Protein tyrosine phosphatase α mediates profibrotic signaling in lung fibroblasts through TGF-ß responsiveness.

Fibrotic lung diseases represent a diverse group of progressive and often fatal disorders with limited treatment options. Although the pathogenesis of these conditions remains incompletely understood, receptor type protein tyrosine phosphatase α (PTP-α encoded by PTPRA) has emerged as a key regulator of fibroblast signaling. We previously reported that PTP-α regulates cellular responses to cytokines and growth factors through integrin-mediated signaling and that PTP-α promotes fibroblast expression of matrix metalloproteinase 3, a matrix-degrading proteinase linked to pulmonary fibrosis. Here, we sought to determine more directly the role of PTP-α in pulmonary fibrosis. Mice genetically deficient in PTP-α (Ptpra(-/-)) were protected from pulmonary fibrosis induced by intratracheal bleomycin, with minimal alterations in the early inflammatory response or production of TGF-ß. Ptpra(-/-) mice were also protected from pulmonary fibrosis induced by adenoviral-mediated expression of active TGF-ß1. In reciprocal bone marrow chimera experiments, the protective phenotype tracked with lung parenchymal cells but not bone marrow-derived cells. Because fibroblasts are key contributors to tissue fibrosis, we compared profibrotic responses in wild-type and Ptpra(-/-) mouse embryonic and lung fibroblasts. Ptpra(-/-) fibroblasts exhibited hyporesponsiveness to TGF-ß, manifested by diminished expression of αSMA, EDA-fibronectin, collagen 1A, and CTGF. Ptpra(-/-) fibroblasts exhibited markedly attenuated TGF-ß-induced Smad2/3 transcriptional activity. We conclude that PTP-α promotes profibrotic signaling pathways in fibroblasts through control of cellular responsiveness to TGF-ß.

Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin signaling.

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated ß-catenin signaling, and this was verified by real-time PCR, nuclear translocation of ß-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of ß-catenin signaling in response to neutrophil transmigration. Knockdown of ß-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of ß-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of ß-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that ß-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Role of ß-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that ß-catenin signaling promotes epithelial repair after inflammatory injury, but the ß-catenin target genes that mediate this effect are unknown. Herein, we examined which ß-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of ß-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the ß-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that ß-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

Matrix metalloproteinase 3 is a mediator of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) may be triggered by epithelial injury that results in aberrant production of growth factors, cytokines, and proteinases, leading to proliferation of myofibroblasts, excess deposition of collagen, and destruction of the lung architecture. The precise mechanisms and key signaling mediators responsible for this aberrant repair process remain unclear. We assessed the importance of matrix metalloproteinase-3 (MMP-3) in the pathogenesis of IPF through i) determination of MMP-3 expression in patients with IPF, ii) in vivo experiments examining the relevance of MMP-3 in experimental models of fibrosis, and iii) in vitro experiments to elucidate possible mechanisms of action. Gene expression analysis, quantitative RT-PCR, and Western blot analysis of explanted human lungs revealed enhanced expression of MMP-3 in IPF, compared with control. Transient adenoviral vector-mediated expression of recombinant MMP-3 in rat lung resulted in accumulation of myofibroblasts and pulmonary fibrosis. Conversely, MMP-3-null mice were protected against bleomycin-induced pulmonary fibrosis. In vitro treatment of cultured lung epithelial cells with purified MMP-3 resulted in activation of the ß-catenin signaling pathway, via cleavage of E-cadherin, and induction of epithelial-mesenchymal transition. These processes were inhibited in bleomycin-treated MMP-3-null mice, as assessed by cytosolic translocation of ß-catenin and cyclin D1 expression. These observations support a novel role for MMP-3 in the pathogenesis of IPF, through activation of ß-catenin signaling and induction of epithelial-mesenchymal transition.

Myeloid differentiation protein-2-dependent and -independent neutrophil accumulation during Escherichia coli pneumonia.

Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS-induced lung inflammation and E. coli-induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2(-/-) mice. MD-2(-/-) mice challenged with LPS displayed attenuated neutrophil influx, NF-kappaB activation, cytokine/chemokine expression, and lung histopathology. MD-2(-/-) mice transplanted with MD-2(+/+) bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2(-/-) mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2(-/-) mice infected with E. coli. Furthermore, MD-2(-/-) mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5(-/-) mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5(-/-) mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow- and non-bone marrow cell-derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2-dependent and MD-2-independent (TLR5) signaling in E. coli-induced neutrophil accumulation and pulmonary host defense.

A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction.

Neutrophils are a critical component of the innate immune response to invading microbial pathogens. However, an excessive and/or prolonged neutrophil response can result in tissue injury that is thought to underlie the pathogenesis of various inflammatory diseases. The development of novel therapeutic strategies for inflammatory diseases depends on an improved understanding of regulation of neutrophil function. However, investigations into neutrophil function have been constrained in part by the difficulty of genetically modifying neutrophils using current techniques. To overcome this, we have developed a novel method for the genetic modification of murine bone marrow derived progenitor cells using retroviral transduction followed by long term bone marrow culture to generate mature neutrophils. These neutrophils are functionally mature as determined by morphology, surface marker (Gr1, CD11b, CD62L and CXCR2) expression, and functional attributes including the ability to generate superoxide, exocytose granule contents, chemotax, and phagocytose and kill bacteria. Further, the in vitro matured neutrophils are capable of migrating to an inflammatory site in vivo. We utilized this system to express the Bcl-2 transgene in mature neutrophils using the retroviral vectors pMIG and pMIT. Bcl-2 overexpression conferred a substantial delay in spontaneous apoptosis of neutrophils as assessed by annexin V and 7-amino-actinomycin D (7AAD) staining. Moreover, Bcl-2 overexpression did not alter granulopoiesis, as assessed by morphology and surface marker expression. This system enables the genetic manipulation of progenitor cells that can be differentiated in vitro to mature neutrophils that are functional in vitro and in vivo.

c-Jun NH2-terminal kinase regulates lipopolysaccharide-induced pulmonary mononuclear cell recruitment via CCL2.

Subsequent to the initial recruitment of neutrophils, monocytes are recruited to the lung after an injurious insult. Previously the authors have shown that inhibition of either p38 or c-Jun NH(2)-terminal kinase (JNK) decreased pulmonary neutrophil recruitment in mice exposed to lipopolysaccharide (LPS). As the signaling pathways regulating the influx of mononuclear cells to the lung are poorly understood, the authors undertook the present study to examine the roles of p38 and JNK. In a model of LPS-induced lung inflammation, systemic inhibition of JNK, but not p38, decreased the recruitment of mononuclear cells to the lung. Levels of CCL2 (monocyte chemoattractant protein 1 [MCP-1]) were decreased in the setting of JNK inhibition, with LPS-induced pulmonary mononuclear cell recruitment in CCL2-deficient mice similar to that found with JNK inhibition. The decrease in LPS-induced CCL2 levels in the lung seen with JNK inhibition, however, was independent of neutrophil recruitment, as systemic depletion of neutrophils had no effect on pulmonary CCL2 levels after LPS exposure. In sum, these results suggest that JNK, but not p38, regulates LPS-induced mononuclear cell recruitment to the lung, that this occurs through a CCL2-dependent pathway, and that LPS-induced pulmonary CCL2 expression is dependent on JNK but independent of pulmonary neutrophil recruitment.

Phlegmonous gastritis in a patient with myeloid sarcoma: a case report.

