Pulmonary inflammation induced by high-stretch ventilation is mediated by tumor necrosis factor signaling in mice

Although high-stretch mechanical ventilation has been demonstrated to induce lung inflammation, the roles of soluble mediators, in particular TNF, remain controversial. We have previously shown in mice that high-stretch ventilation, in the absence of preceding lung injury, induces expression of bioactive TNF in lung lavage fluid early in the course of injury, but the biological significance of this, if any, has yet to be determined. We therefore investigated the pulmonary inflammatory response to a transient period of high-stretch ventilation in anesthetized mice lacking TNF receptors and mice treated with anti-TNF antibodies. A standardized stretch-induced lung injury (assessed by lung mechanics, blood gases, and lavage protein content), followed by noninjurious low-stretch ventilation for 3 h, produced significant alveolar neutrophil infiltration in wild-type mice. However, neutrophil recruitment was substantially attenuated in TNF receptor double knockout mice and in wild-type mice treated with intratracheal anti-TNF antibody. This attenuation was not associated with decreased concentrations of neutrophil attractant CXC chemokines (macrophage inflammatory protein-2 and keratinocyte-derived chemokine) in lavage fluid. In contrast to intratracheal antibody, intravenous anti-TNF antibody did not reduce neutrophil infiltration, suggesting that the role of TNF signaling is localized within the alveolar space and does not require decompartmentalization of TNF into the circulation. These findings provide the first direct evidence that pulmonary inflammation induced by high-stretch ventilation without underlying lung injury possesses a significant TNF-dependent component. The results suggest a potential for regional anti-TNF treatment in attenuating stretch-induced pulmonary inflammation.     

A low level of TNF-alpha mediates hemorrhage-induced acute lung injury via p55 TNF receptor

Acute lung injury after hemorrhagic shock (HS) is associated with the expression of tumor necrosis factor (TNF)-alpha in the lung. However, the role of TNF-alpha and its receptors in this pulmonary disorder remains obscure. This study examined the temporal relationship of pulmonary TNF-alpha production to neutrophil accumulation during HS and determined the role of TNF-alpha in neutrophil accumulation and lung leak. HS was induced in mice by removal of 30% of total blood volume. Lung TNF-alpha was measured by ELISA. Neutrophil accumulation was detected by immunofluorescent staining, and microvascular permeability was assessed using Evans blue dye. Although HS induced a slight and transient increase in lung TNF-alpha, neutrophil accumulation preceded the increase in TNF-alpha. However, lung neutrophil accumulation and lung leak were abrogated in TNF-alpha knockout mice, and both were restored by administration of recombinant TNF-alpha to TNF-alpha knockout mice before HS. Neutrophil accumulation and lung leak were abrogated in mice lacking the p55 TNF-alpha receptor, but neither was influenced by p75 TNF-alpha receptor knockout. This study demonstrates that a low level of pulmonary TNF-alpha is sufficient to mediate HS-induced acute lung injury during HS and that the p55 TNF-alpha receptor plays a dominant role in regulating the pulmonary inflammatory response to HS.     

Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.     

p75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

A member of the TNF receptor family, the p75 neurotrophin receptor (p75(NTR)) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75(NTR) in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75(NTR) in mouse pulmonary arteries and the putative role of p75(NTR) in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75(NTR) knockout (p75(-/-)) mice. Our data indicated that p75(NTR) is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75(-/-) mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca(2+) influx. Furthermore, the contraction due to capacitative Ca(2+) entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca(2+) stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75(-/-) rings. Active tension induced by serotonin, U-46619 (a thromboxane A(2) analog), thrombin, 4-aminopyridine (a K(+) channel blocker), and high extracellular K(+) in p75(-/-) rings was similar to that in WT rings. Deletion of p75(NTR) did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75(NTR) signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.     

LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor

Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent on endogenously produced tumor necrosis factor (TNF)-alpha. The present study was undertaken to determine whether membrane-associated or secreted TNF-alpha signaling through the p55 or p75 receptor was responsible for survival and hepatic injury after lipopolysaccharide administration in D-galactosamine-sensitized mice. Transgenic mice expressing null forms of TNF-alpha, the p55 and p75 receptor, and mice expressing only a cell-associated form of TNF-alpha were challenged with 8 mg D-galactosamine and 100 ng lipopolysaccharide. Mortality and apoptotic liver injury were only seen in wild-type and p75 knockout mice. p75 Knockout mice had significantly higher concentrations of plasma TNF-alpha than any other experimental group (P 相似文献   

Divergent roles for p55 and p75 tumor necrosis factor receptors in the pathogenesis of MOG(35-55)-induced experimental autoimmune encephalomyelitis

To clarify the role of tumor necrosis factor (TNF) in the inflammatory aspects of autoimmunity vs its potential role in the apoptotic elimination of autoreactive effector cells, we assessed the roles of the p55 (TNFR1/Tnfrsf1a/CD120a) and p75 (TNFR2/Tnfrsf1b/CD120b) TNF receptors in the pathogenesis of MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE). TNFR p55/p75(-/-) double knockout mice were completely resistant to clinical disease. TNFR p55(-/-) single knockout mice were also totally resistant to EAE, exhibiting reduced MOG(35-55)- specific proliferative responses and Th1 cytokine production, despite displaying equivalent DTH responses. Importantly, IL-5 was significantly increased in p55(-/-) mice. In contrast, p75(-/-) knockout mice exhibited exacerbated EAE, enhanced Th1 cytokine production, and enhanced CD4(+) and F4/80(+) CNS infiltration. Thus, p55/TNFR1 is required for the initiation of pathologic disease, whereas p75/TNFR2 may be important in regulating the immune response. These results have important implications for therapies targeting p55 and p75 receptors for treating autoimmune diseases.     

Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury.

The use of positive pressure mechanical ventilation can cause ventilator-induced lung injury (VILI). We hypothesized that hyperoxia in combination with large tidal volumes (VT) would accentuate noncardiogenic edema and neutrophil infiltration in VILI and be dependent on stretch-induced macrophage inflammatory protein-2 (MIP-2) production. In rats ventilated with VT 20 ml/kg, there was pulmonary edema formation that was significantly increased by hyperoxia. Total lung neutrophil infiltration and MIP-2 in bronchoalveolar lavage (BAL) fluid were significantly elevated, in animals exposed to high VT both on room air (RA) and with hyperoxia. Hyperoxia markedly augmented the migration of neutrophils into the alveoli. Anti-MIP-2 antibody blocked migration of neutrophils into the alveoli in RA by 51% and with hyperoxia by 65%. We concluded that neutrophil migration into the alveoli was dependent on stretch-induced MIP-2 production. Hyperoxia significantly increased edema formation and neutrophil migration into the alveoli with VT 20 ml/kg, although BAL MIP-2 levels were nearly identical to VT 20 ml/kg with RA, suggesting that other mechanisms may be involved in hyperoxia-augmented neutrophil alveolar content in VILI.     

Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-alpha receptors

This study was designed to investigate the mechanisms through which tumor necrosis factor (Tnf) modulates ozone (O(3))-induced pulmonary injury in susceptible C57BL/6J (B6) mice. B6 [wild-type (wt)] mice and B6 mice with targeted disruption (knockout) of the genes for the p55 TNF receptor [TNFR1(-/-)], the p75 TNF receptor [TNFR2(-/-)], or both receptors [TNFR1/TNFR2(-/-)] were exposed to 0.3 parts/million O(3) for 48 h (subacute), and lung responses were determined by bronchoalveolar lavage. All TNFR(-/-) mice had significantly less O(3)-induced inflammation and epithelial damage but not lung hyperpermeability than wt mice. Compared with air-exposed control mice, O(3) elicited upregulation of lung TNFR1 and TNFR2 mRNAs in wt mice and downregulated TNFR1 and TNFR2 mRNAs in TNFR2(-/-) and TNFR1(-/-) mice, respectively. Airway hyperreactivity induced by acute O(3) exposure (2 parts/million for 3 h) was diminished in knockout mice compared with that in wt mice, although lung inflammation and permeability remained elevated. Results suggested a critical role for TNFR signaling in subacute O(3)-induced pulmonary epithelial injury and inflammation and in acute O(3)-induced airway hyperreactivity.     

