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Patients who developed acute respiratory distress syndrome (ARDS) after infection with severe respiratory viruses (e.g., severe acute respiratory syndrome-coronavirus, H5N1 avian influenza virus), exhibited unusually high levels of CXCL10, which belongs to the non-ELR (glutamic-leucine-arginine) CXC chemokine superfamily. CXCL10 may not be a bystander to the severe virus infection but may directly contribute to the pathogenesis of neutrophil-mediated, excessive pulmonary inflammation.We investigated the contribution of CXCL10 and its receptor CXCR3 axis to the pathogenesis of ARDS with nonviral and viral origins.We induced nonviral ARDS by acid aspiration and viral ARDS by intratracheal influenza virus infection in wild-type mice and mice deficient in CXCL10, CXCR3, IFNAR1 (IFN-α/β receptor 1), or TIR domain-containing adaptor inducing IFN-β (TRIF).We found that the mice lacking CXCL10 or CXCR3 demonstrated improved severity and survival of nonviral and viral ARDS, whereas mice that lack IFNAR1 did not control the severity of ARDS in vivo. The increased levels of CXCL10 in lungs with ARDS originate to a large extent from infiltrated pulmonary neutrophils, which express a unique CXCR3 receptor via TRIF. CXCL10-CXCR3 acts in an autocrine fashion on the oxidative burst and chemotaxis in the inflamed neutrophils, leading to fulminant pulmonary inflammation.CXCL10-CXCR3 signaling appears to be a critical factor for the exacerbation of the pathology of ARDS. Thus, the CXCL10-CXCR3 axis could represent a prime therapeutic target in the treatment of the acute phase of ARDS of nonviral and viral origins.

PLoS one 10(3), e0120802 (2015). doi:10.1371/journal.pone.0120802, Published by PLoS, Lawrence, Kan.

The unique voltage- and Ca2+-dependent K+ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased open-probability of voltage-gated Ca2+ channels. While carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially more dependent on L-type Ca2+ current in mutant than in WT trachea, even though L-type Ca2+ channels were not up-regulated. In the absence of L-type Ca2+ current, half-maximal contraction of trachea was shifted from 0.51 to 1.7 microM. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated-perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline-mediated airway relaxation was enhanced in mutants. In-depth analysis of cAMP and cGMP signaling revealed up-regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up-regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway tone.

Background Patients with multiple organ failure (MOF) require mechanical ventilation for several days. The enormous significance of the ventilation strategy for the outcome of these patients is well appreciated. However, most studies have focused on the onset and the early phase of MOF. It was the aim of the current study to investigate the effect of ventilation in the course of MOF. Methods Using a model where mice develop MOF 7-14 days after intraperitoneal injection of zymosan, the authors analyzed lung functions, signaling pathways, and mediator release in response to protective ventilation (end-expiratory pressure -3 cm H2O; end-inspiratory pressure -10 cm H2O) and overventilation (-22.5 cm H2O) in isolated lungs ex vivo. Results On day 7, pulmonary compliance, pulmonary resistance, and tidal volume were normal, but vascular resistance was elevated compared with untreated animals. During ex vivo ventilation, these lungs showed enhanced nuclear factor-kappaB activation, Akt kinase phosphorylation, and cytokine release, and this was further aggravated by overventilation. After 14 days, zymosan-treated animals were characterized by pulmonary hypertension, reduced tidal volume, elevated pulmonary resistance, and increased mediator production. However, in these lungs, neither nuclear factor-kappaB activation nor cytokine production where enhanced by overventilation. Conclusions The zymosan model is characterized by pulmonary inflammation, diminished lung functions, and chronic hypertension. Mechanical ventilation with high distending pressures further augmented cytokine production in this chronic model of MOF only if it significantly augmented tidal volume. The authors speculate that these findings may be explained on the basis of different degrees of lung stretch.

Pneumonitis is a dose-limiting side effect of radiotherapy. However, the underlying mechanisms of irradiation-induced pneumonitis are unclear. Several observations suggest that the CD95/CD95-ligand (CD95L) system is involved in this process. Therefore, we examined the development of pneumonitis in CD95- and CD95L-deficient and wild-type mice after single irradiation with 0 or 12.5 Gy by measuring breathing frequency, pulmonary resistance, and histopathologic changes. Although wild-type mice developed pathognomonic alterations characteristic of pneumonitis (judged by alveolar wall thickness, interstitial edema, and interstitial and peribronchial inflammation) that paralleled increased breathing frequency ratio on days 5-70 (P < .03) with a maximum at day 37 (12.5 Gy, mean ratio = 1.05, 95% confidence interval [CI] = 1.01 to 1.08; P = .004 versus 0 Gy, mean ratio = 0.997, 95% CI = 0.976 to 1.02; P = .05) and pulmonary resistance (day 42, 12.5 Gy, mean = 0.51, 95% CI = 0.44 to 0.58 versus 0 Gy, mean = 0.40, 95% CI = 0.32 to 0.47; P = .03) after irradiation, no such changes were detected in CD95- or CD95L-deficient mice. This report demonstrates for the first time, to our knowledge, that the CD95/CD95L system is important for the development of irradiation-induced pneumonitis.

