Showing papers in "American Journal of Respiratory Cell and Molecular Biology in 2002"

Role of NRF2 in protection against hyperoxic lung injury in mice

Abstract: NRF2 is a transcription factor important in the protection against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes. This study was designed to determine the role of NRF2 in the pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95-98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2-/-) and wild-type mice (Nrf2+/+). Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury in Nrf2-/- mice were 7.6-fold, 47%, and 43% greater, respectively, compared with Nrf2+/+ mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of NRF2 in the lungs of Nrf2+/+ mice. mRNA expression for ARE- responsive lung antioxidant and phase 2 enzymes was evaluated in both genotypes of mice to identify potential downstream molecular mechanisms of NRF2 in hyperoxic lung responses. Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice. Consistent with differential mRNA expression, NQO1 and total GST activities were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in mice, possibly through transcriptional activation of lung antioxidant defense enzymes.

Heme oxygenase-1: the "emerging molecule" has arrived.

Abstract: Organisms on our planet have evolved in an oxidizing environment that is intrinsically inimical to life, and cells have been forced to devise means of protecting themselves. One of the defenses used most widely in nature is the enzyme heme oxygenase-1 (HO-1). This enzyme performs the seemingly lackluster function of catabolizing heme to generate bilirubin, carbon monoxide, and free iron. Remarkably, however, the activity of this enzyme results in profound changes in cells' abilities to protect themselves against oxidative injury. HO-1 has been shown to have anti-inflammatory, antiapoptotic, and antiproliferative effects, and it is now known to have salutary effects in diseases as diverse as atherosclerosis and sepsis. The mechanism by which HO-1 confers its protective effect is as yet poorly understood, but this area of invetsigation is active and rapidly evolving. This review highlights current information on the function of HO-1 and its relevance to specific pulmonary and cardiovascular diseases.

Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease.

Abstract: Destruction of lung elastin is critical for development of emphysema associated with chronic obstructive pulmonary disease (COPD). Lung macrophages release elastolytic enzymes, including matrix metalloproteinase (MMP)-9, along with tissue inhibitors of MMP (TIMP). We examined the production and activity of macrophage-derived MMP-9 and TIMP-1 from alveolar macrophages (AM) from smokers with COPD, healthy smokers (HS), and nonsmokers (NS). AM were stimulated with either lipopolysaccharide (LPS), interleukin (IL)-1 beta, or cigarette smoke-conditioned culture medium (CSM). AM from patients with COPD released greater amounts of MMP-9 with greater enzymatic activity than HS and NS. In contrast, AM from NS released more TIMP-1 than cells from HS and subjects with COPD. LPS and IL-1 beta caused a dose-dependent increase in MMP-9 release and activity, together with increased levels of TIMP-1. Dexamethasone prevented the increase in MMP-9 release, and increased TIMP-1 release. CSM increased MMP-9 and TIMP-1 release from AM of all groups. Dexamethasone decreased CSM-stimulated MMP-9 release, but had no effect on MMP-9 activity This study suggests that macrophages might be important in the development of COPD because these cells exhibit increased levels of elastolytic activity.

Interleukin-17 Stimulates the Expression of Interleukin-8, Growth-Related Oncogene- α , and Granulocyte–Colony-Stimulating Factor by Human Airway Epithelial Cells

Abstract: Interleukin (IL)-17 is a recently discovered cytokine, which is proposed to play a role in neutrophilic airway inflammation via the release of proinflammatory cytokines and chemokines. To evaluate the role of IL-17 in inflammatory protein production from the airway epithelium, we have analyzed the effects of IL-17 on primary human bronchial epithelial cells (HBECs). Using gene arrays, changes in gene expression in response to IL-17 stimulation were investigated and only IL-8, growth-related oncogene (Gro) α , and granulocyte colony-stimulating factor (G-CSF) were found to be upregulated. Secretion of IL-8, Gro α , and G-CSF in response to IL-17 was measured in HBEC cell culture supernatants by enzyme-linked immunosorbent assay. Upregulation of Gro α , IL-8, and G-CSF was observed to be 8-, 5-, and 8-fold, respectively, after 48 h stimulation with IL-17. When tested at equivalent concentrations, IL-17 was found to be 2- to 3-fold more potent than tumor necrosis factor (TNF)- α in stimulating release of Gro...

