AJP: Lung Cellular and Molecular Physiology recent issues

Animal models of chronic obstructive pulmonary disease

Wright, J. L., Cosio, M., Churg, A. — 2008-07-03

The mechanisms involved in the genesis of chronic obstructive pulmonary disease (COPD) are poorly defined. This area is complicated and difficult to model because COPD consists of four separate anatomic lesions (emphysema, small airway remodeling, pulmonary hypertension, and chronic bronchitis) and a functional lesion, acute exacerbation; moreover, the disease in humans develops over decades. This review discusses the various animal models that have been used to attempt to recreate human COPD and the advantages and disadvantages of each. None of the models reproduces the exact changes seen in humans, but cigarette smoke-induced disease appears to come the closest, and genetically modified animals also, in some instances, shed light on processes that appear to play a role.

Challenges in translating plasma proteomics from bench to bedside: update from the NHLBI Clinical Proteomics Programs

2008-07-03

The emerging scientific field of proteomics encompasses the identification, characterization, and quantification of the protein content or proteome of whole cells, tissues, or body fluids. The potential for proteomic technologies to identify and quantify novel proteins in the plasma that can function as biomarkers of the presence or severity of clinical disease states holds great promise for clinical use. However, there are many challenges in translating plasma proteomics from bench to bedside, and relatively few plasma biomarkers have successfully transitioned from proteomic discovery to routine clinical use. Key barriers to this translation include the need for "orthogonal" biomarkers (i.e., uncorrelated with existing markers), the complexity of the proteome in biological samples, the presence of high abundance proteins such as albumin in biological samples that hinder detection of low abundance proteins, false positive associations that occur with analysis of high dimensional datasets, and the limited understanding of the effects of growth, development, and age on the normal plasma proteome. Strategies to overcome these challenges are discussed.

Cystic fibrosis: ironing out the problem of infection?

Reid, D. W., Anderson, G. J., Lamont, I. L. — 2008-07-03

The {Delta}F508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability

2008-07-03

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing F508-CFTR significantly enhanced P. aeruginosa biofilm formation, and F508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on F508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing F508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The F508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Pulmonary alveolar epithelial uptake of S-nitrosothiols is regulated by L-type amino acid transporter

2008-07-03

Nitric oxide (NO) effects are often mediated via S-nitrosothiol (SNO) formation; SNO uptake has recently been shown to be mediated in some cell types via system L-type amino acid transporters (LAT-1, 2). Inhaled NO therapy may exert some biological effects via SNO formation. We therefore sought to determine if pulmonary epithelial SNO uptake depended on LAT or peptide transporter 2 (PEPT2). Both LAT-1 and PEPT2 proteins were detected by immunoblot and immunocytochemistry in L2 cells and rat lung. We tested SNO uptake through the transporters by exposing rat alveolar epithelial cells (L2 and type II) to RSNOs: S-nitrosoglutathione, S-nitrosocysteinylglycine (SNO-Cys-Gly), S-nitrosocysteine (CSNO), and to NO donor diethylamine NONOate (DEA-NONOate). SNO was detected in cell lysates by ozone chemiluminescence. NO uptake was detected by fluorescence in alveolar epithelial cells loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate cultured in submersion and exposed to RSNOs and DEA NONOate. Addition of l-Cys but not d-Cys to RSNOs or DEA NONOate increased SNO and DAF-FM signal that was inhibited by coincubation with LAT competitors. Incubation of cells with PEPT2 substrate SNO-Cys-Gly showed no increase in SNO or DAF-FM signal unless incubated with l-Cys. This was unaffected by PEPT2 inhibition. We conclude that RSNOs (thionitrites, S-nitrosothiols) and NO enter alveolar epithelial cells predominantly by S-nitrosation of l-Cys, which is then imported through LAT.

Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide

2008-07-03

The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer_12:0 or Cer_8:0; 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramide's proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy

2008-07-03

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) ~2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-µm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 µm but increased significantly between 10 and 15 µm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.

Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells

Firth, A. L., Yuill, K. H., Smirnov, S. V. — 2008-07-03

Voltage-gated K⁺ (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents (I_Kv) revealed the presence of a mitochondria-mediated Mg²⁺ and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.

Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone

2008-07-03

RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested whether DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats and whether inhibition of RhoA/ROCK signaling was involved in the protective effect of DHEA. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I as well as increases in activities of RhoA and ROCK and increases in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wk) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 33 ± 5 and 16 ± 1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-wk DHEA treatment (1%) started 3 wk after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 47 ± 3 and 30 ± 3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of the impressive protection of DHEA against MCT-induced PH in pneumonectomized rats.

Particulate matter exposure induces persistent lung inflammation and endothelial dysfunction

2008-07-03

Epidemiologic and animal studies have shown that exposure to particulate matter air pollution (PM) is a risk factor for the development of atherosclerosis. Whether PM-induced lung and systemic inflammation is involved in this process is not clear. We hypothesized that PM exposure causes lung and systemic inflammation, which in turn leads to vascular endothelial dysfunction, a key step in the initiation and progression of atherosclerosis. New Zealand White rabbits were exposed for 5 days (acute, total dose 8 mg) and 4 wk (chronic, total dose 16 mg) to either PM smaller than 10 µm (PM₁₀) or saline intratracheally. Lung inflammation was quantified by morphometry; systemic inflammation was assessed by white blood cell and platelet counts and serum interleukin (IL)-6, nitric oxide, and endothelin levels. Endothelial dysfunction was assessed by vascular response to acetylcholine (ACh) and sodium nitroprusside (SNP). PM₁₀ exposure increased lung macrophages (P < 0.02), macrophages containing particles (P < 0.001), and activated macrophages (P < 0.006). PM₁₀ increased serum IL-6 levels in the first 2 wk of exposure (P < 0.05) but not in weeks 3 or 4. PM₁₀ exposure reduced ACh-related relaxation of the carotid artery with both acute and chronic exposure, with no effect on SNP-induced vasodilatation. Serum IL-6 levels correlated with macrophages containing particles (P = 0.043) and ACh-induced vasodilatation (P = 0.014 at week 1, P = 0.021 at week 2). Exposure to PM₁₀ caused lung and systemic inflammation that were both associated with vascular endothelial dysfunction. This suggests that PM-induced lung and systemic inflammatory responses contribute to the adverse vascular events associated with exposure to air pollution.

Transforming growth factor-{beta} signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung

2008-07-03

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-β is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-β signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-β type II receptor (DNTGFβRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O₂) or air from birth to 14 days of age. Expression of DNTGFβRII was induced by 20 µg/g ZnSO₄ given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-β and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFβRII mice given ZnSO₄ compared with WT control mice and DNTGFβRII mice not given ZnSO₄. The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFβRII mice given ZnSO₄. These data support the conclusion that TGF-β plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.

Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

2008-07-03

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO^–/– mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO^–/– mice unexpectedly had improved survival compared with wild-type (WT) mice within 5–12 h after intraperitoneal E. coli challenge. Lungs of MPO^–/– mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO^–/– mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO^–/– mice. Inhibition of iNOS in MPO^–/– mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO^–/– mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia

Lu, W., Wang, J., Shimoda, L. A., Sylvester, J. T. — 2008-07-03

Hypoxic pulmonary vasoconstriction (HPV) requires Ca²⁺ influx through store-operated Ca²⁺ channels (SOCC) in pulmonary arterial smooth muscle cells (PASMC) and is greater in distal than proximal pulmonary arteries (PA). SOCC may be composed of canonical transient receptor potential (TRPC) proteins and activated by stromal interacting molecule 1 (STIM1). To assess the possibility that HPV is greater in distal PA because store-operated Ca²⁺ entry (SOCE) is greater in distal PASMC, we measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca²⁺-sensitive dye fura 2. Both hypoxia (4% O₂) and KCl (60 mM) increased [Ca²⁺]_i. Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca²⁺-free solutions containing cyclopiazonic acid to deplete Ca²⁺ stores in sarcoplasmic reticulum and nifedipine to prevent Ca²⁺ entry through L-type voltage-operated Ca²⁺ channels. Under these conditions, the increase in [Ca²⁺]_i caused by restoration of extracellular Ca²⁺ and the decrease in fura 2 fluorescence caused by Mn²⁺ were greater in distal PASMC, indicating greater SOCE. Moreover, the increase in SOCE caused by hypoxia was also greater in distal cells. Real-time quantitative polymerase chain reaction analysis of PASMC and freshly isolated deendothelialized PA tissue demonstrated expression of STIM1 and five of seven known TRPC isoforms (TRPC1 > TRPC6 > TRPC4 >> TRPC3 TRPC5). For both protein, as measured by Western blotting, and mRNA, expression of STIM1, TRPC1, TRPC6, and TRPC4 was greater in distal than proximal PASMC and PA. These results provide further support for the importance of SOCE in HPV and suggest that HPV is greater in distal than proximal PA because greater numbers and activation of SOCC in distal PASMC generate bigger increases in [Ca²⁺]_i.

Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

2008-07-03

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E₂ (PGE₂) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF–/– donors (GM-CSF–/– BMT) with untransplanted mice. GM-CSF–/– BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF–/– BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF–/– BMT mice overproduced PGE₂, but expression of the inhibitory EP₂ receptor was diminished. As a consequence of decreased EP₂ receptor expression, we found diminished accumulation of cAMP in response to PGE₂ stimulation in GM-CSF–/– BMT AMs compared with control BMT AMs. In addition, GM-CSF–/– BMT AMs retained cysteinyl leukotriene production and normal TNF- response compared with AMs from control BMT mice. GM-CSF–/– BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF–/– recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.

Transforming growth factor-{beta}1 protects against pulmonary artery endothelial cell apoptosis via ALK5

Lu, Q. — 2008-07-03

Transforming growth factor (TGF)-β1 has been reported to cause endothelial cell apoptosis. However, conflicting data have also demonstrated that TGF-β1 promotes endothelial cell survival. In this study, the effect of TGF-β1 on apoptosis of cultured bovine pulmonary artery endothelial cells (PAEC) induced by multiple stimuli was investigated. TGF-β1 protected against apoptosis of bovine PAEC induced by serum deprivation or the VEGF receptor inhibitor SU-5416, but not by UV light exposure or TNF. Neither caspase-8 nor caspase-12 was activated by serum deprivation or the VEGF receptor blocker. However, blockade of VEGF receptors activated caspase-9, an effect that was abolished by TGF-β1. Furthermore, serum deprivation and inhibition of VEGF receptors significantly decreased the protein level of Bcl-2, an effect that was also abrogated by TGF-β1. In addition, the baseline level of Bcl-2 was enhanced by TGF-β1 and reduced by inhibition of activin receptor-like kinase 5 (ALK5), a TGF-β1 type I receptor. Furthermore, inhibition of ALK5 caused apoptosis of bovine PAEC. These results suggest that TGF-β1 signaling is critical for maintenance of bovine PAEC survival. Finally, the protective effects of TGF-β1 on bovine PAEC apoptosis and Bcl-2 reduction were abolished by ALK5 inhibition, but not by inhibition of non-SMAD signaling pathways. Also, TGF-β1 activated SMAD2 and SMAD1/5, an effect that was abolished by ALK5 inhibition. The results of this study suggest that TGF-β1 protects against bovine PAEC apoptosis, possibly through ALK5-mediated Bcl-2 induction and subsequent inhibition of the mitochondria-mediated intrinsic pathway of apoptosis. Understanding the mechanism by which TGF-β1 promotes endothelial cell survival may provide a better treatment for apoptosis-dependent vascular diseases, such as emphysema.

Leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro

Haufe, D., Koenigshausen, E., Knels, L., Wendel, M., Stehr, S. N., Koch, T. — 2008-07-03

Application of liquid, aerosolized, and vaporized perfluorocarbons (PFC) in acute lung injury has shown anti-inflammatory effects. Although this may be beneficial in states of pulmonary hyperinflammation, it also could increase susceptibility to nosocomial lung infection. We hypothesized that PFC impair cellular host defense and therefore investigated in an in vitro model the influence of perfluorohexane (PFH) on crucial mechanisms of bacterial elimination in human neutrophils and monocytes. Using scanning and transmission electron microscopy, we could show membrane-bound and ingested PFH particles that morphologically did not alter adherence and phagocytosis of Escherichia coli or leukocyte viability. The amount of adherent and phagocytosed bacteria as determined by flow cytometry was not influenced in cells only pretreated with PFH for 1 and 4 h. When PFH was present during E. coli challenge, bacterial adherence was decreased in polymorphonuclear neutrophils, but respective intracellular uptake was not impaired and was even significantly promoted in monocytes. Overall, E. coli-induced respiratory burst capacity was not reduced by PFH. Our findings provide evidence that key functions of innate host defense are not compromised by PFH treatment in vitro.

EGF antagonizes TGF-{beta}-induced tropoelastin expression in lung fibroblasts via stabilization of Smad corepressor TGIF

Yang, S., Nugent, M. A., Panchenko, M. P. — 2008-07-03

We previously reported that neutrophil elastase (NE) downregulates transforming growth factor-β (TGF-β)-maintained tropoelastin mRNA levels in lung fibroblasts through transactivation of the epidermal growth factor (EGF) receptor (EGFR)/Mek/Erk pathway, which is dependent on the NE-initiated release of soluble EGFR ligands. In the present study, we investigated the mechanism by which EGF downregulates tropoelastin expression. We found that EGF downregulates tropoelastin expression through inhibition of TGF-β signaling. We show that EGF does not prevent the TGF-β-induced nuclear accumulation of Smad2/3; rather, EGF stabilizes the short-lived Smad transcriptional corepressor TG-interacting factor (TGIF) via EGFR/Mek/Erk-mediated phosphorylation of TGIF. Elevation of TGIF levels, either by TGIF overexpression or prevention of TGIF degradation, is sufficient to inhibit TGF-β-induced tropoelastin expression. Moreover, TGIF is essential for EGF-mediated downregulation of tropoelastin expression, inasmuch as small interfering RNA knockdown of TGIF blocked EGF-induced downregulation of tropoelastin. Finally, we demonstrated that NE treatment, which releases EGF-like growth factors, causes stabilization of TGIF through the EGFR/Mek/Erk pathway. These results suggest that EGFR/Mek/Erk signaling specifically antagonizes the proelastogenic action of TGF-β in lung fibroblasts by stabilizing the Smad transcriptional corepressor TGIF.

Utility of magnetic resonance imaging and nuclear magnetic resonance-based metabolomics for quantification of inflammatory lung injury