Phlegmonous gastritis is a rare acute bacterial infection of the gastric wall with an extremely high mortality rate. Early diagnosis is crucial for immediate treatment that could improve the outcomes. Here we report a case in which a patient with underlying chronic myelomonocytic leukemia was diagnosed with phlegmonous gastritis on biopsy. This 57-year-old man presented with shortness of breath and intermittent upper quadrant abdominal pain for 4 days. Laboratory tests showed markedly increased white blood cell. A diagnosis of chronic myelomonocytic leukemia was made based on a peripheral blood smear and flow cytometry. Gastric biopsy showed suppurative inflammation in the submucosal region, prompting the diagnosis of phlegmonous gastritis. The patient was given empirical antibiotic treatment, and the white blood cell decreased dramatically. Surgical intervention was discussed but deferred. Despite continued antibiotics treatment, the patient died. The limited autopsy confirmed the diagnosis of phlegmonous gastritis. Immunohistochemical studies further revealed the occurrence of myeloid sarcoma that involved the gastrointestinal tract.

Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-mediated signaling contributes to innate immune responses in the lung during Escherichia coli pneumonia.

Bacterial pneumonia remains a serious disease and is associated with neutrophil recruitment. Innate immunity is pivotal for the elimination of bacteria, and TLRs are essential in this process. Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) is an adaptor for TLR3 and TLR4, and is associated with the MyD88-independent cascade. However, the importance of TRIF in immune responses against pulmonary bacterial pathogens is not well understood. We investigated the involvement of TRIF in a murine model of Escherichia coli pneumonia. TRIF(-/-) mice infected with E. coli display attenuated neutrophil migration; NF-kappaB activation; and TNF-alpha, IL-6, and LPS-induced C-X-C chemokine production in the lungs. In addition, E. coli-induced phosphorylation of JNK, ERK, and p38 MAPK was detected in bone marrow-derived macrophages (BMMs) of TRIF(+/+) mice, but attenuated in BMMs of TRIF(-/-) mice. Furthermore, E. coli-induced TNF-alpha and IL-6 production was attenuated in BMMs of TRIF(-/-) mice. E. coli LPS-induced late MAPK activation, and TNF-alpha and IL-6 production were abolished in BMMs of TRIF(-/-) mice. Moreover, TRIF is not required for LPS-induced neutrophil influx, and keratinocyte cell-derived chemokine, MIP-2, and LPS-induced C-X-C chemokine production in the lungs. Using TLR3(-/-) mice, we ruled out the role of TLR3-mediated TRIF-dependent neutrophil influx during E. coli pneumonia. A TLR4-blocking Ab inhibited E. coli-induced TNF-alpha and IL-6 in BMMs of both TRIF(-/-) and TRIF(+/+) mice, suggesting that TRIF-mediated signaling involves TLR4. We also found that TRIF is critical to control E. coli burden in the lungs and E. coli dissemination. Thus, rapid activation of TRIF-dependent TLR4-mediated signaling cascade serves to augment pulmonary host defense against a Gram-negative pathogen.

Toll/IL-1R domain-containing adaptor protein (TIRAP) is a critical mediator of antibacterial defense in the lung against Klebsiella pneumoniae but not Pseudomonas aeruginosa.

Bacterial pneumonia is a leading cause of mortality and is associated with extensive neutrophil accumulation. Major pathogens associated with this disease include nonflagellated Klebsiella pneumoniae (Kp) and flagellated Pseudomonas aeruginosa (Pa). TLRs are essential for innate immune defense. TIRAP (Toll/IL-1R domain-containing adaptor protein) is an adaptor in TLR1, TLR2, TLR4, and TLR6 signaling, whereas MyD88 is an adaptor for all TLRs. However, the importance of TIRAP in pulmonary defense against Kp or Pa has not been examined. To demonstrate the role of TIRAP, TIRAP-deficient and wild-type littermates were intratracheally inoculated with Kp or Pa. We found that TIRAP(-/-) mice had substantial mortality, higher bacterial burden in the lungs, and enhanced dissemination following Kp challenge. Furthermore, Kp-induced neutrophil sequestration, histopathology, and MIP-2, TNF-alpha, IL-6, and LIX (lipopolysaccharide-induced CXC chemokine) production were attenuated in the lungs of TIRAP(-/-) mice. In contrast, TIRAP is not required for Pa-induced mortality, pulmonary bacterial burden, bacterial dissemination, neutrophil accumulation, or histopathology, yet it is necessary for MIP-2, TNF-alpha, and IL-6 production, but not LIX production. However, both Kp- and Pa-induced neutrophil influxes are MyD88 dependent. To determine the mechanisms associated with Pa-induced neutrophil accumulation, we inoculated mice with a flagellin C mutant of Pa (PaDeltafliC) or purified flagellin, a TLR5 agonist. PaDeltafliC-induced neutrophil sequestration and LIX expression are dependent on TIRAP, whereas flagellin-induced neutrophil influx and LIX expression are independent of TIRAP. These novel findings illustrate a pathogen-specific role for TIRAP in pulmonary defense and suggest that TLR5 plays an essential role for Pa-induced neutrophil influx via LIX production.