Long-Term Intratracheal Lipopolysaccharide Exposure in Mice Results in Chronic Lung Inflammation and Persistent Pathology

Abstract: Lipopolysaccharide (LPS), a major proinflammatory glycolipid component of the gram-negative bacterial cell wall, is one of the agents ubiquitously present as contaminant on airborne particles, including air pollution, organic dusts, and cigarette smoke. Chronic exposure to significant levels of LPS is reported to be associated with the development and/or progression of many types of lung diseases, including asthma, chronic bronchitis, and progressive irreversible airflow obstruction, that are all characterized by chronic inflammatory processes in the lung. In the present study, pathologic effects of long-term LPS exposure to the lung were investigated in detail. To this end, a murine model in which mice were exposed to repeated intratracheal instillation of Escherichia coli LPS was developed. We show that long-term LPS instillation in mice results in persistent chronic pulmonary inflammation, characterized by peribronchial and perivascular lymphocytic aggregates (CD4(+), CD8(+), and CD19(+)), parenchymal accumulation of macrophages and CD8(+) T cells, and altered cytokine expression. Furthermore, airway and alveolar alterations such as mucus cell metaplasia, airway wall thickening, and irreversible alveolar enlargement accompanied the chronic inflammatory response. Interestingly, the observed inflammatory and pathologic changes mimic changes observed in human subjects with chronic inflammatory lung diseases, especially chronic obstructive pulmonary disease (COPD), suggesting that this murine model could be applicable to dissect the role of inflammation in the pathogenesis of these disease conditions.

Asthmatic Bronchial Epithelium Is More Susceptible to Oxidant-Induced Apoptosis

Abstract: Abnormal apoptotic mechanisms are associated with disease pathogenesis. Because the asthmatic bronchial epithelium is characteristically damaged with loss of columnar epithelial cells, we postulated that this is due to unscheduled apoptosis. Using an antibody directed toward the caspase cleavage product of poly(ADP-ribose) polymerase, immunohistochemistry applied to endobronchial biopsies showed higher levels of staining in the bronchial epithelium of subjects with asthma as compared with normal control subjects (% epithelial staining [median (range) = 10.5 (1.4–24.5) versus 0.4 (0.0–9.7)]; P < 0.001). Because we were unable to determine whether this difference was due to ongoing inflammation in vivo, cultures of normal and asthmatic bronchial epithelial cells were used to study apoptosis in vitro. In complete growth medium, these cells showed no difference in their rate of proliferation or viability. However, cells from subjects with asthma were more susceptible to the apoptotic effects of H2O2 than cell...

The Th2 lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness through direct effects on resident airway cells.

Abstract: Airway inflammation and airway hyperresponsiveness (AHR) are hallmarks of asthma. Cytokines produced by T helper type 2 (Th2) lymphocytes have been implicated in both processes. There is strong support for the idea that Th2 cytokines can produce AHR indirectly by promoting the recruitment of inflammatory cells. Less attention has been given to the possibility that Th2 cytokines might induce AHR by acting directly on resident airway cells. To investigate this, we polarized and activated CD4(+) T cells in vitro and analyzed airway function after administration of lymphocyte-conditioned media to the airways of naive mice. Th2-lymphocyte-conditioned medium induced AHR within 6 h. This finding was reproduced in mast-cell-deficient and in T- and B-lymphocyte-deficient mice. AHR did not occur when Th2-lymphocyte-conditioned medium was administered to mice lacking the IL-4 receptor alpha subunit or Stat6, suggesting a critical role for interleukin (IL)-4 and/or IL-13. This was confirmed by the finding that recombinant IL-4 and IL-13 both induced AHR within 6 h. The induction of AHR occurred in the absence of inflammatory cell recruitment or mucus production. These results strongly suggest that products of activated Th2 lymphocytes can rapidly perturb airway function through direct effects on resident airway cells.

Differential effects of stable prostacyclin analogs on smooth muscle proliferation and cyclic AMP generation in human pulmonary artery.

Abstract: Primary pulmonary hypertension is characterized by increased pulmonary vascular resistance and smooth muscle proliferation. Stable analogs are increasingly being used to treat this disease, although no data exists comparing their effects on proliferation. We therefore investigated the antiproliferative activity of several prostacyclin (PGI(2)) analogs on human pulmonary arterial smooth muscle cells, including UT-15 and iloprost, analogs that have recently completed successful clinical trials. Serum-induced proliferation, as assessed by [(3)H]thymidine incorporation (30 h) or cell number (48 h), was significantly inhibited with a 10-fold difference in potency, ranking in effectiveness UT-15 > iloprost > cicaprost > beraprost. Effects were reversed by the adenylyl cyclase inhibitor, 2,5'dideoxyadenosine (DDA) but not SQ22536. Intracellular cyclic AMP (cAMP) was elevated by all analogs and inhibited by DDA, although SQ22536 was a highly variable inhibitor, suggesting that different pathways might mediate cAMP generation. UT-15 produced a significantly larger and more sustained increase in cAMP compared with other analogs, with iloprost being the weakest elevator. Thus, PGI(2) analogs potently inhibit proliferation of human pulmonary artery, probably via a cAMP-dependent pathway, although cAMP elevation in itself is not a good predictor of antiproliferative potency.