Background Allergic asthma typically shows activated, allergen-specific CD4 + T cells in the early phase and airway remodeling in the late phase of the disease. Although TGF-β plays a crucial role in airway remodeling, it is only marginally induced in CD4 + T cells in the early allergen-dependent activation of the immune system. Objective To elucidate the transition between early- and late-phase events, we investigated the role of activin A, a close family member of TGF-β. Methods Activin A and TGF-β 1 levels were measured systemically in the serum and in CD4 + T cells of asthmatic patients, as well as locally in the lung. Results Activin A serum levels were increased in patients with severe asthma compared with levels in patients with moderate asthma and healthy control subjects, whereas all patients showed significantly increased TGF-β 1 serum levels independent of disease severity. In T cells only patients with moderate asthma showed increased activin A mRNA expression, whereas TGF-β 1 expression was equal to that seen in healthy subjects. Accordingly, ovalbumin sensitization in a mouse model of allergic asthma could induce activin A mRNA expression, but not TGF-β 1 expression, in the lung. Immunohistochemistry of mice and human specimens revealed an abundant expression of activin A by infiltrating lymphocytes and structural cells of the lung. Although TGF-β 1 more potently enhanced proliferation and Smad 2/3–dependent reporter genes in fibroblasts, activin A was capable of inducing TGF-β 1 and vice versa. Conclusion Activin A provides a link between acute allergen-specific T-cell responses and chronic TGF-β 1 –mediated airway remodeling in asthma.

Acute and chronic lung inflammation is driven and controlled by several endogenous mediators that undergo proteolytic conversion from surface-expressed proteins to soluble variants by a disintegrin and metalloproteinase (ADAM)-family members. TNF and epidermal growth factor receptor ligands are just some of the many substrates by which these proteases regulate inflammatory or regenerative processes in the lung. ADAM10 and ADAM17 are the most prominent members of this protease family. They are constitutively expressed in most lung cells and, as recent research has shown, are the pivotal shedding enzymes mediating acute lung inflammation in a cell-specific manner. ADAM17 promotes endothelial and epithelial permeability, transendothelial leukocyte migration, and inflammatory mediator production by smooth muscle and epithelial cells. ADAM10 is critical for leukocyte migration and alveolar leukocyte recruitment. ADAM10 also promotes allergic asthma by driving B cell responses. Additionally, ADAM10 acts as a receptor for Staphylococcus aureus ( S. aureus) α-toxin and is crucial for bacterial virulence. ADAM8, ADAM9, ADAM15, and ADAM33 are upregulated during acute or chronic lung inflammation, and recent functional or genetic analyses have linked them to disease development. Pharmacological inhibitors that allow us to locally or systemically target and differentiate ADAM-family members in the lung suppress acute and asthmatic inflammatory responses and S. aureus virulence. These promising results encourage further research to develop therapeutic strategies based on selected ADAMs. These studies need also to address the role of the ADAMs in repair and regeneration in the lung to identify further therapeutic opportunities and possible side effects.

Ischemia-Reperfusion (I/R) injury after lung transplantation (LTx) can lead to significant morbidity and mortality in recipients. In an attempt to improve our understanding of molecular mechanisms of I/R injury, we examined the changes in gene expression levels in a rat lung transplant model using oligonucleotide microarrays. Lewis rat lung grafts were stored for 6 or 24 h followed by transplantation and reperfusion for 2 h. Lung tissues were taken before and after flushing the grafts, before implantation, and after 2 h of reperfusion. RNA samples were examined with Affymetrix rat microarray chips and RT-PCR was performed to validate significant changes in gene expression. Microarray analysis showed 404 genes that were up-regulated more than 2-fold after reperfusion compared to cold ischemic lungs, and 187 genes that were down-regulated. Using RT-PCR, we confirmed the response pattern of several specific gene transcripts from the microarray analysis. Among these, up-regulation in transcripts of transcription factors, adhesion molecules, pro-coagulant factors and pro-inflammatory cytokines were identified. The differential gene regulation during the I/R process can be considered as molecular signatures for the changes of cellular metabolism, functions and injury. Reperfusion-induced genes related to inflammatory response may contribute to graft dysfunction in LTx.

Ceramide is a lipid second messenger that was recently identified as mediator of pulmonary edema in vivo. Here, we investigated the effect of ceramide on the permeability of confluent endothelial cell monolayers. In monolayers of bovine pulmonary artery and human microvascular pulmonary endothelial cells, incubation with C6-ceramide for 3 h elevated permeability in a concentration-dependent manner, whereas dihydroceramide was without effect. After 3 h of incubation with ceramide, we found no signs of necrosis (release of lactate dehydrogenase, loss of thiazylyl blue reduction) or apoptosis (ssDNA, caspase-8 activity). The increased endothelial permeability in response to ceramide was attenuated by the Ser/Thr protein kinase inhibitors K252a, K252b and H-7, as well as by the phosphatidylinositol-specific phospholipase C inhibitor L108. Since in some systems sphingosine-1-phosphate (S1P) acts antagonistic to ceramide, the effect of S1P was studied. S1P transiently increased endothelial cell resistance, whether it was given together with ceramide or 90 min thereafter. These data provide a novel example of the antagonism between S1P and ceramide. Our findings further suggest that ceramide alters vascular permeability by activation of pathways dependent on unidentified phospholipase C and Ser/Thr kinase isoenzymes.

Recent evidence suggests that alveolar epithelial cells (AECs) may contribute to the development, propagation, and resolution of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Proinflammatory cytokines, pathogen products, and injurious mechanical ventilation are important contributors of excessive inflammatory responses in the lung. In the present study, we used cDNA microarrays to define the gene expression patterns of A549 cells (an AEC line) in the early stages of three models of pulmonary parenchymal cell activation: cells treated with tumor necrosis factor-alpha (TNFalpha) (20 ng/ml), lipopolysaccharide (LPS, 1 microg/ml), or cyclic stretch (20% elongation) for either 1 h or 4 h. Differential gene expression profiles were determined by gene array analysis. TNFalpha induced an inflammatory response pattern, including induction of genes for chemokines, inflammatory mediators, and cell surface membrane proteins. TNFalpha also increased genes related to pro- and anti-apoptotic proteins, signal transduction proteins, and transcriptional factors. TNFalpha further induced a group of genes that may form a negative feedback loop to silence the NFkappaB pathway. Stimulation of AECs with mechanical stretch changed cell morphology and activated Src protein tyrosine kinase. The combination of TNFalpha plus stretch enhanced or attenuated expression of multiple genes. LPS decreased microfilament polymerization but had less impact on NFkappaB translocation and gene expression. Results from this study indicate that AECs can tailor their response to different stimuli or/and combination of stimuli and subsequently play an important role in acute inflammatory responses in the lung.

Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP3)-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP3receptor (1–10 μM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 μM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 μM ML-7) or of calmodulin kinase kinase (26 μM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 μM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 μM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.

Tumor necrosis factor-alpha (TNF-alpha) plays an important role in innate immunity. Recent in vitro studies have shown that TNF-alpha may also serve as a growth factor for some bacteria. We examined the physiologic relevance of this phenomenon both in vitro and in vivo. Recombinant mouse TNF-alpha increased in vitro proliferation of Escherichia coli but not Pseudomonas aeruginosa in a concentration-dependent manner, and this effect was attenuated by anti-TNF-alpha antibodies. However, in vivo, TNF-alpha gene-deficient (TNF-alpha-/-) mice showed higher mortality than wild-type (TNF-alpha+/+) mice after inoculation of intranasal bacteria. An impaired bacterial clearance in TNF-alpha-/- mice was associated with decreased systemic concentrations of chemokine macrophage inflammatory protein-2, reduced pulmonary neutrophil recruitment, and depressed expression of neutrophil CD11b and CD16/CD32, suggesting that the effect of TNF-alpha on E. coli growth was outweighed by the recruited neutrophils. We also demonstrated that neutropenic TNF-alpha+/+ mice had approximately 100-fold higher E. coli counts in their lungs than TNF-alpha-/- mice, although survival rates in both groups were similar. We conclude that TNF-alpha augments E. coli growth in vitro and in vivo. However, in vivo, this effect becomes only apparent in neutropenic animals. The relevance of these findings for immune compromised patients remains to be investigated.

Summary In vivo experiments showed no increased production of tumour necrosis factor (TNF) in response to injurious ventilation strategies in otherwise untreated animals. Because interleukin-6 (IL-6) and macrophage inflammatory protein-2 (MIP-2) are more sensitive markers of ventilation-induced cytokine release, serum and bronchoalveolar lavage (BAL) samples were examined for these mediators. Eighty-five adult rats were randomized to three different ventilation strategies. Rats were ventilated with low pressures and low tidal volumes [13/3; peak inspiratory pressure (PIP)/positive end-expiratory pressure (PEEP) in cmH2O], the second group of rats was ventilated with high pressures and low PEEP resulting in high tidal volumes (32/6), and the third group was ventilated with the same high pressures but without PEEP (32/0). Animals were ventilated either for 90 or 240 min, subsequently serum and BAL were collected for analyses on IL-6 and MIP-2 content. Non-ventilated animals served as healthy controls. Ventilation with 32/0 for 90 or 240 min, led to increased serum IL-6 levels. Serum MIP-2 levels were increased by ventilation with 32/6 (90 min) and 32/0 (240 min). Ventilation under any condition, even at 13/3, resulted in elevated MIP-2 levels in the BAL fluid. Even at normal pressures pulmonary MIP-2 levels were increased, suggesting that ventilation may promote pro-inflammatory responses in healthy subjects.

Mainly among children incidental ingestion of conventional lamp oils (paraffin oil) is responsible for casual intoxications with severe pulmonary toxicity and fatal consequences. On the basis of the isolated blood-free perfused rat lung we developed a model to study the acute toxic effects of lamp oil. Three oils were studied: conventional paraffin lamp oil (kinematic viscosity 1.62 10(-6) m(2)/sec), the methyl ester Edenor ME C12 70 (2.7 10(-6) m(2)/s) and another ester Edenor LPL (4.5 10(-6) m(2)/s). The oils were instilled into the trachea of isolated rat lungs and the changes in lung mechanics (tidal volume, pulmonary compliance and pulmonary conductance) as well as edema formation (increase in lung weight) studied. Instillation of as little as 10 microl paraffin oil caused complete failure of lung functions within 20 min and even 2 microl caused noticeable edema. Similar results were obtained with Edenor ME C12 70, which appeared to be even more toxic than paraffin oil, while Edenor LPL was less toxic. We conclude that tracheal instillation of oils into isolated perfused rat lungs may be a useful model to study the toxicity of lamp oils in vitro. Our findings further suggest that Edenor LPL may be a safer alternative for use as a lamp oil than paraffin oil.

The human acute respiratory distress syndrome (ARDS) is a severe pulmonary complication with high mortality rates. To support their vital functions, patients suffering from ARDS are mechanically ventilated. Recently it was shown that low tidal volume ventilation reduces mortality and pro-inflammatory mediator release in these patients, suggesting biotrauma as a side effect of mechanical ventilation. Because the application of exogenous surfactant has been proposed as a treatment for ARDS, we investigated the effect of surfactant on ventilation-induced release of tumor necrosis factor (TNF), interleukin-6 (IL-6) and 6-keto-PGF(1 alpha) (the stable metabolite of prostacyclin) in isolated perfused mouse lungs ventilated with high end-inspiratory pressures. Instillation of 100mg/kg surfactant into the lungs was well tolerated and improved tidal volume, pulmonary compliance and alveolar expansion. Exogenous surfactant increased the ventilation-induced liberation of TNF and IL-6 into the perfusate, but had no effect on the release of 6-keto-PGF(1 alpha). The surfactant preparation used reduced baseline TNF production by murine alveolar macrophages, indicating that the exaggeration of ventilation-induced TNF release cannot be explained by a direct effect of surfactant on these cells. We hypothesize that ventilation-induced mediator release is explained by stretching of lung cells, which is reinforced by surfactant. The findings that in this model of ventilation-induced lung injury exogenous surfactant at the same time improved lung functions and enhanced mediator release suggest that surfactant treatment may prevent barotrauma and augment biotrauma.

