bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS-induced bronchial hyperreactivity: interference by mIL-10 differs according to site of delivery
Authors: Deleuze Virginie , Lefort Jean , Bureau Michel F. , Scherman Daniel , Vargaftig B. Boris
Published: January 2004

When administered to mice systemically or via the airways, LPS induces bronchoconstriction (BC) and/or bronchopulmonary hyperreactivity (BHR), associated with inflammation. Accordingly, a relationship between inflammation and allergic and nonallergic BHR can be hypothesized. We therefore studied the interference of the anti-inflammatory cytokine murine IL-10 (mIL-10) with LPS-induced lung inflammation, BC, and BHR. mIL-10 was administered directly into the airways by intranasal instillation or generated in vivo after muscle electrotransfer of mIL-10-encoding plasmid. Electrotransfer led to high mIL-10 circulating levels for a longer time than after the injection of recombinant mIL-10 (rmIL-10). rmIL-10 administered intranasally reduced lung inflammation and BHR after LPS administration into airways. It also reduced the ex vivo production of TNF-α by LPS-stimulated lung tissue explants. Two days after electrotransfer, mIL-10 blood levels were elevated, but lung inflammation, BC, and BHR persisted unaffected. Blood mIL-10 reaches the airways poorly, which probably accounts for the ineffectiveness of mIL-10-encoding plasmid electrotransfer. When LPS was aerosolized 15 days after electrotransfer, lung inflammation persisted but BHR was significantly reduced, an effect that may be related to the longer exposure of the relevant cells to mIL-10. The dissociation between inflammation and BHR indicates that both are not directly correlated. In conclusion, this study shows that mIL-10 is efficient against BHR when present in the airway compartment. Despite this, the muscle electrotransfer with mIL-10-encoding plasmid showed a protective effect against BHR after a delay of 2 wk that should be further investigated.
Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity
Authors: Wheelock Åsa M. , Zhang Lu , Tran Mai-Uyen , Morin Dexter , Penn Sharron , Buckpitt Alan R. , Plopper Charles G.
Published: January 2004

Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins that facilitates in vivo proteomics studies of two target-cell pheno-types of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.
Expression and functional implications of CCR2 expression on murine alveolar epithelial cells
Authors: Christensen Paul J. , Du Ming , Moore Bethany , Morris Susan , Toews Galen B. , Paine Robert
Published: January 2004

Acute lung injury results in damage to the alveolar epithelium, leading to leak of proteins into the alveolar space and impaired gas exchange. Lung function can be restored only if the epithelial layer is restored. The process of reepithelialization requires migration of lung epithelial cells to cover denuded basement membranes. The factors that control the migration of lung epithelial cells are incompletely understood. We examined isolated murine type II alveolar epithelial cells (AECs) for expression of CC chemokine receptor 2 (CCR2) and functional consequences of the binding of the main CCR2 ligand monocyte chemoattractant protein-1 (MCP-1). We found that primary AECs bound MCP-1 and expressed CCR2 mRNA. These cells demonstrated functional consequences of CCR2 expression with migration in response to MCP-1 in chemotaxis/haptotaxis assays. Primary AECs cultured from mice lacking CCR2 did not respond to MCP-1. Monolayers of AECs lacking CCR2 demonstrated delayed closure of mechanical wounds compared with AEC monolayers expressing CCR2. Delayed closure of mechanical wounds of wild-type AECs was also demonstrated in the presence of anti-MCP-1 antibody. These data demonstrate for the first time that AECs express CCR2 and are capable of using this receptor for chemotaxis and healing of wounds. CCR2-MCP-1 interactions may be important in the process of reepithelialization after lung injury.
Modulation of PDGF-C and PDGF-D expression during bleomycin-induced lung fibrosis
Authors: Zhuo Ying , Zhang Jian , Laboy Miguel , Lasky Joseph A.
Published: January 2004

PDGF isoforms are a family of polypeptides that bind to cell surface receptors and induce fibroblast proliferation and chemotaxis. The PDGF-A and -B chain isoforms have been implicated in fibroproliferative lung injury in animal models and in human disease. Two recently recognized PDGF polypeptides, PDGF-C and -D, differ from the PDGF-A and -B isoforms in that they require proteolytic cleavage before they can bind and activate the PDGF receptors. Our findings demonstrate that administration of bleomycin to murine lungs leads to a significant increase in PDGF-C mRNA expression and a significant decrease in PDGF-D mRNA expression. PDGF-C expression was localized to areas of lung injury by in situ hybridization, and PDGF-C expression was not upregulated in the lungs of BALB/c mice that are resistant to bleomycin-induced lung fibrosis. Moreover, there is in vivo phosphorylation of the PDGF-receptor that binds PDGF-C in response to bleomycin administration. These observations strongly suggest a role for PDGF-C in bleomycin-induced pulmonary fibrosis.
Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts
Authors: Blaisdell Carol J. , Morales Marcelo M. , Andrade Ana Carolina Oliveira , Bamford Penelope , Wasicko Michael , Welling Paul
Published: January 2004

