated in vivo. To investigate in vivo the consequences of dysregulated Activin-A expression in the lung and identify key Activin-A-induced processes that contribute to respiratory pathology. Activin-A was ectopically expressed in murine lung, and functional, structural, and molecular alterations were extensively analyzed. The validity of Activin-A as a therapeutic target was demonstrated in animals overexpressing Activin-A or treated with intratracheal instillation of LPS. Relevancy to human pathology was substantiated by demonstrating high Activin-A levels in bronchoalveolar lavage (BAL) samples from patients with acute respiratory distress syndrome (ARDS). Overexpression of Activin-A in mouse airways caused pulmonary pathology reminiscent of acute lung injury (ALI)/ARDS. Activin-A triggered a lasting inflammatory response characterized by acute alveolar cell death and hyaline membrane formation, sustained up-regulation of high-mobility group box 1, development of systemic hypercoagulant state, reduction of surfactant proteins SpC, SpB, and SpA, decline of lung compliance, transient fibrosis, and eventually emphysema. Therapeutic neutralization of Activin-A attenuated the ALI/ARDS-like pathology induced either by ectopic expression of Activin-A or by intratracheal instillation of LPS. In line with the similarity of the Activin-A-induced phenotype to human ARDS, selective up-regulation of Activin-A was found in BAL of patients with ARDS. Our studies demonstrate for the first time in vivo the pathogenic consequences of deregulated Activin-A expression in the lung, document novel aspects of Activin-A biology that provide mechanistic explanation for the observed phenotype, link Activin-A to ALI/ARDS pathophysiology, and provide the rationale for therapeutic targeting of Activin-A in these disorders.

Endotoxin-induced cardiovascular dysfunction in mice: effect of simvastatin

Lung infections are associated with acute lung injury (ALI), systemic inflammation, and vascular events. Clinical studies suggest that statins improve health outcomes of patients with pneumonia and ALI. The mechanisms by which this occurs are unknown. The aim of this study was to determine whether statins attenuate systemic inflammation and cardiovascular dysfunction related to ALI in mice. Simvastatin (SS; 20 mg/kg) or vehicle solution was instilled intraperitoneally into mice 24 h before and again just prior to intratracheal LPS instillation (1 μg/g). These mice were then anesthetized with 2.0% isoflurane and underwent a short surgical procedure to instill LPS. Four hours later, IL-6 levels in bronchoalveolar lavage fluid and in arterial and venous serum were measured. Cardiac function was evaluated using 2-D echocardiography, and endothelial function was determined using wire myography on aortic sections. LPS induced a significant increase in serum IL-6 levels. SS reduced venous (P = 0.040) but not arterial concentrations of IL-6 (P = 0.112). SS improved the maximal vasodilatory response of the aorta to ACh (P = 0.004) but not to sodium nitroprusside (P = 1.000). SS also improved cardiac output (P = 0.023). Vasodilatory response to ACh was impaired when aorta from untreated mice was incubated with ex vivo IL-6 (P = 0.004), whereas in the aorta from mice pretreated with SS, the vasodilatory response did not change with IL-6 incubation (P = 0.387). SS significantly improved LPS-induced cardiovascular dysfunction possibly by reducing systemic expression of IL-6 and its downstream signaling pathways. These findings may explain how statins improve health outcomes in patients with ALI.

Protection of LPS-Induced Murine Acute Lung Injury by Sphingosine-1-Phosphate Lyase Suppression

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

Involvement of mannose receptor in the preventive effects of mannose in lipopolysaccharide-induced acute lung injury