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.

Regulation of lipopolysaccharide-induced lung inflammation by plasminogen activator Inhibitor-1 through a JNK-mediated pathway.

The neutrophil is of undoubted importance in lung inflammation after exposure to LPS. We have shown recently that systemic inhibition of JNK decreased neutrophil recruitment to the lung after exposure to LPS, although the mechanisms underlying this inhibition are incompletely understood. As plasminogen activator inhibitor-1 (PAI-1) accentuates cell migration, with JNK activation recently shown to up-regulate PAI-1 expression, this suggested that systemic JNK inhibition may down-regulate LPS-induced pulmonary neutrophil recruitment through a decrease in PAI-1 expression. We show in this study that exposure of mice to aerosolized LPS increased PAI-1 expression in the lung and alveolar compartment, which was decreased by pretreatment with the JNK inhibitor SP600125. Exogenous, intratracheally administered PAI-1 prevented the inhibition of pulmonary neutrophil recruitment in the setting of systemic JNK inhibition, thereby suggesting a role for PAI-1 in the JNK-mediated pathway regulating LPS-induced neutrophil recruitment. In addition, PAI-1(-/-) mice had a decrease in neutrophil recruitment to the alveolar compartment after exposure to LPS, compared with wild-type controls, further suggesting a role for PAI-1 in LPS-induced lung inflammation. An increase in the intravascular level of KC is a likely mechanism for the inhibition of pulmonary neutrophil recruitment after LPS exposure in the setting of decreased PAI-1 expression, as systemic KC levels after exposure to LPS were increased in PAI-1-deficient mice and in mice pretreated with SP600125, with augmentation of intravascular KC levels inhibiting neutrophil recruitment to the lung after exposure to LPS.

Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury.

Acute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-microg/ml) LPS-induced microvascular leakage, NF-kappaB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-dependent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.

Toll-IL-1 receptor domain-containing adaptor protein is critical for early lung immune responses against Escherichia coli lipopolysaccharide and viable Escherichia coli.

Pulmonary bacterial diseases are a leading cause of mortality in the U.S. Innate immune response is vital for bacterial clearance from the lung, and TLRs play a critical role in this process. Toll-IL-1R domain-containing adaptor protein (TIRAP) is a key molecule in the TLR4 and 2 signaling. Despite its potential importance, the role of TIRAP-mediated signaling in lung responses has not been examined. Our goals were to determine the role of TIRAP-dependent signaling in the induction of lung innate immune responses against Escherichia coli LPS and viable E. coli, and in lung defense against E. coli in mice. LPS-induced neutrophil sequestration; NF-kappaB translocation; keratinocyte cell-derived chemokine, MIP-2, TNF-alpha, and IL-6 expression; histopathology; and VCAM-1 and ICAM-1 expression were abolished in the lungs of TIRAP-/- mice. A cell-permeable TIRAP blocking peptide attenuated LPS-induced lung responses. Furthermore, immune responses in the lungs of TIRAP-/- mice were attenuated against E. coli compared with TIRAP+/+ mice. TIRAP-/- mice also had early mortality, higher bacterial burden in the lungs, and more bacterial dissemination following E. coli inoculation. Moreover, we used human alveolar macrophages to examine the role of TIRAP signaling in the human system. The TIRAP blocking peptide abolished LPS-induced TNF-alpha, IL-6, and IL-8 expression in alveolar macrophages, whereas it attenuated E. coli-induced expression of these cytokines and chemokines. Taken together, this is the first study illustrating the crucial role of TIRAP in the generation of an effective early immune response against E. coli LPS and viable E. coli, and in lung defense against a bacterial pathogen.