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology ( 26 ). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage ( Vt) of lung explants transfected with antisense CLC-2 was inhibited with Vt= -1.5 ± 0.2 mV (means + SE) compared with -3.7 ± 0.3 mV (means + SE) for mock-transfected controls and -3.3 ± 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.
NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-α
Authors: Gertzberg Nancy , Neumann Paul , Rizzo Victor , Johnson Arnold
Published: January 2004

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion ([Formula: see text]) mediates tumor necrosis factor-α (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phoxand p22phoxwere assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species [Formula: see text] was measured by the fluorescence of 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phoxtranslocation, 2) an increase in p22phoxprotein, 3) increased localization of p47phoxwith p22phox, 4) [Formula: see text] generation, and 5) increased permeability to albumin. p22phoxantisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, [Formula: see text], and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in [Formula: see text] and permeability to albumin was also prevented by the [Formula: see text] scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of [Formula: see text], mediates TNF-induced barrier dysfunction in PMEM.
Stimulation of human bronchial epithelial cells by IgE-dependent histamine-releasing factor
Authors: Yoneda Kazuo , Rokutan Kazuhito , Nakamura Yoichi , Yanagawa Hiroaki , Kondo-Teshima Shigetada , Sone Saburo
Published: January 2004

An IgE-dependent histamine-releasing factor (HRF p23; also known as translationally controlled tumor protein or p23) stimulates the release of histamine, IL-4, and IL-13 from a subpopulation of highly allergic donor basophils. It has also been shown to act as a chemoattractant for eosinophils. To elucidate novel functions of HRF p23 in airway inflammation, we examined the effects of human recombinant HRF p23 (hrHRF) on bronchial epithelium and found that hrHRF stimulated the secretions of IL-8 and granulocyte/macrophage colony-stimulating factor by both primary cultures of human bronchial epithelial cells and BEAS-2B cells. In response to hrHRF, these cells induced IL-8 mRNA expression within 4 h. H2O2, but not IL-1β or tumor necrosis factor-α, stimulated secretion of HRF p23 by BEAS-2B cells, suggesting that oxidative stress may trigger the release of HRF p23 from bronchial epithelial cells. Bronchoalveolar lavage (BAL) from healthy volunteers contained only trivial or undetectable amounts of HRF p23. Significantly higher amounts of HRF p23 were recovered from BAL fluid taken from asthmatic patients, and the amounts of HRF p23 were further elevated in patients with idiopathic eosinophilic pneumonia. Our results demonstrate for the first time that HRF p23 can stimulate nonimmune epithelium. HRF p23 derived from bronchial epithelial cells may regulate complex cytokine networks in eosinophil-dependent inflammation of the human airway.
Multiple contributing roles for NOS2 in LPS-induced acute airway inflammation in mice
Authors: Okamoto Tatsuya , Gohil Kishorchandra , Finkelstein Erik I. , Bove Peter , Akaike Takaaki , van der Vliet Albert
Published: January 2004

Acute lung inflammation and injury were induced by intranasal instillation of lipopolysaccharide (LPS) in normal and type 2 nitric oxide synthase (NOS2)-deficient (NOS2-/-) C57BL/6 mice. LPS-induced increases in extravasated airway neutrophils and in lung lavage fluid of TNF-α and macrophage inflammatory protein-2 were markedly lower in NOS2-/-than in wild-type mice, indicating that NOS2-derived nitric oxide (NO·) participates in inflammatory cytokine production and neutrophil recruitment. Instillation of LPS also increased total lung lavage protein and induced matrix metalloproteinase-9 and mucin 5AC, as indexes of lung epithelial injury and/or mucus hyperplasia, and increased tyrosine nitration of lung lavage proteins, a marker of oxidative injury. All these responses were less pronounced in NOS2-/-than in wild-type mice. Inhibition of NOS activity also suppressed production of TNF-α and macrophage inflammatory protein-2 by LPS-stimulated mouse alveolar MH-S macrophages, and this was restored by NO· donors, illustrating involvement of NO· in macrophage cytokine signaling. Oligonucleotide microarray (GeneChip) analysis of global lung gene expression revealed that LPS inhalation induced a range of transcripts encoding proinflammatory cytokines and chemokines, stress-inducible factors, and other extracellular factors and suppressed mRNAs encoding certain cytoskeletal proteins and signaling proteins, responses that were generally attenuated in NOS2-/-mice. Comparison of both mouse strains revealed altered expression of several cytoskeletal proteins, cell surface proteins, and signaling proteins in NOS2-/-mice, changes that may partly explain the reduced responsiveness to LPS. Collectively, our results suggest that NOS2 participates in the acute inflammatory response to LPS by multiple mechanisms: involvement in proinflammatory cytokine signaling and alteration of the expression of various genes that affect inflammatory-immune responses to LPS.
Calcium and ROS-mediated activation of transcription factors and TNF-α cytokine gene expression in macrophages exposed to ultrafine particles
Authors: Brown D. M. , Donaldson K. , Borm P. J. , Schins R. P. , Dehnhardt M. , Gilmour P. , Jimenez L. A. , Stone V.
Published: January 2004