Platelet-activating factor (PAF) is an important endogenous mediator of pulmonary edema in many models of acute lung injury. PAF triggers edema formation by simultaneous activation of two independent pathways; one is mediated by a cyclooxygenase metabolite, and the other is blocked by quinine. We examined the hypothesis that the cyclooxygenase-dependent part of PAF-induced edema is mediated by prostaglandin E(2) (PGE(2)). In isolated rat lungs, PAF administration stimulated release of PGE(2) into the venous effluate and increased lung weight as a measure of edema formation. Perfusion with a neutralizing PGE(2) antibody attenuated the PAF-induced edema formation. In vivo, E-prostanoid 3-receptor-deficient mice showed less pulmonary Evans blue extravasation in response to PAF injection than did mice deficient in EP1, EP2, or EP4 receptors. Perfusion of rat lungs with PGE(2) caused pulmonary edema, which was largely prevented by inhibition of voltage-gated potassium channels (25 nM beta-dendrotoxin), but not by blocking calcium-dependent potassium currents (100 micro M paxilline). In line with its effects on PGE(2)-induced edema formation, beta-dendrotoxin attenuated PAF-induced edema partly if given alone, and completely in combination with quinine. Our findings suggest that PAF-triggered edema is partly mediated by the release of PGE(2), activation of EP3 receptors, and activation of voltage-gated potassium channels.

Objective. To determine the effect of pretreatment with exogenous surfactant on ventilator-induced decompartmentalization of TNF-α. Design and setting. Prospective, randomized, animal study in the experimental laboratory of a university. Subjects and interventions. Male Sprague-Dawley rats (n=102) received lipopolysaccharide either intratracheally or intraperitoneally to stimulate TNF-α production; one-half of the animals were pretreated with surfactant. Animals were ventilated for 20 min with a peak inspiratory pressure/positive end-expiratory pressure (PEEP) ratio of either 45/0 or 45/10 (frequency 30 bpm, I/E ratio 1:2, FIO2=1). Measurements and results. Blood gas tension and arterial pressures were recorded 1, 10, and 20 min after the start of mechanical ventilation. After the animals were killed pressure-volume curves were recorded, and bronchoalveolar lavage was performed for assessment of protein content and the small/large surfactant aggregate ratio. TNF-α was determined in serum and bronchoalveolar lavage. Pretreatment with surfactant decreased decompartmentalization of TNF-α during 45/0 ventilation. Addition of a PEEP level of 10 cm H2O reduced decompartmentalization even further. In addition, surfactant prevented deterioration in oxygenation and decreased accumulation of protein in the bronchoalveolar lavage in the zero-PEEP group. Conclusions. An excess of active surfactant decreases transfer of cytokines across the alveolar-capillary membrane similar to PEEP. The combination of PEEP and surfactant reduces decompartmentalization of TNF-α even further.

Inhibitors of phosphodiesterase 4 (PDE4) possess bronchospasmolytic and anti-inflammatory properties, which make them very attractive drugs for the treatment of asthma and COPD. Unfortunately, many PDE4 inhibitors also produce central nervous and gastrointestinal side effects, which have limited their clinical application. PDE4 has two binding sites for the archetypal PDE4 inhibitor rolipram, and it has been suggested that binding to the high-affinity rolipram binding site (HARBS) is responsible for the side effects of PDE4 inhibitors. Recently, we have synthesised the PDE4 inhibitor CC3 which shows low affinity to the HARBS. In the present study we investigated the bronchospasmolytic and anti-inflammatory properties of this novel compound in comparison to rolipram and the PDE3 inhibitor motapizone. The airway-relaxant properties of the PDE inhibitors were analysed in rat precision-cut lung slices (PCLS) in which airways were contracted by methacholine or in passively sensitised PCLS exposed to ovalbumin. The anti-inflammatory properties were investigated by measuring the release of TNF from endotoxin-treated human monocytes.Up to concentrations of 10 microM none of the PDE inhibitors significantly affected bronchoconstriction elicited by 10 microM methacholine. However, if rolipram or CC3 were given in combination with motapizone, methacholine-induced bronchoconstriction was concentration-dependently attenuated. Allergen-induced bronchoconstriction in passively sensitised PCLS was attenuated by CC3 (IC(50) 2.7 microM), rolipram (0.23 microM) and motapizone (8 microM). Combination of equimolar concentrations of motapizone and CC3 (0.34 microM) or rolipram (0.005 microM) showed an additive effect. Endotoxin-induced TNF release from human monocytes was attenuated by all three PDE inhibitors, i.e. CC3 (IC(50) 4.6 microM), rolipram (0.18 microM) and motapizone (5.8 microM). Our findings suggest that PDE4 inhibitors with only low affinity for the HABRS have bronchospasmolytic and anti-inflammatory properties.

Platelet activating factor (PAF) induces vascular barrier breakdown and intestinal failure that contribute to the development of sepsis. The exact cellular mechanisms are not well understood.