Induction of CXCL5 during inflammation in the rodent lung involves activation of alveolar epithelium.

The lung is continuously exposed to bacteria and their products, and has developed a complex defense mechanism, including neutrophil recruitment. In mice, keratinocyte cell-derived chemokine and macrophage inflammatory protein-2 are the major chemokines for neutrophil recruitment into the lung. We have previously described a role for C-X-C chemokine (CXCL5) in neutrophil trafficking during lipopolysaccharide (LPS)-induced lung inflammation in mice. The aims of the present study were to identify the cellular origin of CXCL5 and to determine the signaling cascades that regulate its expression in the lung during LPS-induced inflammation and in isolated LPS-stimulated CXCL5-expressing cells. Our immunohistochemical analysis indicates that alveolar epithelial type II (AEII) cells are the primary source of CXCL5 in the rodent lung. These in vivo observations were confirmed with primary AEII cells. In addition, our data indicate that the Toll-like receptor 4 (TLR4) signaling cascade involving TLR4, myeloid differentiation factor 88, and Toll-IL-1R domain-containing adapter protein is required to induce CXCL5 expression in the lung. Furthermore, p38 and c-Jun N-terminal kinases are involved in lung CXCL5 expression. Similarly, TLR4, and p38 and c-Jun N-terminal kinases, are associated with LPS-induced CXCL5 expression in AEII cells. These novel observations demonstrate that activation of AEII cells via TLR4-dependent signaling is important for the production of CXCL5 in the lung exposed to LPS.

Inhibition of c-Jun N-terminal kinase limits lipopolysaccharide-induced pulmonary neutrophil influx.

The influx of neutrophils into the lung is a sentinel event in LPS-induced acute lung inflammation. Previous studies have shown that systemic inhibition of p38 decreases LPS-induced neutrophil influx into the alveolar space but has no effect on pulmonary parenchymal neutrophil accumulation or on microvascular leak, indicating other pathways are important in LPS-induced acute lung inflammation. This study examined the role of c-Jun N-terminal kinase in LPS-induced acute lung inflammation. Systemic inhibition of c-Jun N-terminal kinase, with the specific c-Jun N-terminal kinase inhibitor SP600125, decreased the LPS-induced accumulation of neutrophils into the lung parenchyma and alveolar space. In addition, increases in microvascular leak after LPS exposure were diminished by c-Jun N-terminal kinase inhibition. To determine mechanisms by which systemic c-Jun N-terminal kinase inhibition decreased pulmonary neutrophil influx, LPS and tumor necrosis factor alpha (TNF-alpha-)-induced neutrophil actin assembly and retention were examined. Neutrophil actin assembly was decreased after LPS and TNF-alpha stimulation with SP600125 pretreatment, as well as LPS-induced neutrophil retention. Finally, c-Jun N-terminal kinase inhibition decreased Cdc42 activation after LPS or TNF-alpha stimulation, thereby providing one mechanism by which c-Jun N-terminal kinase inhibition decreased actin assembly, and thereby pulmonary neutrophil accumulation.

A role for hydroxy-methylglutaryl coenzyme a reductase in pulmonary inflammation and host defense.