Ultrafine (Uf) particles are a component of particulate air pollution suggested to be responsible for the health effects associated with elevations of this pollutant. We have previously suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals. Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease. The effect of Uf particles on rat alveolar macrophages and human blood monocytes was investigated with reference to the roles of calcium and reactive oxygen species (ROS). TNF-α protein release, intracellular calcium concentration, TNF-α mRNA expression, and transcription factor activation were studied as end points after treatment of rat alveolar macrophages or peripheral blood monocytes. The calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, the calmodulin inhibitor W-7, and the antioxidants Trolox and Nacystelin (NAL) were included in combination with Uf particles. Verapamil reduced intracellular calcium concentration in rat alveolar macrophages on stimulation with Uf particles. This effect was also apparent with transcription factor AP-1 activation. All antagonists and antioxidants reduced Uf-stimulated nuclear localization of the p50 and p65 subunits of NF-κB in human monocytes. Verapamil, BAPTA-AM, and NAL reduced Uf-stimulated TNF-α protein release, whereas only verapamil reduced Uf-stimulated mRNA expression in rat alveolar macrophages. In human monocytes, verapamil, Trolox, BAPTA-AM, and W-7 reduced Uf-stimulated TNF-α protein release. These findings suggest that Uf particles may exert proinflammatory effects by modulating intracellular calcium concentrations, activation of transcription factors, and cytokine production through a ROS-mediated mechanism.
Lung retinol storing cells synthesize and secrete retinoic acid, an inducer of alveolus formation
Authors: Dirami Ghenima , Massaro Gloria DeCarlo , Clerch Linda Biadasz , Ryan Una S. , Reczek Peter R. , Massaro Donald
Published: January 2004

Retinoids play a key role in the formation of pulmonary alveoli. Lipid interstitial cells (LICs) of the alveolar wall store retinol and are concentrated at sites of alveolus formation, suggesting they are an endogenous source of retinoids for alveolus formation. We show in cultured rat lung cells that LICs synthesize and secrete all- trans retinoic acid (ATRA); its secretion is halved by dexamethasone, an inhibitor of alveolus formation. In a second alveolar wall cell, the pulmonary microvascular endothelial cell (PMVC), ATRA increases expression of the mRNA of cellular retinol binding protein-I (CRBP-I), a protein involved in ATRA synthesis. Serum-free, exogenous ATRA-free medium conditioned by LICs rich in retinol storage granules caused a 10-fold greater increase of CRBP-I mRNA in PMVCs than media conditioned by LICs with few retinol storage granules. This action of medium conditioned by retinol storage granule-rich LICs is decreased by a retinoic acid receptor pan-antagonist and by a retinoid X receptor pan-antagonist, suggesting the responsible molecule(s) is a retinoid and that retinoid signaling occurs in a paracrine fashion.
Granulocyte/macrophage colony-stimulating factor treatment improves alveolar epithelial barrier function in alcoholic rat lung
Authors: Pelaez Andres , Bechara Rabih I. , Joshi Pratibha C. , Brown Lou Ann S. , Guidot David M.
Published: January 2004