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are formed simultaneously under inflammatory conditions such as asthma and acute respiratory distress syndrome. Here we investigated the effects of TNF-alpha (10 ng/ml) and/or IL-1beta (10 ng/ml) in isolated blood-free perfused rat lungs. In lungs precontracted with methacholine, IL-1beta alone and IL-1beta/TNF-alpha decreased airway resistance 10 min after administration, whereas TNF-alpha alone had no effect. In untreated lungs, airway resistance was unaltered by either cytokine alone but started to increase 40 min after treatment with both cytokines together, indicating bronchoconstriction. The bronchoconstriction was accompanied by a steroid-sensitive increase in cyclooxygenase (COX)-2 mRNA expression and thromboxane formation. The cytokine-induced bronchoconstriction was blocked by the thromboxane receptor antagonist SQ-29548, indomethacin, the selective COX-2 inhibitor NS-398, and the steroid dexamethasone. We conclude that IL-1beta has an early bronchodilatory effect (after 10 min) that is unchanged by TNF-alpha. However, at later time points (after 40 min), IL-1beta and TNF-alpha in concert cause a COX-2- and thromboxane-dependent bronchoconstriction. Our findings show that TNF-alpha and IL-1beta exert complex and time-dependent effects on lung functions that cannot be predicted by studying each cytokine alone.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is used to ameliorate neutropenia in patients after antineoplastic treatment. It has also been suggested as an adjunct treatment in septic patients; however, the recruitment and priming of leukocytes by GM-CSF bears the hazard of a hyperinflammatory response. In particular, the role of GM-CSF in pulmonary functions in septic lungs is still unclear. Therefore, we pretreated rats in vivo with GM-CSF (50 microg/kg, intravenous) and assessed the pulmonary functions of their subsequently prepared isolated perfused lungs when exposed to subtoxic concentrations of lipopolysaccharide (LPS, 2 microg/ml). These lungs showed enhanced expression of cyclooxygenase 2 (COX-2), a significant increase in thromboxane (TX) and tumor necrosis factor (TNF) release into the venous perfusate, and bronchoconstriction. COX-2 inhibition or blocking of the TX receptor abolished the GM-CSF/LPS-induced bronchoconstriction, but not the TNF release. Neutralizing antibodies against TNF did not prevent GM-CSF/LPS-induced bronchoconstriction. After GM-CSF pretreatment, massive neutrophil invasion into the lung occurred. Neutropenic rats were protected against GM-CSF/ LPS-induced lung injury. Similar results were obtained in rats pretreated with G-CSF instead of GM-CSF. We conclude that GM-CSF pretreatment exacerbates pulmonary injury by low-dose LPS via COX-2 expression, TX release, and bronchoconstriction by initiating neutrophil invasion and activation.

In acute pulmonary inflammation, danger is first recognized by epithelial cells lining the alveolar lumen and relayed to vascular responses, including leukocyte recruitment and increased endothelial permeability. We supposed that this inflammatory relay critically depends on the immunological function of lung interstitial cells such as smooth muscle cells (SMC). Mice with smooth muscle protein-22α promotor-driven deficiency of the disintegrin and metalloproteinase (ADAM) 17 (SM22-Adam17−/−) were investigated in models of acute pulmonary inflammation (LPS, cytokine, and acid instillation). Underlying signaling mechanisms were identified in cultured tracheal SMC and verified by in vivo reconstitution experiments. SM22-Adam17−/− mice showed considerably decreased cytokine production and vascular responses in LPS- or acid-induced pulmonary inflammation. In vitro, ADAM17 deficiency abrogated cytokine release of primary SMC stimulated with LPS or supernatant of acid-exposed epithelial cells. This was explained by a loss of ADAM17-mediated growth factor shedding. LPS responses required ErbB1/epidermal growth factor receptor transactivation by TGFα, whereas acid responses required ErbB4 transactivation by neuregulins. Finally, LPS-induced pulmonary inflammation in SM22-Adam17−/− mice was restored by exogenous TGFα application, confirming the involvement of transactivation pathways in vivo. This highlights a new decisive immunological role of lung interstitial cells such as SMC in promoting acute pulmonary inflammation by ADAM17-dependent transactivation.

Two new ether lipids, 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine and its lyso form, 1-O-alkyl/alkenyl-glycero-3-phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC-MS, MALDI-TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1′ (plasmalogen-type lipid) or 9′ in a ratio ≈ 1 : 1. This is the first report of the presence of alkyl and vinyl (alk-1′-enyl) ether lipids in the cell membrane of aerobically grown mycoplasmas. Lipids of this type have been found in some Gram-positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2-O-acetylated lyso form lipid can mimic platelet-activating factor activity in isolated perfused and ventilated rat lungs.

Endothelins are a family of potent endogenous mediators that have been implicated in a number of airway and other diseases. Recently, the non-peptide mixed ET(A)/ET(B) endothelin receptor antagonist bosentan has been successfully tested in the treatment of cardiovascular diseases. It was the aim of the present study to characterize the effects of bosentan on the pulmonary actions of endothelin- (ET-1), endothelin-3 (ET-3) and the ET(B)-receptor agonist IRL1620 in the isolated perfused and ventilated rat lung (IPL) and in precision-cut lung slices (PCLS). In the IPL, bosentan completely prevented the IRL1620-induced vasoconstriction (IC50 3 microM). The inhibition by bosentan of ET-1-elicited vasoconstriction showed a biphasic course, reflecting the inhibition of ET(A)-and ET(B)-mediated vasoconstriction (IC50 0.2 microM and 19 microM, respectively). In addition, bosentan prevented the ET-1- (IC50 6 microM) and IRL1620-induced (IC50 3 microM) prostacyclin release. Bosentan also completely prevented the bronchoconstriction induced by IRL1620 in the IPL (IC50 20 microM) and in PCLS (IC50 13 microM). In PCLS, the pD2-values were ET-1 7.20+/-0.23, ET-3 7.51+/-0.27 and IRL1620 7.33+/-0.29. Bosentan at 100 microM caused a rightward shift of the concentration-response curve of ET-1, ET-3 and IRL1620 by a factor of 5, 46 and 64, respectively. In all cases the slope of the Schild regression was lower than unity, disregarding a simple interaction of bosentan with one receptor. With respect to ET-1-induced bronchoconstriction, in the IPL bosentan in concentrations of up to 10 microM aggravated ET-1-induced bronchoconstriction probably due to the blockade of bronchodilatory ET(A)-receptors (IC50 0.3 microM) and even at 100 microM showed only very little protection from ET- -induced bronchoconstriction in the IPL and in the PCLS. The similar IC50-values for ET-1-induced vasoconstriction and bronchodilation suggest that only one type of ET(A)-receptor is involved. The differing IC50-values between IRL1620-induced bronchoconstriction and prostacyclin release, the slope of the Schild regression and the failure of bosentan to prevent the ET-1-induced bronchoconstriction suggest a complex interaction between the known ET-receptors or the existence of unknown ET(B)-receptor subtypes.