Involvement of microbial components and toll-like receptors 2 and 4 in cytokine responses to air pollution particles.

Abstract: Inhalation of particulate matter (PM) may result in exacerbation of inflammatory airways disease, including asthma. Results from this laboratory have shown that the coarse inhalable particle fraction (PM2.5–10) is responsible for most of the PM effects on human airway macrophages (AM), including induction of cytokine production. Endotoxins associated with these particles account for a large part of their potency, as activity of PM can be inhibited by polymixin B and an activating moiety bound by lipopolysaccharide (LPS)-binding protein (LBP). The hypothesis behind the present study was that not only particle-bound LPS, but also Gram-negative (Gram−) and Gram-positive (Gram+) bacteria are responsible for PM-induced stimulation of AM, and therefore that PM are likely to activate receptors involved in recognition of microbes. Low level contamination of model pollution particles with environmental Staphyloccocus, Streptococcus, and Pseudomonas species was found to confer cytokine-inducing activity on inactive...

Modeling allergic asthma in mice: pitfalls and opportunities.

Abstract: Studies in murine experimental models have contributed greatly to understanding the mechanisms of allergic inflammation underlying asthma. However, models involving short-term high-level exposure of sensitized animals to antigen have significant limitations for investigating the pathogenesis of the lesions of chronic asthma. Modeling chronic asthma is problematic, because long-term antigenic challenge often triggers widespread pulmonary parenchymal inflammation or leads to eventual downregulation of inflammation and airway hyperreactivity. We have developed an improved murine model in which animals are exposed to low mass concentrations of aerosolized antigen for 6-8 wk. The mice exhibit airway-specific acute-on-chronic inflammation and changes of airway wall remodeling as seen in human asthma, together with hyperreactivity to a cholinergic agonist which can be specifically attributed to airway disease. This more realistic model of asthma offers a number of opportunities for investigation of pathogenetic mechanisms and novel therapeutic agents.

Neutrophil elastase induces MUC5AC gene expression in airway epithelium via a pathway involving reactive oxygen species.

Abstract: Neutrophil-predominant airway inflammation and mucus obstruction of the airways are major pathologic features of chronic airway diseases, including cystic fibrosis and chronic bronchitis. Neutrophils release elastase, a serine protease that impairs mucociliary clearance and stimulates goblet cell metaplasia and mucin production. We previously reported that neutrophil elastase increases expression of a major respiratory mucin gene, MUC5AC, by enhancing mRNA stability. However, the molecular mechanisms of elastase-regulated MUC5AC expression are not known. We hypothesized that reactive oxygen species, generated by elastase treatment, mediate MUC5AC gene expression. To test this hypothesis, A549, a respiratory epithelial cell line, was treated with elastase in the presence or absence of the oxygen radical scavenger, dimethylthiourea, or the iron chelator, desferrioxamine. MUC5AC mRNA levels were assessed by Northern analysis. Both antioxidants significantly inhibited elastase-induced MUC5AC gene expression. ...

Acute cigarette smoke-induced connective tissue breakdown requires both neutrophils and macrophage metalloelastase in mice.

Abstract: The cells/proteases responsible for the development of smoke-induced emphysema is an area of intense investigation. Mice with knockout of macrophage metalloelastase genes (MME−/−) do not develop emphysema after smoke exposure, but we also observed that neutrophils (PMN) in lavage appeared to be a requirement for acute connective tissue breakdown. In this study we exposed mice to cigarette smoke and examined lavage PMN, macrophages (MAC), desmosine (DES, a measure of elastin breakdown) and hydroxyproline (HP, a measure of collagen breakdown) 24 h afterwards. MME+/+ mice exposed to smoke showed elevations in PMN, DES, and HP, but no elevations were seen in MME-deficient mice. Both PMN influx and increased levels of DES/HP could be restored by administering MAC from MME+/+ mice to MME-deficient mice and then exposing them to smoke. RS113456, a metalloprotease inhibitor, also prevented PMN influx and connective tissue breakdown. Western blots against mouse α1-antitrypsin (α1AT) showed that α1AT was not protec...

Shear Stress-Mediated Cytoskeletal Remodeling and Cortactin Translocation in Pulmonary Endothelial Cells

Abstract: Hemodynamic forces in the form of shear stress (SS) and mechanical strain imposed by circulating blood are recognized factors involved in the control of systemic endothelial cell (EC) cytoskeletal structure and function. However, the effects of acute SS on pulmonary endothelium have not been precisely characterized, nor the mechanism of rapid SS-induced EC cytoskeletal rearrangement understood. We exposed bovine and human pulmonary EC monolayers to laminar SS (10 dynes/ cm2) in a parallel plate flow chamber and observed increased actin stress fiber formation 15 min after application of flow. Acute SS-induced pronounced cortical cytoskeletal rearrangement characterized by myosin light chain kinase (MLCK)- and Rho-associated kinase (RhoK)-dependent accumulation of diphosphorylated regulatory myosin light chains (MLC) in the cortical actin ring, junctional protein tyrosine phosphorylation, and transient peripheral translocation of cortactin, an actin-binding protein involved in the regulation of actin polyme...