Chronic alcohol abuse increases the risk of developing acute lung injury approximately threefold in septic patients, and ethanol ingestion for 6 wk in rats impairs alveolar epithelial barrier function both in vitro and in vivo. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a trophic factor for the alveolar epithelium, and a recent phase II clinical study suggests that GM-CSF therapy decreases sepsis-mediated lung injury. Therefore, we hypothesized that GM-CSF treatment could improve ethanol-mediated defects in the alveolar epithelium during acute stresses such as endotoxemia. In this study, we determined that recombinant rat GM-CSF improved lung liquid clearance (as reflected by lung tissue wet:dry ratios) in ethanol-fed rats anesthetized and then challenged with 2 ml of saline via a tracheostomy tube. Furthermore, GM-CSF treatment improved lung liquid clearance and decreased epithelial protein leak in both control-fed and ethanol-fed rats after 6 h of endotoxemia induced by Salmonella typhimurium lipopolysaccharide given intraperitoneally, but with the greater net effect seen in the ethanol-fed rats. Our previous studies indicate that chronic ethanol ingestion decreases lung liquid clearance by increasing intercellular permeability. Consistent with this, GM-CSF treatment in vitro decreased permeability of alveolar epithelial monolayers derived from both control-fed and ethanol-fed rats. As in the endotoxemia model in vivo, the effect of GM-CSF was most dramatic in the ethanol group. Together, these results indicate that GM-CSF treatment has previously unrecognized effects in promoting alveolar epithelial barrier integrity and that these salutary effects may be particularly relevant in the setting of chronic alcohol abuse.
Role of EETs in regulation of endothelial permeability in rat lung
Authors: Alvarez Diego F. , Gjerde Eli-Anne B. , Townsley Mary I.
Published: January 2004

This study tested the hypothesis that epoxyeicosatrienoic acids (EETs) derived from arachidonic acid via P-450 epoxygenases are soluble factors linking depletion of endoplasmic reticulum Ca2+ stores and store-dependent regulation of endothelial cell (EC) permeability in rat lung. EC permeability was measured via the capillary filtration coefficient ( Kf,c) in isolated, perfused rat lungs. 14,15-EET and 5,6-EET increased EC permeability, a response that was significantly different from that of 8,9-EET, 11,12-EET, and vehicle control. The permeability response to 14,15-EET was not significantly attenuated by the nonspecific Ca2+ channel blocker Gd3+ ( P = 0.068). In lungs perfused with low [Ca2+], 14,15-EET tended to increase EC permeability, although a significant increase in Kf,c was observed only following Ca2+ add-back. As positive control, we showed that the 3.7-fold increase in Kf,c evoked by thapsigargin (TG), a known activator of store depletion-induced Ca2+ entry, was blocked by both Gd3+ and low [Ca2+] buffer. Nonetheless, the permeability response to TG could not be blocked by the phospholipase A2 inhibitors mepacrine or methyl arachidonyl fluorophosphonate or the P-450 epoxygenase inhibitors 17-octadecynoic acid or propargyloxyphenyl hexanoic acid. Similarly, combined pretreatment with ibuprofen and dicyclohexylurea to block EET metabolism had no effect on the permeability response to TG. We conclude that EETs have a heterogeneous impact on EC permeability. Despite a requirement for Ca2+ entry with both TG and 14,15-EET, our data suggest that distinct signaling pathways or heterogeneity in EC responsiveness is responsible for the observed EC injury evoked by EETs and store depletion in the isolated rat lung.
Post-BMT lung injury occurs independently of the expression of CCL2 or its receptor, CCR2, on host cells
Authors: Panoskaltsis-Mortari Angela , Hermanson John R. , Taras Elizabeth , Wangensteen O. Douglas , Charo Israel F. , Rollins Barrett J. , Blazar Bruce R.
Published: January 2004

Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality post-bone marrow transplant (BMT) in humans. In our murine model, lethal pre-BMT conditioning and allogeneic T cells result in the recruitment of host antigen-presenting cells (APC) and donor T cells into the lung post-BMT concomitant with development of severe lung dysfunction. CCL2 induction is found in bronchoalveolar lavage fluid (BALF) before host monocyte influx. The major receptor for CCL2 is CCR2 present on monocytes; this interaction can play a crucial role in monocyte recruitment in inflammation. To determine whether blockade of the CCL2/CCR2 pathway could hinder host monocyte influx, lethally conditioned wild-type (WT), CCL2-/-, or CCR2-/-mice were transplanted with allogeneic marrow and spleen cells. WT and-/-recipients exhibited equivalent lung dysfunction post-BMT. The frequencies of host macrophages as well as donor CD4+and CD8+T cells in lungs post-BMT did not differ between WT and-/-recipients. However, the T cell dependency of the host CD11b+major histocompatibility complex class II+cell influx was lost in CCR2-/-recipients. In CCR2-/-mice, this influx was accompanied by elevated levels of CCL20. Post-BMT BALF and sera of-/-mice did not reveal any decrease in cytokines or chemokines compared with WT mice. CCL2-/-mice had a deficiency of CCL2 in their BALF and sera post-BMT, confirming our hypothesis that CCL2 is predominantly host derived. Therefore, IPS can occur independently of host expression of CCL2 or CCR2, and compensatory mechanisms exist for regulating APC recruitment into the lung during the early post-BMT period.
Retinoic acid reverses the airway hyperresponsiveness but not the parenchymal defect that is associated with vitamin A deficiency
Authors: McGowan Stephen E. , Holmes Amey Jo , Smith Jennifer
Published: January 2004