Little is known about interstrain variations in baseline lung functions or smooth muscle contractility in murine lungs. We therefore examined basal lung mechanics and airway, as well as vascular reactivity to methacholine, thromboxane (using U-46619), and endothelin-1 (ET-1), A/J, AKR, BALB/c, C3H/HeN, C57BL/6, and SCID mice. All experiments were performed with isolated perfused mouse lungs. Except AKR mice (which were excluded from further analysis), all other strains showed stable pulmonary compliance, pulmonary resistance, and pulmonary arterial pressure within a control period of 45 min. Among these strains, C3H/HeN mice exhibited higher dynamic pulmonary compliance and lower pulmonary resistance, whereas SCID mice had higher baseline pulmonary resistance than the other strains. Concentration-response experiments with methacholine showed a lower airway reactivity for C57BL/6 mice compared with the other strains. Perfusion with 1 μM U-46619 or 100 nM ET-1 revealed a similar pattern: the agonist-inducible broncho- and vasoconstriction was lower in C57BL/6 mice than in all other strains, whereas it tended to be higher in SCID mice. The present study demonstrates a correlation between airway and vascular responsiveness in all tested strains. SCID mice are hyperreactive, whereas C57BL/6 mice are hyporeactive, to smooth muscle constrictors. Lung mechanics, as well as airway and vascular responsiveness, appear to be genetically controlled.

Objectives: To determine the effect on compartmentalization of the tumor necrosis factor (TNF)-α response in the lung and systemically after ventilation with high peak inspiratory pressure with and without positive end-expiratory pressure (PEEP). Design and setting: Prospective, randomized, animal study in an experimental laboratory of a university. Subjects and interventions: 85 male Sprague-Dawley rats. Lipopolysaccharide was given intratracheally or intraperitoneally to stimulate TNF-α production; control animals received a similar amount of saline. Animals were subsequently ventilated for 20 min in a pressure control mode with peak inspiratory pressure/PEEP ratio of either 45/0 or 45/10 (frequency 30 bpm, I/E ratio 1:2, FIO2 = 1). Measurements and results: Blood gas tension and arterial pressures were recorded at 1, 10, and 20 min after start of mechanical ventilation. After killing of the animals pressure-volume curves were recorded, and bronchoalveolar lavage (BAL) was performed for assessment of protein content and the small/large surfactant aggregate ratio. TNF-α was determined in serum and BAL. TNF-α levels were significantly increased after lipopolysaccharide stimulation; furthermore ventilation without PEEP resulted in a significant shift of TNF-α to the nonstimulated compartment as opposed to ventilation with a PEEP level of 10 cmH2O. Conclusions: Ventilation strategies which are known to induce ventilation-induced lung injury (VILI) disturb the compartmentalization of the early cytokines response in the lung and systemically. Furthermore, the loss of compartmentalization is a two-way disturbance, with cytokines shifting from the vascular side to the alveolar side and vice versa. A ventilation strategy (PEEP level of 10 cmH2O) which prevents VILI significantly diminished this shift in cytokines.

In the present study we have investigated the mechanisms of pulmonary edema caused by platelet-activating factor (PAF) in isolated rat lungs as well as in mice in vivo. In blood-free perfused and ventilated rat lungs, PAF increased lung weight by 0.59 +/- 0.18 g. The cyclooxygenase inhibitor aspirin (500 microM) blocked this response by one-third, and the quinolines quinine (330 microM), quinidine (100 microM), and chloroquine (100 microM) by two-thirds. Lipoxygenase inhibition (10 microM AA861) alone or in combination with thromboxane receptor antagonism (10 microM SQ29548) had no effect on PAF-induced weight gain. In combination with aspirin, quinine or quinidine completely prevented PAF-induced weight gain and the concomitant increase of the capillary filtration coefficient (K(f,c)). Pretreatment with quinine in vivo prevented not only PAF-, but also endotoxin-induced edema formation as assessed by Evans Blue extravasation. In addition, in vivo quinine prevented the endotoxin-induced release of tumor neurosis factor (TNF). Furthermore, in perfused lungs quinine reduced the PAF-induced increases in airway and vascular resistance, as well as thromboxane release. These findings demonstrate the following anti-inflammatory properties of quinolines: reduction of thromboxane and TNF formation; reduction of PAF-induced vasoconstriction and bronchoconstriction; and attenuation of PAF- and lipopolysaccharide (LPS)-induced edema formation. We conclude that the PAF- induced edema consists of two separate mechanisms, one dependent on an unknown cyclooxygenase metabolite, the other one sensitive to quinolines.

We characterized the responses of murine airways and pulmonary vessels to a variety of endogenous mediators in the isolated perfused and ventilated mouse lung (IPL) and compared them with those in precision-cut lung slices. Airways: The EC50 (μM) for contractions of airways in IPL/slices was methacholine (Mch), 6.1/1.5>serotonin, 0.7/2.0>U46619 (TP-receptor agonist), 0.1/0.06>endothelin-1, 0.1/0.05. In the IPL, maximum increase in airway resistance (RL) was 0.6, 0.4, 0.8 and 11 cmH2O s ml−1, respectively. Adenosine (⩽1 mM), bombesin (⩽100 μM), histamine (⩽10 mM), LTC4 (⩽1 μM), PAF (0.25 μM) and substance P (⩽100 μM) had only weak effects (U46619, 0.05