Interaction of IL-13 and C10 in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Abstract: The initial stimulus for inflammatory cell recruitment and the mechanisms responsible for the perpetuation and evolution of chronic inflammation, granulation tissue formation, and fibrosis have not been fully elucidated. Although interleukin (IL)-13, a Th2 cytokine, has been shown to have direct effects on fibroblasts that support fibroproliferation, it is also a potent inducer of a novel CC chemokine, C10, which is chemotactic for mononuclear phagocytes. The macrophage/mononuclear phagocyte has been shown to have a role in the pathogenesis of pulmonary fibrosis, serving as an important source of growth factors that regulate extracellular matrix synthesis. In this study we demonstrate that IL-13 and C10 are elevated in the pathogenesis of bleomycin-induced pulmonary fibrosis. Neutralization of IL-13, but not IL-4, attenuated bleomycin-induced pulmonary fibrosis and levels of C10, suggesting that IL-13 has an important role in the development of pulmonary fibrosis. IL-13 is a potent inducer of C10 in vivo, and neutralization of C10 attenuated bleomycin-induced pulmonary fibrosis and intrapulmonary macrophage numbers. This suggests that IL-13 has a role in the development of pulmonary fibrosis that is independent of its direct effect on fibroblasts and is evidence for an interaction between Th2 cytokines and specific CC chemokines.

Interaction of alveolar macrophages and airway epithelial cells following exposure to particulate matter produces mediators that stimulate the bone marrow.

Abstract: Exposure to ambient air pollution particles with a diameter of < 10 microm (PM(10)) has been associated with increased cardiopulmonary morbidity and mortality. We postulate that these adverse health effects are related to proinflammatory mediators produced in the lung and released into the circulation where they initiate a systemic inflammatory response. The present study was designed to determine if alveolar macrophages (AMs) and primary human bronchial epithelial cells (HBECs) interact to amplify the production of certain cytokines when exposed to ambient PM(10) (EHC-93). Candidate cytokines were measured at the mRNA level using a RNase protection assay and at the protein level by enzyme-linked immunosorbent assay (ELISA). When AM/HBEC cocultures were exposed to 100 microg/ml of PM(10), levels of tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)-1beta, IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), and IL-8 mRNA increased within 2 h (P < 0.05) and 8 h following exposure compared with control cells. GM-CSF mRNA expression was more rapidly induced in cocultured cells compared with HBECs or AMs alone. The concentrations of TNF-alpha, GM-CSF, IL-1beta, IL-6, and IL-8 in the cocultured supernatants collected after 24 h PM(10) exposure increased significantly compared with control cells. There was a significant synergistic effect between AMs and HBECs in the production of GM-CSF and of IL-6 (P < 0.05). Instillation of supernatants from HBECs cultured with PM(10) into lungs of rabbits failed to increase circulating band cell counts or stimulate the bone marrow. However, those from AM/HBEC cocultures exposed to PM(10) increased circulating band cell counts (P < 0.05) and shortened the transit time of polymorphonuclear leukocytes (PMNs) through the bone marrow compared with control co-cultures (P < 0.01). These results suggest that the interaction between AMs and HBECs during PM(10) exposure contributes to the production of mediators that induce a systemic inflammatory response.

Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth muscle cells: modulation by glucocorticoids and beta-agonists.

Abstract: Recent studies have demonstrated that tumor necrosis factor (TNF)-alpha stimulates the secretion of interleukin (IL)-6 and regulated on activation, normal T cells expressed and secreted (RANTES) from airway smooth muscle (ASM) cells, with the induction of each molecule being differentially regulated (IL-6 increased, RANTES inhibited) by cyclic adenosine monophosphate (cAMP)-elevating agents. In this study we identify the mechanisms mediating IL-6 and RANTES gene transcription in human ASM cells. We found that TNF-alpha induced IL-6 gene expression in ASM cells via a nuclear factor (NF)-kappaB-dependent pathway, whereas RANTES gene expression was mediated via activation of activator protein (AP)-1 and nuclear factor of activated T cells (NF-AT). TNF-alpha-induced IL-6 secretion was only partially inhibited by dexamethasone, yet TNF-alpha-induced RANTES secretion was abolished. beta-Agonists induced IL-6 secretion from ASM via activation of the CRE region of the IL-6 promoter. beta-Agonists augmented TNF-alpha-induced IL-6 secretion, reflecting an additive effect of NF-kappaB and CRE response elements on IL-6 gene expression. In contrast, beta-agonists inhibited TNF-alpha-induced RANTES secretion via an AP-1-independent pathway. Collectively, these data elucidate transcriptional mechanisms mediating TNF-alpha-induced IL-6 and RANTES secretion from ASM cells, and identify the specific cis- or trans-acting elements that determine the differential effects of glucocorticoids and cAMP-elevating agents on the expression of these genes.