Role of LPS and receptor subtypes in the uptake of TNF by the murine lung

Tumor necrosis factor alpha (TNF) is an important mediator in lung injury. The kinetics of TNF uptake by the lung are not completely understood. In this study, we evaluated the role that lipopolysaccharide (LPS) and the two types of TNF receptor (p55 and p75) play in the uptake of circulating murine TNF by the murine lung. TNF radioactively labeled with 125I (I-mTNF) was administered intravenously (2 x 10(6) cpm/mouse) to mice with both receptors (wild-type) or to mice missing one (p55-/- or p75-/-) or both (p55-/- and p75-/-) TNF receptors. Blood to lung non-reversible sequestration (Ki) and reversible uptake (Vi) were measured with multiple-time regression analysis. Uptake by lung of I-mTNF in wild-type mice had reversible and non-reversible components. This uptake was decreased by intratracheal, but not by intravenous, LPS, suggesting modulation by local, rather than systemic, inflammation. The p75-/- deficient mice retained the Ki (saturable, non-reversible) component of TNF uptake, whereas p55-/- deficient mice retained the Vi (saturable, reversible) component of TNF uptake. Both Ki and Vi components of TNF uptake were absent in the lungs of p55-/- p75-/- deficient mice. These studies show that local inflammation inhibits the uptake of circulating I-mTNF by lung and that uptake consists of two distinguishable compartments: reversible uptake mediated by the p75 receptor and non-reversible sequestration mediated by the p55 receptor.     

Epidermal growth factor receptor (EGFR) contributes to fetal lung fibroblast injury induced by mechanical stretch

Abstract

Context: Epidermal growth factor receptor (EGFR) is critical for normal fetal lung development. However, the role of this receptor in lung injury induced by mechanical ventilation is controversial. Objective: To investigate in vitro whether EGFR plays a protective role or contributes to stretch-induced lung injury. Methods: Fetal lung fibroblasts were isolated from wild-type and EGFR knockout mice and exposed to physiologic stretch (2.5% elongation) or injurious stretch (20% distention). Cells were evaluated for necrosis, apoptosis, proliferation and inflammation. Results: Injurious stretch increased lactate dehydrogenase (LDH) release to similar degree in wild-type and knockout cells. In contrast, 20% stretch increased cleaved caspase-3 and decreased proliferating cell nuclear antigen (PCNA) only in wild-type cells. Furthermore, 20% stretch increased macrophage inflammatory protein-2 (MIP-2) and monocyte chemotactic protein-1 (MCP-1) by 3–5 fold in wild-type cells. In contrast, in knockout cells MIP-2 decreased by 50% and MCP-1 only increased by 60% when compared to physiologic stretch. Conclusion: Our data show a decrease of apoptosis and inflammation and absence of decreased proliferation after injurious stretch of fetal fibroblasts lacking EGFR. These data suggest that EGFR contributes to lung injury mediated by stretch. We speculate that EGFR may contribute to the arrest of lung development observed after mechanical ventilation by decreasing the population of lung fibroblasts.     

Reduced IL-10 Production in Fetal Type II Epithelial Cells Exposed to Mechanical Stretch Is Mediated via Activation of IL-6-SOCS3 Signaling Pathway

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury.     