RATIONALE: A growing literature indicates that hydroxy-methylglutaryl coenzyme A reductase inhibitors (statins) modulate proinflammatory cellular signaling and functions. No studies to date, however, have addressed whether statins modulate pulmonary inflammation triggered by aerogenic stimuli or whether they affect host defense. OBJECTIVES: To test whether lovastatin modulates LPS-induced pulmonary inflammation and antibacterial host defense. METHODS: To address these questions, and to confirm any effect of statins as dependent on inhibition of hydroxy-methylglutaryl coenzyme A reductase, we treated C57Bl/6 mice with three oral doses of 10 mg/kg lovastatin (or vehicle) and three intraperitoneal doses of 10 mg/kg mevalonic acid (or saline), and then exposed them to the following: (1) aerosolized LPS, (2) intratracheal keratinocyte-derived chemokine (KC), or (3) intratracheal Klebsiella pneumoniae. MEASUREMENTS AND MAIN RESULTS: LPS- and KC-induced airspace neutrophils were reduced by lovastatin, an effect that was blocked by mevalonic acid cotreatment. Lovastatin was also associated with reduced parenchymal myeloperoxidase and microvascular permeability, and altered airspace and serum cytokines after LPS. Native pulmonary clearance of K. pneumoniae was inhibited by lovastatin and extrapulmonary dissemination was enhanced, both reversibly with mevalonic acid. Ex vivo studies of neutrophils isolated from lovastatin-treated mice confirmed inhibitory effects on Rac activation, actin polymerization, chemotaxis, and bacterial killing. CONCLUSION: Lovastatin attenuates pulmonary inflammation induced by aerosolized LPS and impairs host defense.

Transcriptional profiling of lipopolysaccharide-induced acute lung injury.

Mortality associated with acute lung injury (ALI) induced by lipopolysaccharide (LPS) remains high in humans, warranting improved treatment and prevention strategies. ALI is characterized by the expression of proinflammatory mediators and extensive neutrophil influx into the lung, followed by severe lung damage. Understanding the pathogenesis of LPS-induced ALI is a prerequisite for designing better therapeutic strategies. In the present study, we used microarrays to gain a global view of the transcriptional responses of the lung to LPS in a mouse model of ALI that mimics ALI in humans. A total of 71 inflammation-associated genes were up-regulated in LPS-treated lungs, including a chemokine, LPS-induced CXC chemokine (LIX), whose role in the induction of ALI is unknown. Most of the inflammatory genes peaked at 2 h post-LPS treatment. Real-time reverse transcription-PCR confirmed the LPS-induced up-regulation of selected genes identified by microarray analysis, including LIX. The up-regulation of LIX, tumor necrosis factor alpha, and macrophage inflammatory protein 2 was confirmed at the protein level by enzyme-linked immunosorbent assays. To determine the role of LIX in the induction of ALI, we used both exogenous LIX and a LIX blocking antibody. Exogenous LIX alone elicited a neutrophil influx in the lungs, and the anti-LIX antibody attenuated the LPS-induced neutrophil accumulation in the lungs. Taken together, the results of our study demonstrate for the first time the temporal expression of inflammatory genes during LPS-induced ALI and suggest that early therapeutic intervention is crucial to attenuate lung damage. Moreover, we identified a role for LIX in the induction of ALI, and therefore LIX may serve as a novel therapeutic target for the minimization of ALI.

Extracellular superoxide dismutase attenuates lipopolysaccharide-induced neutrophilic inflammation.

Extracellular superoxide dismutase (EC-SOD) is an abundant antioxidant in the lung and vascular walls. Previous studies have shown that EC-SOD attenuates lung injury in a diverse variety of lung injury models. In this study, we examined the role of EC-SOD in mediating lipopolysaccharide (LPS)-induced lung inflammation. We found that LPS-induced neutrophilic lung inflammation was exaggerated in EC-SOD-deficient mice and diminished in mice that overexpressed EC-SOD specifically in the lung. Similar patterns were seen for bronchoalveolar lavage cytokines, such as tumor necrosis factor-alpha, keratinocyte-derived chemokines, and macrophage inflammatory protein-2 as well as expression of lung intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial cell selectin, and platelet selectin. In a macrophage cell line, EC-SOD inhibited LPS-induced macrophage cytokine release, but did not alter expression of intercellular adhesion molecules in endothelial cells. These results suggest that EC-SOD plays an important role in attenuating the inflammatory response in the lung most likely by decreasing release of proinflammatory cytokines from phagocytes.