Production of interleukin-6 by skeletal myotubes: role of reactive oxygen species.

Abstract: In the present study we have tested the ability of reactive oxygen species (ROS) to stimulate the production of interleukin (IL)- 6 from skeletal myocytes. Differentiated C2C12 murine skeletal muscle cells (myotubes) exposed to pyrogallol (PYR), xanthine/ xanthine-oxidase (X/XO), or H2O2 for 24 h exhibited a concentration-dependent increase in IL-6 production. Unlike myotubes, incubation of myoblasts and endothelial cells with X/XO or PYR did not result in increased IL-6 release. In myotubes, superoxide dismutase and catalase blocked the ROS-induced IL-6 release. Exposure of myotubes to H2O2 increased steady-state IL-6 mRNA levels, and pretreatment of myotubes with actinomycin D or cycloheximide abolished the ROS-induced IL-6 production. In addition, pretreatment of cells with N-acetyl-cysteine blocked tumor necrosis factor (TNF)- α –induced IL-6 release, suggesting that endogenously produced ROS participate in IL-6 production. Myotubes stimulated with H2O2 exhibited increased I κ B- α phosphorylation and...

Interleukin-13 mediates a fundamental pathway for airway epithelial mucus induced by CD4 T cells and interleukin-9.

Abstract: Mucus hyperproduction in asthma results from Th2-induced airway inflammation. Controversy exists about the precise mechanism of this Th2 effect. Although we showed that mucus can be induced by Th2 cells in the absence of interleukin (IL)-4, IL-5, eosinophils, and mast cells, but not without IL-4Ralpha signaling, others demonstrated that IL-4 and IL-9 can directly stimulate airway epithelial mucus. Using a system in which in vitro-generated T cell receptor transgenic Th2 cells are transferred into recipient mice and activated in the respiratory tract with inhaled antigen, we now show that CD4 Th cells can stimulate mucus only through a common, IL-13-mediated pathway. All Th cytokines depend on IL-13 for this effect and IL-13 acts, not through intermediate inflammatory cells, but on structural cells within the lung, likely the airway epithelium itself. The potency of IL-13 is shown, requiring its complete blockade for a significant reduction in mucus production. We show that mucus induction by Th2 cells does not require nuclear factor-kappaB, unlike mucins induced by gram-negative infection. These studies define in vivo pathways that lead to mucus induction and indicate that, whereas IL-13 mediates a dominant pathway for CD4 Th induced inflammation, other inflammatory stimuli activate the epithelium to produce mucus by different pathways.

Dysfunction and Remodeling of the Mouse Airway Persist after Resolution of Acute Allergen-Induced Airway Inflammation

Abstract: The mechanisms underlying airway hyperresponsiveness remain unclear, although airway inflammation and remodeling are likely important contributing factors. We hypothesized that airway physiology would differ between mice subjected to brief or chronic allergen exposure, and that these differences would be associated with characteristic inflammatory markers and indices of airway remodeling. BALB/c mice were sensitized to ovalbumin and studied at several time points following brief or chronic allergen challenge protocols. By measuring airway responses to methacholine, we demonstrated increases in maximal inducible bronchoconstriction that persisted for 8 wk following either brief or chronic allergen challenge; we also observed increases in airway reactivity, although it was only in chronically challenged mice that these changes persisted beyond the resolution of allergen-induced inflammation. Using airway morphometry, we further demonstrated that increases in maximal bronchoconstriction were associated with increases in airway contractile tissue in both models, and that chronic, but not brief, allergen challenge resulted in subepithelial fibrosis. Our observations that different aspects of sustained airway dysfunction and remodeling persist beyond the resolution of acute inflammatory events support the concept that remodeling occurs as a consequence of allergic airway inflammation, and that these structural changes contribute independently to the persistence of airway hyperresponsiveness.

Maternal nicotine exposure upregulates collagen gene expression in fetal monkey lung. Association with alpha7 nicotinic acetylcholine receptors.