Although macrophage mannose receptor contributes to the anti-inflammatory procedure, its mechanisms of action are incompletely understood in acute lung injury. We recently found that mannose which could bind to mannose receptor, prevented acute lung injury in rats. Here, we profiled the involvement of mannose receptor in the preventive effects of mannose in lipopolysaccharide-induced acute lung injury in mice. We found that pulmonary edema, protein exudation, and lung histopathology were significantly improved in a dose-dependent manner among the mannose (50, 150, and 450 mg/kg) mice (a bolus tail vein injection of mannose 5 min before and 3 h after intratracheal instillation of LPS) compared to the LPS mice. Mannose also prevented the inflammatory cell accumulation, and inhibited production of cytokines. Further, in the in vitro alveolar macrophages, treatment with mannose resulted in decreased phagocytic activity and production of cytokines, and its anti-inflammatory effects were associated with up-regulation of mannose receptor. Importantly, we found that competitive inhibition of mannose receptor (mannan 2 mg/ml) or targeted short interfering RNA-mediated gene suppression of mannose receptor mRNA was associated with the elimination of the anti-inflammatory effects of mannose. Furthermore, the up-regulation of mannose receptor by mannose administration was associated with inhibited NF-κB nuclear translocation. Taken together, these studies reveal involvement of mannose receptor and impaired NF-κB activation in the mannose prevention of acute lung injury, and implicate mannose receptor as a potential therapeutic target during acute lung injury.

SIGNALING PATHWAYS AND MEDIATORS IN LPS-INDUCED LUNG INFLAMMATION IN DIABETIC RATS

Diabetic patients are more susceptible to infections, and their inflammatory response is impaired. This is restored by insulin treatment. In the present study, we investigated the effect of insulin on LPS-induced signaling pathways and mediators in the lung of diabetic rats. Diabetic male Wistar rats (alloxan, 42 mg/kg i.v., 10 days) and control rats received intratracheal instillation of LPS (750 microg/0.4 mL) or saline. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU s.c.) 2 h before LPS. After 6 h, bronchoalveolar lavage was performed for the release of mediators, and lung tissue was homogenized for analysis of LPS-induced signaling pathways. Relative to control rats, diabetic rats exhibited a significant reduction in the LPS-induced phosphorylation of extracellular signal-regulated kinase (64%), p38 (70%), protein kinase B (67%), and protein kinase C alpha (57%) and delta (65%) and in the expression of iNOS (32%) and cyclooxygenase 2 (67%) in the lung homogenates. The bronchoalveolar lavage fluid concentrations of NO (47%) and IL-6 (49%) were also reduced in diabetic rats, whereas the cytokine-induced neutrophil chemoattractant 2 (CINC-2) levels were increased 23%, and CINC-1 was not different from control animals. Treatment of diabetic rats with insulin completely or partially restored all these parameters. In conclusion, data presented show that insulin regulates mitogen-activated protein kinase, phosphatidylinositol 3'-kinase, protein kinase C pathways, expression of the inducible enzymes, cyclooxygenase 2 and iNOS, and levels of IL-6 and CINC-2 in LPS-induced lung inflammation in diabetic rats. These results suggest that the protective effect of insulin in sepsis could be due to modulation of cellular signal transduction factors.

Abstract 5064: Endothelial-Specific Deletion of Cx40 Promotes Atherosclerosis by Increasing CD73-Dependent Leukocyte Adhesion

Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. Here, we sought to determine whether Cx40 contributes to the development of the disease. Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Tie2CRE+ Cx40fl/fl ApoE−/− mice; EC-Cx40del mice). EC-Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in EC-Cx40del mice after 10 weeks high-cholesterol diet; lipid deposition in the thoracic-abdominal aortas was 21.4±3.5% (N=10, p<0.01) in EC-Cx40del mice as compared to 12.6±1.3% for Tie2CRE+ Cx40wt/wt ApoE−/− and 7.1±1.3% for Tie2CRE- Cx40fl/fl ApoE−/− controls. Moreover, spontaneous lesions were observed in the aortic sinuses of young EC-Cx40del mice without diet. These lesions showed monocyte infiltration into the intima, increased expression of VCAM-1 and decreased expression of the ecto-enzyme CD73 in the endothelium. The pro-inflammatory phenotype of EC-Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. The number of leukocytes that migrated to the alveolar space in response to intratracheal instillation of LPS was markedly enhanced (p<0.001) in EC-Cx40del mice compared to controls, both 3 and 6 hours after the treatment. Endothelial CD73 is known to induce anti-adhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with siRNA or anti-sense decreased CD73 expression and activity, and increased leukocyte adhesion to mouse endothelial cells (p<0.01). These effects were reversed by an adenosine receptor agonist. A control siRNA or Cx40 sense had no effect on leukocyte adhesion. We suggest that Cx40-mediated gap junctional communication contributes to a quiescent non-activated endothelium by propagating adenosine-evoked anti-inflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Attenuation of lipopolysaccharide‐induced acute lung injury by treatment with IL‐10