Airway hyperresponsiveness (AHR) is influenced by structural components of the bronchial wall, including the smooth muscle and connective tissue elements and the neuromuscular function. AHR is also influenced by parenchymally derived tethering forces on the bronchial wall, which maintain airway caliber by producing outward radial traction. Our previous work has shown that vitamin A-deficient (VAD) rats exhibit cholinergic hyperresponsiveness and a decrease in the expression and function of the muscarinic-2 receptors (M2R). We hypothesized that if decreases in radial traction from airway or parenchymal structures contributed to the VAD-related increase in AHR, then the radial traction would normalize more slowly than VAD-related alterations in neurotransmitter signaling. Rats remained vitamin A sufficient (VAS) or were rendered VAD and then maintained on the VAD diet in the presence or absence of supplementation with all- trans retinoic acid (RA). VAD was associated with an approximately twofold increase in respiratory resistance and elastance compared with VAS rats. Exposure to RA for 12 days but not 4 days restored resistance and elastance to control (VAS) levels. In VAD rats, AHR was accompanied by decreases in bronchial M2R gene expression and function, which were restored after 12 days of RA supplementation. Subepithelial bronchial elastic fibers were decreased by ∼50% in VAD rats and were significantly restored by RA. The increase in AHR that is associated with VAD is accompanied by decreases in M2R expression and function that can be restored by RA and a reduction in airway elastic fibers that can be partially restored by RA.
BMP4 signaling induces senescence and modulates the oncogenic phenotype of A549 lung adenocarcinoma cells
Authors: Buckley S. , Shi W. , Driscoll B. , Ferrario A. , Anderson K. , Warburton D.
Published: January 2004

Lung cancer is the most common visceral malignancy in males, with rapidly increasing incidence in females, and a devastatingly poor prognosis. Transforming growth factor (TGF)-β has been shown to induce senescence in A549 lung cancer cells, and both TGF-β and bone morphogenetic protein (BMP) 2 can suppress the transformed phenotype of A549 cells in vitro. We examined the effects of BMP4, another member of the TGF-β superfamily, on specific oncogenic properties of A549 cancer cells. When A549 cancer cells were treated continuously with 100 ng/ml of BMP4, a senescent phenotype was observed after 2 wk of treatment. The BMP-treated cells appeared larger than untreated cells, grew more slowly, had more senescence-associated β-galactosidase activity, and had less telomerase activity, as measured by the telomeric repeat amplification protocol assay. Invasion through Engelbreth Holm-Swarm matrix was inhibited in the senescent cell population. Senescent BMP4-treated cells had lower ERK activation, VEGF expression, and Bcl2 expression than wild-type cells, consistent with a less proliferative, less angiogenic phenotype with increased susceptibility to death by apoptosis. BMP4 treatment also resulted in sustained elevation of Smad1. In vivo xenograft studies in the flanks of nude mice confirmed that the BMP-treated cells were significantly less tumorigenic than untreated cells. Direct overexpression of Smad1 using adenoviral constructs resulted in cell death within 5 days. These studies suggest that BMP4 pathway signaling can induce senescence and thus negatively regulate the growth of A549 lung cancer cells.
DNA synthesis and Bcl-2 expression during development of mucous cell metaplasia in airway epithelium of rats exposed to LPS
Authors: Tesfaigzi Yohannes , Harris J. Foster , Hotchkiss Jon A. , Harkema Jack R.
Published: January 2004

Exposure of pulmonary airways to environmental toxins and allergens may cause proliferation of airway epithelial cells and mucous cell metaplasia (MCM); however, it is unclear to what extent proliferating cells differentiate into mucus-storing cells and contribute to MCM. Our previous studies demonstrated that Bcl-2, an inhibitor of apoptosis with cell cycle regulatory functions, is expressed in metaplastic mucous cells. The purpose of the present study was to investigate the number of metaplastic mucous cells that are derived from proliferating epithelial cells and whether Bcl-2 has a role in cell cycle entry in these cells. Rats were intratracheally instilled with 100 μg of LPS from Pseudomonas aeruginosa in 500 μl of saline, and proliferating airway cells were labeled with bromodeoxyuridine (BrdU) by implanting a subcutaneous osmotic pump 24 h before instillation. The volume of stored mucosubstance and the number of mucous cells were increased 10- and 3-fold, respectively, from 24–48 h after instillation. The number of total epithelial cells per millimeter of basal lamina increased, and the number of serous cells per millimeter of basal lamina decreased during this time. Approximately 50% of Alcian blue-periodic acid Schiff-stained mucous cells were labeled with BrdU at 48 h after instillation, suggesting that one-half of the secretory cells were derived from proliferating cells. Furthermore, 50% of the Bcl-2-positive mucous cells were BrdU negative and therefore derived from nonproliferating, preexisting cells. Our findings demonstrate that preexisting and proliferating cells differentiate into mucous cells and compose LPS-induced metaplasia and that Bcl-2 does not have cell cycle regulatory function in these cells.
Mitochondrial cytochromecrelease is a key event in hyperoxia-induced lung injury: protection by cyclosporin A
Authors: Pagano Alessandra , Donati Yves , Métrailler Isabelle , Barazzone Argiroffo Constance
Published: January 2004