Abstract: The recent identification of nicotinic acetylcholine receptors (nAChR) in pulmonary fibroblasts suggests that in utero nicotine exposure may alter collagen expression by these cells in the developing lung. To test this hypothesis, timed-pregnant rhesus monkeys were administered nicotine (1-1.5 mg/kg/d, subcutaneously) using osmotic minipumps from Days 26-134 or 26-160 of gestation (term = 165 d). In utero nicotine exposure significantly increased airway wall area per unit epithelial basement membrane. Collagen type I and III mRNA expression and immunostaining were significantly increased in the airway and alveolar walls of the nicotine-treated group. Elastin mRNA expression increased, but protein expression in parenchyma remained unchanged. Dual labeling studies colocalized alpha7 nAChR and collagen to the same cells in airway wall cells, and colocalization of alpha7 nAChR and collagen was confirmed in isolated pulmonary fibroblasts. These findings suggest that nicotine may directly interact with alpha7 nAChR to increase collagen accumulation in airway and alveolar walls following in utero nicotine exposure. These data suggest that passage of nicotine across the placenta to increase collagen deposition and therefore increase airway wall dimensions in fetal lung may partially explain the observed alterations in lung mechanics in the infants of mothers who smoke during pregnancy.

Linkage analysis of susceptibility to hyperoxia. Nrf2 is a candidate gene

Abstract: A strong role for reactive oxygen species (ROS) has been proposed in the pathogenesis of a number of lung diseases. Hyperoxia (> 95% oxygen) generates ROS and extensive lung damage, and has been used as a model of oxidant injury. However, the precise mechanisms of hyperoxia-induced toxicity have not been completely clarified. This study was designed to identify hyperoxia susceptibility genes in C57BL/6J (susceptible) and C3H/HeJ (resistant) mice. The quantitative phenotypes used for this analysis were pulmonary inflammatory cell influx, epithelial cell sloughing, and hyperpermeability. Genome-wide linkage analyses of intercross (F2) and recombinant inbred cohorts identified significant and suggestive quantitative trait loci on chromosomes 2 (hyperoxia susceptibility locus 1 [Hsl1]) and 3 (Hsl2), respectively. Comparative mapping of Hsl1 identified a strong candidate gene, Nfe2l2 (nuclear factor, erythroid derived 2, like 2 or Nrf2) that encodes a transcription factor NRF2 which regulates antioxidant and phase 2 gene expression. Strain-specific variation in lung Nrf2 messenger RNA expression and a T --> C substitution in the B6 Nrf2 promoter that cosegregated with susceptibility phenotypes in F2 animals supported Nrf2 as a candidate gene. Results from this study have important implications for understanding the mechanisms through which oxidants mediate the pathogenesis of lung disease.

Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis.

Abstract: Estimates of the level of transcripts from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene required to develop a CF phenotype range from 4–20% of normal. Due to the importance of obtaining reliable data on this issue for therapeutic strategies, we developed a novel polymerase chain reaction–based method to quantify CFTR transcripts and applied it to the analysis of nasal epithelium RNA of five patients with CF and the 3272-26A>G/F508del genotype. We calculated that 8.2 ± 0.84% of the total CFTR RNA present in these five patients is normal full-length CFTR mRNA. We then demonstrated (in nasal samples from F508del carriers, n = 30) that the abundance of full-length F508del CFTR transcripts is reduced compared with wild-type transcripts, and estimated that the average ratio of F508del/wild-type transcripts is 0.87 ± 0.06. To determine the amount of full-length transcripts relative to levels found in normal individuals, we corrected for the lower abundance of the F508del transcripts a...

Differences in the Fibrogenic Response after Transfer of Active Transforming Growth Factor-β1 Gene to Lungs of “Fibrosis-prone” and “Fibrosis-resistant” Mouse Strains

Abstract: Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium, resulting in impaired lung function and respiratory failure. Investigation of the differences in individual susceptibility to the development of fibrosis may help to detect patients that are at risk to fibrosis when exposed to fibrogenic stimuli. In this study we used adenoviral gene transfer to transiently expose a fibrosis-prone (C57BL/6) and a fibrosis-resistant (Balb/c) mouse strain to high levels of active transforming growth factor (TGF)-beta1, a key profibrotic cytokine. Balb/c mice developed significantly less fibrosis compared with C57BL/6 mice in response to active TGF-beta1 despite higher levels of the transgene protein in the lung. This was not due to a general unresponsiveness of cells to TGF-beta1, because primary fibroblasts of both strains increased collagen synthesis upon stimulation with TGF-beta1 in vitro to the same degree. However, TGF-beta1 induced a strong upregulation of tissue inhibitor of metalloprotease-1 gene in pulmonary fibroblasts as well as in lungs of C57BL/6 mice, in contrast to a weak induction in Balb/c mice. These findings suggest that the differences in susceptibility to pulmonary fibrosis are downstream from TGF-beta1 and that fibrosis-prone individuals may have an altered collagen metabolism in the lungs that is balanced toward a "nondegrading" environment.

HLA-DQB1*0201: a marker for good prognosis in British and Dutch patients with sarcoidosis.