The aim of this study was to characterize the changes in neutrophils and cytokines in BAL fluid following acute lung injury (ALI), and to determine the protective effect of post-injury treatment with IL-10. A rat model of ALI was established by evenly spraying LPS (16 mg/kg) into the lungs followed by observation for 48 h. Histological changes and the kinetics of neutrophil infiltration were evaluated in the injured lungs. The cytokines (TNF-alpha, IL-6, IL-10 and interferon-gamma) and macrophage-inflammatory protein (MIP-2) were measured in BAL fluid by ELISA. The activation of BAL fluid neutrophils was investigated after treatment with IL-10 in vitro. The protective effect on histology and MIP-2 levels of intra-tracheal instillation of IL-10 12 and 16 h after LPS treatment was studied in vivo. Intra-tracheal instillation of LPS caused significant lung injury and the activation of neutrophils. The levels of TNF-alpha and IL-6 in BAL fluid peaked at 8 and 16 h after LPS instillation respectively. IL-10 levels reached a maximum at 16-24 h, at the beginning of resolution of tissue injury. IL-10 inhibited the activation of neutrophils in vitro and MIP-2 induction in vivo. IL-10 had a protective effect if it was administered 12 but not 16 h after LPS. Neutrophils appeared to play an important role in ALI. Time-dependent treatment with IL-10 after intra-tracheal instillation of LPS was effective in protecting rats from ALI, probably by suppressing pulmonary infiltration with activated neutrophils.

Role of p38 mitogen activated protein kinase pathway in attenuation of LPS-induced acute lung injury by Radix Paeoniae Rubra in rats

Objective To investigate the role of p38 mitogen activated protein kinase(MAPK)iNOSI/HO-1 in attenuation of LPS-induced acute lung injury(ALI)by Radix Paeoniae Rubra (RPR) in rats.Methods Forty pathogen-free male Wistar rats weighing 200-250 g were randomly divided into 5 groups(n=8 each):group Ⅰ I control(C);groupⅡLPS;group Ⅲ RPR;group Ⅳ RPR precondtioning and group Ⅴ SB203580 (p38MAPK specific inhibitor).ALI Wag induced by slow intra-tracheal instillation of LPS 2.5 mg/kg in 1 ml of normal saline(NS)in groupⅡ-Ⅴ.BPR 30 mg/kg waft infused iv over 2h simultaneouslv with and at 2 h before intra.tracheal LPS instillation in group Ⅲ and Ⅳ respectively.In groupⅤ SB203580 5,μmol/kg Was infused iv over 2 h at 3 h before intra-tracheal LPS instillation.Arterial blood samples were taken at 6 h after intra-Iracheal LPS instillation for blood gas analysis and determination of serum NO concenwafion.The animals were sacrificed bv exsangulnation.The lunga were immediately removed for microscopic examination and determination of p38MAPK and HO-I and iNOS expression and MDA content in the lung tissue.The left lung was lavaged and broncho- alveolar lavage fluid(BALF)Wag collected for determination of neutrophil count and protein COilcentration.Results LPS intra-tracheal instillation significantly decreased PaO2,PaCO2 and HCO3- concentration and increased serum NO concentration, the number of neutrophils and protein concentration in BALF, and p38MAPK and iNOS and HO-I expression and MDA content in the lung tissue. RPR and RPB preconditioning and SB203580 significandy attenuated the LPS-induced changes in group Ⅲ ,ⅣandⅤ as compared with group Ⅱ . The LPS intratracheal instillation induced pathologic changes of the lung were also attenuated in group ⅢⅣ and Ⅴ.Conclusion RPB can attenuate LPS-indueed ALl through p38MAPK/iNOS/HO-1 signalling pathway. Key words: RADIX PAEONIAE RUBRA; p38 Mitogen-activated protein kinases; Nitric-oxide synthase; Heine oxygenase(decyelizing); Respiratory distress syndrome, adult; Endotoxins