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.
Airway responsiveness after acute exposure to urban particulate matter 1648 in a DO11.10 murine model
Authors: Archer Amy J. , Cramton Jennifer L. H. , Pfau Jean C. , Colasurdo Giuseppe , Holian Andrij
Published: January 2004

Enhanced airway responsiveness (AR) is a well-established characteristic of asthma that epidemiological evidence has linked with inhalation of ambient particulate matter (PM). To determine whether acute exposure to urban particulate matter PM1648 can exacerbate airway responsiveness and alter the early inflammatory state, a unique murine model was created using DO11.10 mice, transgenic for a T cell receptor recognizing ovalbumin323-339. Because these mice are sensitive to ovalbumin, immunization procedures involving adjuvant or long aerosolization procedures are not necessary and, therefore, allow for the study of an acute AR response to particulate and antigen in young animals. AR was assessed by barometric whole body plethysmography and measured by enhanced pause (Penh). PM1648 and ovalbumin were administered intranasally 72 and 4 h before to AR assessment, respectively. A dose-response relationship between PM1648 and Penh was determined, and doses at or above 500 μg had Penh values significantly higher than saline controls. Penh values of control particle titanium dioxide (TiO2) were similar to saline controls demonstrating no nonspecific particulate effect on AR. Lung lavage at time of AR assessment showed no significant inflammation due to particulate exposure or ovalbumin alone; however, PM1648/ovalbumin and TiO2/ovalbumin combinations resulted in significant neutrophilia. In addition, treatment with polymyxin B to remove surface-bound endotoxin did not significantly affect Penh levels. These results indicate that PM1648 specifically increases AR in a dose-dependent manner and that this exacerbation is not a direct response to increased neutrophil concentration, particle-bound endotoxin or nonspecific particle effects.
Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites
Authors: Das Kumuda C. , Dashnamoorthy Ravi
Published: January 2004

Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 (Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 (Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2did not phosphorylate Ser37 in either ATM+/+or ATM-/-cells. Furthermore, H2O2failed to phosphorylate Ser15 in ATM-/-cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM+/+as well as in ATM-/-cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2or UV light.
Protein kinase C inhibits BKCachannel activity in pulmonary arterial smooth muscle
Authors: Barman Scott A. , Zhu Shu , White Richard E.
Published: January 2004

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in pulmonary vascular smooth muscle and cause pulmonary vasodilatation. BKCachannel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCachannel has been implicated in the development of pulmonary vasoconstriction. Protein kinase C (PKC) causes pulmonary vasoconstriction, but little is known about the effect of PKC on BKCachannel activity. Accordingly, studies were done to determine the effect of PKC activation on cAMP-induced BKCachannel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 μM), a stimulator of adenylate cyclase and an activator of cAMP, opened BKCachannels in single FHR PASMC, which were blocked by the PKC activators phorbol 12-myristate 13-acetate (100 nM) and thymeleatoxin (100 nM). The inhibitory response by thymeleatoxin on forskolin-induced BKCachannel activity was blocked by Gö-6983, which selectively blocks the α, β, δ, γ, and ζ PKC isozymes, and Gö-6976, which selectively inhibits PKC-α, PKC-β, and PKC-μ, but not by rottlerin, which selectively inhibits PKC-δ. Collectively, these results indicate that activation of specific PKC isozymes inhibits cAMP-induced activation of the BKCachannel in pulmonary arterial smooth muscle, which suggests a unique signaling pathway to modulate BKCachannels and subsequently cAMP-induced pulmonary vasodilatation.
Nucleotide-mediated inhibition of alveolar fluid clearance in BALB/c mice after respiratory syncytial virus infection
Authors: Davis Ian C. , Sullender Wayne M. , Hickman-Davis Judy M. , Lindsey J. Russell , Matalon Sadis
Published: January 2004