The aim of this study was to characterize early ultrastructural, biochemical, and functional alterations of the pulmonary surfactant system induced by Salmonella minnesota lipopolysaccharide (LPS) in rat lungs. Experimental groups were: (1) control in vitro, 150 min perfusion; (2) LPS in vitro, 150 min perfusion, infusion of 50 microg/ml LPS after 40 min; (3) control ex vivo, 10 min perfusion; (4) LPS ex vivo, lungs perfused for 10 min from rats treated for 110 min with 20 mg/kg LPS intraperitoneally. Morphometry of type II pneumocytes showed that LPS increased stored surfactant. Lamellar bodies were increased in size, but decreased in numerical density, suggesting that giant lamellar bodies observed in LPS-treated lungs may result from fusion of normal bodies. Structural analysis of alveolar surfactant composition showed that LPS elicited an increase in lamellar body-like and multilamellar forms. Bronchoalveolar lavage (BAL) material from LPS-treated lungs was decreased in phospholipids. BAL bubble surfactometer analysis showed a reduction in hysteresis area caused by LPS. We conclude that LPS leads to alterations of intracellular and alveolar surfactant within 2 h: fusion of lamellar bodies, reduction in surfactant secretion, and changes in alveolar surfactant transformation, composition, and function, which may contribute to the development of respiratory distress.

Artificial mechanical ventilation represents a major cause of iatrogenic lung damage in intensive care. It is largely unknown which mediators, if any, contribute to the onset of such complications. We investigated whether stress caused by artificial mechanical ventilation leads to induction, synthesis, and release of cytokines or eicosanoids from lung tissue. We used the isolated perfused and ventilated mouse lung where frequent perfusate sampling allows determination of mediator release into the perfusate. Hyperventilation was executed with either negative (NPV) or positive pressure ventilation (PPV) at a transpulmonary pressure that was increased 2.5-fold above normal. Both modes of hyperventilation resulted in an approximately 1.75-fold increased expression of tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) mRNA, but not of cyclooxygenase-2 mRNA. After switching to hyperventilation, prostacyclin release into the perfusate increased almost instantaneously from 19 +/- 17 pg/min to 230 +/- 160 pg/min (PPV) or 115 +/- 87 pg/min (NPV). The enhancement in TNFalpha and IL-6 production developed more slowly. In control lungs after 150 min of perfusion and ventilation, TNFalpha and IL-6 production was 23 +/- 20 pg/min and 330 +/- 210 pg/min, respectively. In lungs hyperventilated for 150 min, TNFalpha and IL-6 production were increased to 287 +/- 180 pg/min and more than 1,000 pg/min, respectively. We conclude that artificial ventilation might cause pulmonary and systemic adverse reactions by inducing the release of mediators into the circulation.

Treatment of animals with big endothelin-1 (bET) causes pulmonary hypertension and bronchoconstriction, both in vivo and in perfused lungs. The biological activity of bET requires proteolytic cleavage to ET-1 by endothelin converting enzymes (ECE) and possibly other proteases such as neutral endopeptidase 24.11 (NEP 24.11). Since the role of NEP 24.11 in the physiological activation of bET is unclear, we investigated the effects of the selective NEP 24.11 inhibitor thiorphan on bET-induced vaso- and bronchoconstriction in the isolated perfused rat lung. We also studied the effects of phosphoramidon and (S)-2-biphenyl-4-yl-1-(1H-tetraol-5-yl)-ehtylaminomethylphosphonic acid (CGS-26303), i.e. agents which block not only NEP 24.11 but also ECE. The bET-induced vasoconstriction was much less prominent than the bronchoconstriction, i.e. after exposure for 110 min vascular and airway conductance were decreased by 33% and 80% respectively. The small bET-induced vasoconstriction was attenuated to a similar degree by pretreatment with any of the three protease inhibitors. However, thiorphan up to a concentration of 10 microM had only little effect on the bET-induced bronchoconstriction, while 10 microM phosphoramidon or CGS-26303 provided half-maximal and 100 microM phosphoramidon complete protection in this model. This profile of inhibitor action suggests that in rat lung ECE is the major enzyme responsible for activation of bET.

Gravimetric methods are useful for investigating mechanisms of edema formation. In isolated lungs perfused under isogravimetric conditions, important information may be gained by analyzing the weight changes induced by a sudden change in capillary pressure. In the present study, we investigated the possibility to analyze this weight change by a biexponential equation and use the coefficients obtained to derive vascular compliance (Cv), interstitial compliance (Ci) and the capillary filtration coefficient (Kf,c). Fitting the data of the weight gain to a biexponential curve explained the data significantly better than fitting it to a monoexponential curve, suggesting that two phases can be separated. The first phase is thought to represent vascular filling and was completed for 95% after 0.57 +/- 0.21 min (n = 30). In contrast, 95% completion of phase 2, which relates to interstitial filling, took 18.4 +/- 13.9 min (n = 30). The values obtained for Cv, Ci and Kf,c were 0.064 +/- 0.018 ml/cm H2O, 0.067 +/- 0.030 ml/cm H2O, and 0.588 +/- 0.206 ml/min/cm H2O/100 g lung wet weight, respectively. Treatment of the lungs with platelet activating factor caused no changes in Cv or Ci, but increased Kf,c suggesting increased endothelial permeability. We conclude that analysis of weight changes by a biexponential equation offers a quick and reliable way to analyze factors related to edema formation.

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

Mechanical ventilation (MV) is, by definition, the application of external forces to the lungs. Depending on their magnitude, these forces can cause a continuum of pathophysiological alterations ranging from the stimulation of inflammation to the disruption of cell-cell contacts and cell membranes. These side effects of MV are particularly relevant for patients with inhomogeneously injured lungs such as in acute lung injury (ALI). These patients require supraphysiological ventilation pressures to guarantee even the most modest gas exchange. In this situation, ventilation causes additional strain by overdistension of the yet non-injured region, and additional stress that forms because of the interdependence between intact and atelectatic areas. Cells are equipped with elaborate mechanotransduction machineries that respond to strain and stress by the activation of inflammation and repair mechanisms. Inflammation is the fundamental response of the host to external assaults, be they of mechanical or of microbial origin and can, if excessive, injure the parenchymal tissue leading to ALI. Here, we will discuss the forces generated by MV and how they may injure the lungs mechanically and through inflammation. We will give an overview of the mechanotransduction and how it leads to inflammation and review studies demonstrating that ventilator-induced lung injury can be prevented by blocking pathways of mechanotransduction or inflammation.