Systemic inhibition of the angiotensin-converting enzyme limits lipopolysaccharide-induced lung neutrophil recruitment through both bradykinin and angiotensin II-regulated pathways

Recruitment of neutrophils to the lung is a sentinel event in acute lung inflammation. Identifying mechanisms that regulate neutrophil recruitment to the lung may result in strategies to limit lung damage and improve clinical outcomes. Recently, the renin angiotensin system (RAS) has been shown to regulate neutrophil influx in acute inflammatory models of cardiac, neurologic, and gastrointestinal disease. As a role for the RAS in LPS-induced acute lung inflammation has not been described, we undertook this study to examine the possibility that the RAS regulates neutrophil recruitment to the lung after LPS exposure. Pretreatment of mice with the angiotensin-converting enzyme (ACE) inhibitor enalapril, but not the anti-hypertensive hydralazine, decreased pulmonary neutrophil recruitment after exposure to LPS. We hypothesize that inhibition of LPS-induced neutrophil accumulation to the lung with enalapril occurred through both an increase in bradykinin, and a decrease in angiotensin II (ATII), mediated signaling. Bradykinin receptor blockade reversed the inhibitory effect of enalapril on neutrophil recruitment. Similarly, pretreatment with bradykinin receptor agonists inhibited IL-8-induced neutrophil chemotaxis and LPS-induced neutrophil recruitment to the lung. Inhibition of ATII-mediated signaling, with the ATII receptor 1a inhibitor losartan, decreased LPS-induced pulmonary neutrophil recruitment, and this was suggested to occur through decreased PAI-1 levels. LPS-induced PAI-1 levels were diminished in animals pretreated with losartan and in those deficient for the ATII receptor 1a. Taken together, these results suggest that ACE regulates LPS-induced pulmonary neutrophil recruitment via modulation of both bradykinin- and ATII-mediated pathways, each regulating neutrophil recruitment by separate, but distinct, mechanisms.     

NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.     

Increased hypothalamic expression of the p75 tumor necrosis factor receptor in New Zealand obese mice.

Previous studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) production from adipose tissue is elevated in obese animal models and in obese humans. It plays an important role in the induction of insulin resistance in experimental animals. In this study, we examined hypothalamic tissue expression of TNF-alpha and its receptors and TNF-alpha expression of adipose tissue in lean C57BLKSJ+/+ and obese polygenic New Zealand obese (NZO) mice. Obese animals exhibited hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and hypercholesterinemia. Using RT-PCR, we observed increased expression (2.4-fold) of TNF receptor 2 (p75) in the hypothalamus of obese mice. TNF-alpha expression in adipose tissue of obese mice was eight times higher than in controls. TNF-alpha and TNF receptor 1 (p55) expression in hypothalamic tissue was similar in obese and lean animals. These results suggest that the hypothalamic TNF receptor 2 (p75) might play a role in obesity by modulating the actions of TNF-alpha in conditions of leptin resistance.     

Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0-75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR(-/-) mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.     

Tumor necrosis factor and the p55TNF receptor are required for optimal development of the marginal sinus and for migration of follicular dendritic cell precursors into splenic follicles

The development and function of secondary lymphoid tissue require signaling by tumor necrosis factor and lymphotoxins. Mice deficient in LTbetaR show defective organogenesis of lymph nodes and Peyer's patches and a severely disturbed splenic architecture. In contrast, TNF or p55TNF-R deficiency does not affect the organogenesis of peripheral lymphoid organs but interferes with the formation of B cell follicles and the appearance of FDC networks and germinal centers in all secondary lymphoid organs. Based on these differences, we have previously hypothesized that the role of TNF in lymphoid structure is distinct from that of LT and restricted in regulating cellular interactions that allow the differentiation and/or correct positioning of FDCs. In the present study we show that, in addition to the defects in follicular structure, TNF or p55TNF-R knockout mice exhibit defects in the formation of the macrophage populations and of the sinus lining cells of the splenic marginal zone. Interestingly, a large number of dendritic-shaped cells stained with FDC-specific markers and able to trap immune complexes are retained within the defective marginal zone of TNF and p55TNF-R knockout spleens. We conclude that the primary defect in the lymphoid phenotype of TNF or p55TNF-R knockout mice is the failure of FDC precursors to migrate through the disorganized marginal sinus and to home properly into the splenic follicular areas where they would promote the formation of B cell follicles and germinal centers.     

Activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury

Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.     

Neurokinin-1 receptor agonists are involved in mediating neutrophil accumulation in the inflamed, but not normal, cutaneous microvasculature: an in vivo study using neurokinin-1 receptor knockout mice

We have used tachykinin neurokinin-1 receptor (NK1 receptor) knockout mice to learn of the link between NK1 receptors and neutrophil accumulation in normal naive skin, as compared with inflamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK1 knockout mice, as expected. However, in contrast to IL-1beta (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1beta-induced neutrophil accumulation was similar in wild-type and knockout mice, but a significant (p < 0.05) contributory effect of added NK1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inflammatory process. We investigated responses to two inflammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was significantly attenuated (p < 0.001) in carrageenin- but not zymosan-induced inflammation in NK1 knockout mice. The carrageenin (500 microg)-induced response was inhibited (p < 0.05) by a NK1 receptor antagonist, SR140333 (480 nmol/kg i.v. at -5 min), in the wild-type group. The bradykinin B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals (p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK1 receptor-dependent neutrophil accumulation in inflamed mouse skin.     

Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNF-independent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome

The administration of endotoxins from Gram-negative bacteria induces manifestations reminding of acute respiratory distress syndrome. p38 MAPKs have been implicated in this pathology. In this study, we show that the specific p38 alpha,beta MAPK inhibitor, compound 37, prevents LPS-induced bronchoconstriction and neutrophil recruitment into the lungs and bronchoalveolar space in a dose-dependent manner in C57BL/6 mice. Furthermore, TNF induction and TNF signals were blocked. In TNF-deficient mice, bronchoconstriction, but not neutrophil sequestration, in the lung was abrogated after LPS administration. Therefore, TNF inhibition does not explain all of the effects of the p38 MAPK inhibitor. The p38 alpha,beta MAPK inhibitor also prevented LPS-induced neutrophilia in TNF-deficient mice. In conclusion, LPS provokes acute bronchoconstriction that is TNF dependent and p38 MAPK mediated, whereas the neutrophil recruitment is independent of TNF but depends on LPS/TLR4-induced signals mediated by p38 MAPK.     

Cyclic stretch induces alveolar epithelial barrier dysfunction via calpain-mediated degradation of p120-catenin

Lung hyperinflation is known to be an important contributing factor in the pathogenesis of ventilator-induced lung injury. Mechanical stretch causes epithelial barrier dysfunction and an increase in alveolar permeability, although the precise mechanisms have not been completely elucidated. p120-catenin is an adherens junction-associated protein that regulates cell-cell adhesion. In this study, we determined the role of p120-catenin in cyclic stretch-induced alveolar epithelial barrier dysfunction. Cultured alveolar epithelial cells (MLE-12) were subjected to uniform cyclic (0.5 Hz) biaxial stretch from 0 to 8 or 20% change in surface area for 0, 1, 2, or 4 h. At the end of the experiments, cells were lysed to determine p120-catenin expression by Western blot analysis. Immunofluorescence staining of p120-catenin and F-actin was performed to assess the integrity of monolayers and interepithelial gap formation. Compared with unstretched control cells, 20% stretch caused a significant loss in p120-catenin expression, which was coupled to interepithelial gap formation. p120-Catenin knockdown with small interfering RNA (siRNA) dose dependently increased stretch-induced gap formation, whereas overexpression of p120-catenin abolished stretch-induced gap formation. Furthermore, pharmacological calpain inhibition or depletion of calpain-1 with a specific siRNA prevented p120-catenin loss and subsequent stretch-induced gap formation. Our findings demonstrate that p120-catenin plays a critical protective role in cyclic stretch-induced alveolar barrier dysfunction, and, thus, maintenance of p120-catenin expression may be a novel therapeutic strategy for the prevention and treatment of ventilator-induced lung injury.