Abstract: Human leukocyte antigen (HLA)-DQB1 is one of the intriguing candidate genes in sarcoidosis. We performed high resolution molecular HLA-DQB1 typing on two groups of white patients (British [UK] and Dutch [NL]) in order to investigate the relationship between 19 DQB1 alleles and disease severity and progression. A total of 803 individuals were studied (133 UK and 102 NL patients, and 354 UK and 214 NL control subjects). Disease severity data included extrapulmonary disease, chest X-ray stage, lung diffusing capacity for carbon monoxide, and FVC. Progression was evaluated on follow-up chest radiographs (2 and 4 yr). The results showed DQB1*0201 to be strongly protective against severe sarcoidosis in both populations; in other words, it localized to Stage I at all time points (P < 0.0001, P(corrected) (P(c)) = 0.002), whereas another DQB1 allele, *0602, tended to have opposite effects. Further, a clear association was found between the *0201 allele and Lofgren's syndrome (P < 0.0001, P(c) = 0.001). More importantly, carriage of this allele reduced the risk of disease progression. In contrast, the other common split of DQB1*02, *0202, did not affect disease severity but was mildly protective against sarcoidosis in the UK population (P = 0.02, p(c) not significant). In conclusion, this study shows that DQB1*0201 is a strong marker for mild sarcoidosis. Additional mapping across the DQB1*0201-DRB1*0301 haplotype, including specific alleles at genes such as DRB3, tumor necrosis factor, lymphotoxin-alpha, I-kappa-B-like protein, and B-associated transcript 1, is necessary for a final localization of the protective effect on this haplotype.

Emphysematous lung destruction by cigarette smoke. The effects of latent adenoviral infection on the lung inflammatory response.

Abstract: This study was designed to test the hypothesis that cigarette smoke-induced inflammation and emphysema are amplified by the presence of latent adenoviral (Ad) infection, and to determine whether this emphysematous process can be reversed by all-trans-retinoic acid (RA) treatment. The results confirm that in guinea pigs, chronic cigarette-smoke exposure caused lesions similar to human centrilobular emphysema. They also show that latent Ad infection combined with cigarette-smoke exposure caused an excess increase in lung volume (P < 0.001), air-space volume (P < 0.001), and lung weight (P < 0.01), and further decrease in surface-to-volume ratio (P < 0.001) compared with smoke exposure alone. RA treatment failed to reverse these emphysematous changes. Analysis of inflammatory response in parenchymal and airway tissue showed that smoking caused an increase of polymorphonuclear leukocytes (PMNs) (P < 0.0002), macrophages (P < 0.001), and CD4 cells (P < 0.0009), and that latent Ad infection independently increased PMNs (P < 0.001), macrophages (P = 0.003), and CD8 cells (P < 0.001). We conclude that latent Ad infection amplifies the emphysematous lung destruction and increases the inflammatory response produced by cigarette-smoke exposure. In this study, the increase in CD4 was associated with cigarette smoke and the increase in CD8 cells with latent Ad infection.

Selective p38 activation in human non-small cell lung cancer.

Abstract: The mitogen-activated protein kinase (MAPK) pathways transmit signals from the cell membrane to the nucleus. Activation of MAPK cascades may play a role in malignant transformation. We hypothesized that enhanced expression of one or more of these pathways would occur in human lung cancers. Using Western blot analysis of tissue homogenates from resected non– small cell lung cancers and matched non-neoplastic lung tissue, we determined that only activated p38 was consistently increased in tumor compared with normal tissue. In vitro kinase assays confirmed that the levels of activated MAPK correlated with the activity of the enzymes, and immunohistochemical analysis confirmed the cellular localization of the activated MAPKs. We incubated a lung cancer cell line in a hypoxic chamber to simulate the hypoxic environment in solid lung tumors, but found no increase in p38 activation. Contrary to our expectations, ERK and JNK, the MAPK pathways traditionally associated with cell growth and perhaps malignant transf...

Defective Pulmonary Development in the Absence of Heparin-Binding Vascular Endothelial Growth Factor Isoforms

Abstract: Development of the airways, alveoli, and the pulmonary vasculature in the fetus is a process that is precisely controlled. One of the growth factors involved, vascular endothelial growth factor (VEGF), is so critical for embryonic development that in the mouse, elimination of just a single allele is lethal. In the early stages of lung development, the mouse VEGF gene expresses three isoforms (120, 164, and 188) in a distinct temporo-spatial pattern, suggesting a specific function for each. We engineered mice that express only VEGF 120, to study the role of VEGF isoforms in lung development. Lung vessel development in these mice was studied by scanning electron microscopy of Mercox casts of lung vasculature. Airway and air-blood barrier development was analyzed by light microscopy, transmission electron microscopy, immunohistochemistry, and morphometry. In all VEGF120/120 fetuses and pups, lung vascular casts were smaller and less dense compared with 120/+ and wild-type littermates. Although the generation count of pre-acinar vessels was similar in all three genotypes, the most peripheral vessels were dilated and were more widely separated in 120/120 fetuses of all ages, compared with 120/+ and wild-type littermates. In addition, 120/120 animals had fewer air-blood barriers and a decreased airspace-parenchyma ratio compared with 120/+ and wild-type littermates. We concluded that the absence of VEGF 164 and 188 isoforms impairs lung microvascular development and delays airspace maturation, indicating an essential role for heparin-binding VEGF isoforms in normal lung development.