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in infants and children worldwide. Intranasal infection of BALB/c mice with RSV strain A2, but not ultraviolet-inactivated RSV, for 2 or 4 days reduced basal alveolar fluid clearance (AFC), a seminal function of bronchoalveolar epithelium, and caused loss of AFC sensitivity to amiloride inhibition. Reduced AFC was temporally associated with increased lung water content but was not a consequence of increased epithelial permeability or cell death. Reduced AFC was also not due to decreased transcription of epithelial Na+channel subunit genes in lung tissue. RSV-mediated inhibition of AFC 2 days after infection was rapidly prevented by addition to the instillate of P2Y receptor antagonists (suramin and XAMR-0721) or enzymes that degrade UTP, but not those that degrade ATP. After UTP degradation, AFC returned to control levels but was no longer sensitive to amiloride. UTP at nanomolar concentrations recapitulated the AFC inhibitory effect of RSV in normal mice and mice infected with RSV for 6 days, indicating that normalization of AFC at this time point is a consequence of cessation of UTP release, rather than P2Y receptor desensitization. We conclude that RSV infection of the bronchoalveolar epithelium results in reduced AFC as a consequence of autocrine feedback inhibition mediated by UTP. These studies are the first to demonstrate AFC inhibition by an important pulmonary viral pathogen. Reduced AFC may result in formation of an increased volume of fluid mucus, airway congestion, and rhinorrhea, all features of severe RSV disease.
DEP-inducedfra-1expression correlates with a distinct activation of AP-1-dependent gene transcription in the lung
Authors: Zhang Qin , Kleeberger Steven R. , Reddy Sekhar P.
Published: January 2004

Recent studies indicate a potential role for Fra-1, a heterodimeric partner of activator protein (AP)-1, in toxicant-induced epithelial injury, repair, and cellular transformation. Here we have investigated the effects of diesel exhaust particles (DEP) on fra-1 expression in C10 cells, a murine lung epithelial cell line. DEP markedly upregulated fra-1, but not fra-2, expression. The increase in fra-1 mRNA expression correlated well with its protein- and DNA-binding activity. DNA-binding assays also revealed a predominant presence of Jun-B and Jun-D in the AP-1 complex. Interestingly, DEP did not alter Jun-B and Jun-D protein levels. Transcriptional analysis revealed that fra-1 induction is regulated in part at the transcriptional level. The -379 to +32 bp 5′-flanking region mediated this induction. Furthermore, inhibitors of ERK1/2, JNK1, and p38 mitogen-activated protein kinases (MAPKs) significantly suppressed DEP-stimulated fra-1 transcription, suggesting their involvement in the induction process. Consistent with this finding, DEP stimulated phosphorylation of ERK1/2, JNK1, and p38 MAPKs with a distinct activation pattern. Overexpression of Fra-1 downregulated c-Jun and Nrf2 enhanced AP-1- and ARE-mediated reporter gene expression, respectively. In contrast, Fra-1 had the opposite effect on matrix metalloproteinase (MMP)-9 promoter activity. In particular, it bound to the functional AP-1 site of the MMP-9 promoter after DEP stimulation. Consistent with this result, DEP also markedly upregulated MMP-9 promoter activity. Collectively, these findings suggest that fra-1 induction by DEP may play a role in selectively regulating gene expression involved in alveolar epithelial cell injury and repair.
Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins
Authors: Shi Wei , Chen Hui , Sun Jianping , Chen Cheng , Zhao Jingsong , Wang Yan-Ling , Anderson Kathryn D. , Warburton David
Published: January 2004

Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. Bone morphogenetic protein 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant protein C and Clara cell protein 10 expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, protein levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth.
Glutathione regulates transforming growth factor-β-stimulated collagen production in fibroblasts
Authors: Liu Rui-Ming , Liu Yanwen , Forman Henry Jay , Olman Mitchell , Tarpey Margaret M.
Published: January 2004

Transforming growth factor-β (TGF-β) is a potent fibrogenic cytokine. The molecular mechanism underlying TGF-β fibrogenesis, however, has not been completely elucidated. In this study, we showed that TGFβ decreased the intracellular GSH content in murine embryo fibroblasts (NIH 3T3), which was followed by an increase in collagen I mRNA content and collagen protein production. Prevention of GSH depletion with N-acetylcysteine (NAC), GSH, or GSH ester abrogated TGF-β-stimulated collagen production, whereas a decrease in intracellular GSH content with l-buthionine- S,R-sulfoximine, an inhibitor of de novo GSH synthesis, enhanced TGF-β-stimulated collagen production. These results suggest that GSH depletion induced by TGF-β may mediate TGF-β-stimulated collagen production. In addition, we showed that TGF-β stimulated superoxide production and increased release of H2O2from the cells, whereas GSH ester decreased basal and TGF-β + glucose oxidase-stimulated H2O2release. H2O2, exogenously added or continuously generated by glucose oxidase, enhanced TGF-β-stimulated collagen production, whereas suppression of superoxide production by diphenyliodonium, an NAD(P)H oxidase inhibitor, blocked TGF-β-stimulated collagen production. These data further suggest that reactive oxygen species are involved in TGF-β-stimulated collagen production and that the effect of GSH depletion on TGF-β-stimulated collagen production may be mediated by facilitating reactive oxygen species signaling.
Continuous subcutaneous injection reduces polymorphonuclear leukocyte activation by granulocyte colony-stimulating factor
Authors: Sato Yukio , Goto Yukinobu , Sato Shoko , Endo Shunsuke , Sohara Yasunori
Published: January 2004