Activation of Rho Kinase Isoforms in Lung Endothelial Cells during Inflammation

Rho kinase (ROCK) is a downstream effector of Rho family GTPases, and two highly homologous isoforms, ROCK1 and ROCK2, are similarly inhibited by the widely used pharmacologic inhibitors. In endothelial cells (ECs), activation of ROCK regulates myosin L chain (MLC) phosphorylation, stress fiber formation and permeability increases during inflammation. This study examined isoform-specific ROCK activation in lung ECs in vitro using human pulmonary microvascular ECs and ex vivo using freshly isolated lung ECs from mice. In unstimulated human as well as mouse lung ECs, ROCK2 activity was greater than ROCK1 activity. TNF-alpha stimulation induced activation of both ROCK1 and ROCK2 in cultured human ECs. Studies using lung ECs freshly isolated from mice showed that intratracheal instillation of LPS induced ROCK activation in lung ECs that was inhibited by treating animals with fasudil, a pharmacologic ROCK inhibitor, and that both ROCK1 and ROCK2 were activated. Small interference RNA targeting ROCK1 or ROCK2 was used to examine their functions in regulating MLC phosphorylation and permeability increases induced by TNF-alpha in human ECs. TNF-alpha-induced MLC phosphorylation required ROCK activation. Inhibition of ROCK1 alone was not sufficient to prevent TNF-alpha-induced MLC phosphorylation, whereas inhibition of ROCK2 prevented TNF-alpha-induced late MLC phosphorylation at 24 h. Although ROCK1 was dispensable for TNF-alpha-induced MLC phosphorylation, ROCK1 was required for TNF-alpha-induced early permeability increases. Therefore, ROCK1 and ROCK2 are both activated by TNF-alpha and can be functionally separated in the signaling pathways leading to TNF-alpha-induced MLC phosphorylation and permeability increases.

Macrolides attenuate mucus hypersecretion in rat airways through inactivation of NF‐κB

To examine the effect of a 14-membered ring macrolide on airway mucus hypersecretion in rats treated with LPS. Mucus hypersecretion in rat airways was induced by intratracheal instillation of LPS. Rats treated with or without LPS were administered roxithromycin (1-10 mg/kg), josamycin (10 mg/kg) or amoxicillin (40 mg/kg), orally for 4 days. Expression of Muc5ac, nuclear factor (NF)-kappaB, and the mitogen-activated protein (MAP) kinases p38 and ERK1/2 in bronchial epithelium were detected by RT-PCR, immunohistochemistry or western blotting. Mucins, IL-1beta, IL-8 and tumour necrosis factor (TNF)-alpha in BAL fluid were assayed by enzyme-linked lectin assay and ELISA. LPS significantly induced the expression of Muc5ac mRNA and protein in bronchial epithelium, increased the release of mucins, IL-1beta, IL-8 and TNF-alpha, and increased neutrophil numbers in BAL. Moreover, LPS increased staining for NF-kappaB in the cytoplasm as well as nuclear translocation of NF-kappaB in airway epithelial cells. Upregulated expression of Muc5ac mRNA correlated positively with NF-kappaB activation and the levels of cytokines (P < 0.05). Roxithromycin (5 and 10 mg/kg) significantly attenuated bronchial Muc5ac expression and NF-kappaB nuclear translocation stimulated by LPS, and reduced neutrophil numbers, mucins and inflammatory cytokines in BAL (P < 0.05). However, LPS-stimulated expression of p38 and ERK1/2 in airway epithelium was not affected by roxithromycin. Josamycin and amoxicillin had no effects on Muc5ac expression, NF-kappaB activation or cytokine release. Roxithromycin inhibits the pulmonary inflammatory response and airway mucus hypersecretion induced by LPS. The inhibitory effect of roxithromycin on airway mucus hypersecretion may be mediated through reduction of NF-kappaB activation, neutrophil infiltration and release of inflammatory cytokines in the lung.