PLoS one 9(1), e87685 (2014). doi:10.1371/journal.pone.0087685, Published by PLoS [u.a.], Lawrence, Kan.

Acute lung injury is a life-threatening disease that is characterized by pulmonary inflammation, loss of barrier functions, and hypoxemia. Sphingolipids are critically involved in the disease process that they can both expedite and extenuate: They expedite inflammation by promoting chemotaxis (neutral sphingomyelinase), increased endothelial permeability (acid sphingomyelinase, S1P3-receptors), increased epithelial permeability (S1P2- and S1P3-receptors), and delaying neutrophil apoptosis (neutral sphingomyelinase, S1P1-receptors). They extenuate inflammation by attenuating chemotaxis (S1P) and by stabilizing the endothelial and the epithelial barrier (S1P1-receptor). This chapter discusses the multiple roles and therapeutic options that sphingolipids offer with respect to acute lung injury.

International audience; This paper presents an improved identification method of the constitutive properties of lung parenchyma. We aim to determine the non-linear viscoelastic behavior of lung parenchyma with a particular focus on the compressible properties - i.e. the ability to change volume. Uniaxial tensile tests are performed on living precision-cut rat lung slices. Image registration is used to compute the displacement field at the surface of the sample. The constitutive model consists of a hyperelastic potential split into volumetric and isochoric contributions and a viscous contribution. This allows for the description of the experimentally observed hysteresis loop. The identification is performed numerically: each test is simulated using the realistic geometry of the sample; the difference between the measured and computed displacements is minimized with an optimization algorithm. We compare several hyperelastic potentials and we can determine the most suitable law for rat lung parenchyma. An exponential potential or a polynomial potential with a first order term and a third or higher order term give similarly satisfactory results. The identified parameters are: for the volumetric contribution: κ =7.25e4 Pa, for the exponential form: k1=4.34e3 Pa, k2=5.92, for the polynomial form: C1=2.87e3 Pa, C3=3.83e4 Pa. The identification of the time parameter for the viscous contribution shows that it depends on the loading frequency (0.2 Hz: τ =0.257 s, 0.4 Hz: τ=0.123 s, 0.8 Hz: τ=0.050 s). Adding a viscous contribution significantly increases the accuracy of the identification.

Acute lung injury is a life-threatening disease that is characterized by pulmonary inflammation, loss of barrier functions, and hypoxemia. Sphingolipids are critically involved in the disease process that they can both expedite and extenuate: They expedite inflammation by promoting chemotaxis (neutral sphingomyelinase), increased endothelial permeability (acid sphingomyelinase, S1P3-receptors), increased epithelial permeability (S1P2- and S1P3-receptors), and delaying neutrophil apoptosis (neutral sphingomyelinase, S1P1-receptors). They extenuate inflammation by attenuating chemotaxis (S1P) and by stabilizing the endothelial and the epithelial barrier (S1P1-receptor). This chapter discusses the multiple roles and therapeutic options that sphingolipids offer with respect to acute lung injury.

D -Galactosamine-sensitized mice challenged with tumor necrosis factor α (TNF) developed severe apoptotic and secondary necrotic liver injury as assessed by histology, measurement of cytosolic DNA fragments and determination of liver-specific enzymes in plasma. Pretreatment of mice with interleukin-1/3 (IL-1) resulted in elevated levels of nitrite/nitrate in serum and rendered mice insensitive towards TNF toxicity. Pharmacological doses of the nitric oxide (NO) donor sodium nitroprusside (SNP) also conferred complete protection against TNF toxicity, suggesting a possible link between IL-1- and NO-induced protection. However, NO-synthesis inhibition by N G -monomethyl- L -arginine failed to abrogate IL-1-induced tolerance against TNF toxicity. We conclude that IL-1 and NO protect against TNF-induced liver injury through distinct pathways.

Lung cell culture may be useful as an in vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2 x 10(8) cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC50 = 5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC50 = 0.6 mM). All cell types were equally sensitive to the more potent toxicant tert-butylhydroperoxide (EC50 = 0.1 mM). Paraquat was more toxic to lung cells (EC50 = 0.03 mM) than to rat (EC50 = 2.8 mM) and mouse (EC50 = 0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC50 = 2.6 mM) compared to rat (EC50 = 0.2 mM) and mouse (EC50 = 0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC50 = 0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC50 = 0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants.

Weight measurement represents a means of quantitating edema formation in isolated lungs and following its time course. During ventilation by negative pressure measurements with commonly used weight transducers are impossible because the pressure changes inside the artificial thoracic chamber affect the weight reading. Therefore, we have developed a weight transducer that can be used during negative pressure ventilation. Its design and applicability for assessing weight gain induced by various agents in the negative-pressure-ventilated isolated perfused rat lung is presented here.

In isolated rat lungs, perfusion with either endothelin-1 (ET-1) or the ETB receptor agonist IRL 1620 caused a slowly developing and long-lasting bronchoconstriction. This bronchoconstriction was partly attenuated by the thromboxane receptor antagonist BM 13177. Our data suggest that thromboxane partly mediates this process, together with further unknown mechanisms responsible for ET-induced bronchoconstriction.

PLoS one 7(10), e47344 (2012). doi:10.1371/journal.pone.0047344, Published by PLoS [u.a.], Lawrence, Kan.