Polymers of alpha(1)-antitrypsin are chemotactic for human neutrophils: a new paradigm for the pathogenesis of emphysema.

Abstract: Plasma deficiency of alpha(1)-antitrypsin is most commonly due to the Z mutation ((342)Glu--> Lys) and is associated with early-onset panlobular emphysema. The lung disease in these patients is attributed to the relative deficiency of circulating alpha(1)-antitrypsin resulting in uncontrolled neutrophil-derived proteolytic activity. We have previously demonstrated that the local deficiency of Z alpha(1)-antitrypsin is exacerbated by the formation of polymers within the lung and now show that this polymerization not only inactivates alpha(1)-antitrypsin but also converts the molecule to a chemoattractant for human neutrophils. The chemotactic action of polymeric alpha(1)-antitrypsin was substantially greater than that seen with other conformers, was of similar magnitude to C5a, and was apparent over a range of physiologically relevant concentrations (EC(50) 0.0045 +/- 0.002 mg/ml). The biologic activity of polymeric alpha(1)-antitrypsin was confirmed by the demonstration that polymers, but not native alpha(1)-antitrypsin, induced neutrophil shape change and stimulated myeloperoxidase release and neutrophil adhesion. Polymeric alpha(1)-antitrypsin had no effect on basal or N-formyl-Met-Leu-Phe- stimulated superoxide anion release or constitutive apoptosis. The chemotactic properties of polymeric alpha(1)-antitrypsin may provide an explanation for the excessive neutrophils found in the lungs of Z alpha(1)-antitrypsin homozygotes and suggests a new paradigm for the pathogenesis of emphysema in these patients.

Expression profiling of the developing mouse lung: insights into the establishment of the extracellular matrix.

Abstract: We have undertaken a comprehensive gene expression profiling of the entire process of murine lung development using oligonucleotide-based microarrays. Our data reveals the expression pattern of � 11,000 genes throughout the morphologic stages of lung development. This includes known genes with unappreciated pulmonary expression and novel genes with undefined functions. Traditional gene expression analysis techniques verify a high degree of confidence in the microarray data. Examination of the data confirms previously known patterns of expression for extracellular matrix genes and provides new information regarding relationships in temporal expression among groups of these genes. Large-scale cluster analysis reveals associations in the expression profile of specific genes with defined developmental processes. For instance, we identify groups of genes, which are coordinately expressed with extracellular matrix genes during lung development. These data should serve as a resource for the pulmonary research community and assist in deciphering the molecular mechanisms governing normal lung development as well as those involved in aberrant developmental pathology. Mammalian lung development is a complex morphogenetic process, which initiates near mid-gestation and continues through early postnatal life. The lung arises as two lateral buds that emerge from the ventral foregut endoderm at � 9 days after fertilization (in mouse) and undergo nu

Prostaglandin E2 synthesis and suppression of fibroblast proliferation by alveolar epithelial cells is cyclooxygenase-2-dependent.

Abstract: Alveolar epithelial cells (AECs) may influence neighboring fibroblasts by the elaboration of prostaglandin E(2) (PGE(2)). This prostanoid can be synthesized via "constitutive" cyclooxygenase (COX)-1 and "inducible" COX-2 enzyme isoforms. We compared AECs isolated from wild-type (WT), COX-1 knockout (KO), and COX-2 KO mice to determine the contribution of COX isoforms to AEC PGE(2) synthesis and capacity for suppression of fibroblast proliferation in co-cultures. WT AECs constitutively expressed both COX-1 and COX-2 isoforms by immunoblot analysis. COX-1 KO cells and WT cells comparably augmented PGE(2) synthesis following incubation with lipopolysaccharide or interleukin-1, whereas COX-2 KO cells were unable to do so. Surprisingly, however, constitutive generation of PGE(2) was also dramatically reduced only in COX-2 KO cells. When co-cultured with WT murine lung fibroblasts, AECs from WT and COX-1 KO animals suppressed serum-induced fibroblast proliferation, whereas COX-2-deficient AECs caused a modest enhancement in fibroblast proliferation. These results indicate that PGE(2) synthetic capacity in AECs is predominantly COX-2-dependent under both basal and stimulated conditions. They also demonstrate conclusively that AECs can modulate fibroblast function by the elaboration of suppressive prostanoids. These alterations in AEC phenotype likely contribute to the propensity for pulmonary fibrosis observed in COX-2-deficient mice.