The use of granulocyte colony stimulating factor (G-CSF) for recovery from neutropenia has been established; however, acute lung injury due to G-CSF-induced polymorphonuclear leukocyte (PMN) activation is a serious complication. This study was designed to compare the activation of PMN with single bolus administration and continuous administration of G-CSF. Healthy volunteers (age 33.8 ± 1.4 yr; n = 6) received a single bolus injection of 50 μm/m2of G-CSF (SI; n = 6) or continuous subcutaneous injection of 50 μm/m2of G-CSF for 24 h (CI; n = 6) and were followed for 48 h. Circulating leukocyte counts, markers of activation on PMN, and circulating levels of G-CSF, IL-6, and PMN elastase were measured. SI rapidly increased serum G-CSF levels, which peaked at 4 h, whereas CI gradually increased G-CSF levels, which remained at a steady level from 8 to 24 h. SI caused a rapid decrease in PMN counts at 0.5 h followed by sustained increase to peak at 12 h. CI gradually increased PMN counts, which peaked at 24 h, but the peak values were not significantly different between the groups. SI-induced activation of PMN, which was characterized by increased expression of CD11b, decreased expression of L-selectin, and increased F-actin content, led to increases in serum IL-6 and PMN elastase level. Such changes were all attenuated with CI ( P < 0.05). We conclude that continuous subcutaneous injection of G-CSF resulted in a marrow response similar to that to a single injection but yielded reduced PMN activation.
G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation
Authors: Walker J. K. L. , Gainetdinov R. R. , Feldman D. S. , McFawn P. K. , Caron M. G. , Lefkowitz R. J. , Premont R. T. , Fisher J. T.
Published: January 2004

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M2muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene ( GRK5- /-) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5- /-and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from β2-adrenergic receptor activation were diminished in GRK5- /-mice. These data suggest that M2muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5- /-mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.
Cytokine mRNA expression in unilateral ischemic-reperfused rat lung with salt solution supplemented with low-endotoxin or standard bovine serum albumin
Authors: Teng Shu , Kurata Shunichi , Katoh Iyoko , Georgieva Gabriela S. , Nosaka Toshihisa , Mitaka Chieko , Imai Takasuke
Published: January 2004

Our aim was to determine whether cytokine mRNA expression is induced by experimental manipulation including artificial perfusate or ischemia-reperfusion (I/R) in an isolated, perfused rat lung model. Constant pulmonary flow [Krebs-Henseleit solution supplemented with lowendotoxin (LE) or standard (ST) bovine serum albumin 4%, 0.04 ml/g body wt] and ventilation were maintained throughout. Right and left pulmonary arteries were isolated, and the left pulmonary artery was occluded for 60 min and then reperfused for 30 min. Analysis of tumor necrosis factor-α, IL-1β, IL-6, IL-10, and IFN-γ mRNA expression by RT-PCR and evaluation of vascular permeability by bronchoalveolar lavage (BAL) fluid albumin content were conducted separately in right and left lung. Both LE and ST groups (each 12 rats) showed increases in vascular permeability by I/R (BAL fluid albumin content: 5.53 ± 1.55 vs. 15.63 ± 8.87 and 4.76 ± 2.71 vs. 16.72 ± 4.85 mg·ml BAL fluid-1·g lung dry wt-1, mean ± SD; right vs. left lung in LE and ST groups, P < 0.05 between right and left). Cytokine mRNA expression was significantly higher in the I/R lung than in the control lung in the LE group, whereas it was higher in the control lung in the ST group ( P < 0.05). mRNAs of not only proinflammatory but also anti-inflammatory cytokines were expressed in I/R lung, which are expected to aggravate I/R injury. The reversed pattern of cytokine mRNA expression in the ST group was possibly due to the longer perfusion of control lung with perfusate containing endotoxin, which caused no lung damage without I/R.