2008-07-03

Magnetic resonance imaging (MRI) and metabolic nuclear magnetic resonance (NMR) spectroscopy are clinically available but have had little application in the quantification of experimental lung injury. There is a growing and unfulfilled need for predictive animal models that can improve our understanding of disease pathogenesis and therapeutic intervention. Integration of MRI and NMR could extend the application of experimental data into the clinical setting. This study investigated the ability of MRI and metabolic NMR to detect and quantify inflammation-mediated lung injury. Pulmonary inflammation was induced in male B6C3F1 mice by intratracheal administration of IL-1β and TNF- under isoflurane anesthesia. Mice underwent MRI at 2, 4, 6, and 24 h after dosing. At 6 and 24 h lungs were harvested for metabolic NMR analysis. Data acquired from IL-1β+TNF--treated animals were compared with saline-treated control mice. The hyperintense-to-total lung volume (HTLV) ratio derived from MRI was higher in IL-1β+TNF--treated mice compared with control at 2, 4, and 6 h but returned to control levels by 24 h. The ability of MRI to detect pulmonary inflammation was confirmed by the association between HTLV ratio and histological and pathological end points. Principal component analysis of NMR-detectable metabolites also showed a temporal pattern for which energy metabolism-based biomarkers were identified. These data demonstrate that both MRI and metabolic NMR have utility in the detection and quantification of inflammation-mediated lung injury. Integration of these clinically available techniques into experimental models of lung injury could improve the translation of basic science knowledge and information to the clinic.

Metalloelastase in lungs and alveolar macrophages is modulated by extracellular substance P in mice

Xu, J., Xu, F., Barrett, E. — 2008-07-03

Metalloelastase (MMP-12), mainly produced by macrophages, has been shown to play a key role in the pathogenesis of emphysema in animal models. Chronic cigarette smoke increases pulmonary MMP-12, which is closely correlated with an elevation of pulmonary substance P (SP). Because alveolar macrophages (AMs) contain the neurokinin-1 receptor (NK1R), we tested whether SP was able to trigger the upregulation of MMP-12 synthesis in AMs by acting on the NK1R. AMs isolated from bronchoalveolar lavage cells in C3H/HeN mice were cultured with control medium or SP that was coupled without or with NK1R antagonists (CP-99,994 or aprepitant) for 24 h. We found that SP significantly increased the mRNA of MMP-12 and NK1R by 11-fold and 82%, respectively, in AMs (P < 0.05), and these responses were abolished by NK1R antagonists with little change in the cells' viability. Because pulmonary SP is primarily released by bronchopulmonary C-fibers (PCFs), we further asked whether destruction of PCFs would reduce SP and MMP-12. Two groups of mice were pretreated with vehicle and neonatal capsaicin (NCAP) to degenerate PCFs, respectively. Our results show that NCAP treatment significantly decreased mRNA and protein levels of SP associated with a reduction NK1R and MMP-12 in the lungs and AMs. These findings suggest that SP has a modulatory effect on pulmonary MMP-12 by acting on NK1R to trigger MMP-12 syntheses in the AMs.

MAP kinases mediate interleukin-13 effects on calcium signaling in human airway smooth muscle cells

2008-07-03

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing Ca²⁺ release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the Ca²⁺ response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased Ca²⁺ responses to histamine. The augmentation of Ca²⁺ signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of Ca²⁺ responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms.

Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis

Li, X., Molina-Molina, M., Abdul-Hafez, A., Uhal, V., Xaubet, A., Uhal, B. D. — 2008-07-03

Earlier work from this laboratory showed that local generation of angiotensin (ANG) II is required for the pathogenesis of experimental pulmonary fibrosis and that ANG peptides are expressed robustly in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Angiotensin converting enzyme-2 (ACE-2) degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation. On this basis, we hypothesized that ACE-2 would be protective against experimental lung fibrogenesis and might be downregulated in human and experimental lung fibrosis. In lung biopsy specimens from patients with IPF, ACE-2 mRNA and enzyme activity were decreased by 92% (P < 0.01) and 74% (P < 0.05), respectively. ACE-2 mRNA and activity were also decreased similarly in the lungs of bleomycin-treated rats and C57-BL6 mice. In mice exposed to low doses of bleomycin, lung collagen accumulation was enhanced by intratracheal administration of either ACE-2-specific small interfering RNAs (siRNAs) or the peptide DX₆₀₀, a competitive inhibitor of ACE-2 (P < 0.05). Administration of either ACE-2 siRNA or DX₆₀₀ significantly increased the ANG II content of mouse lung tissue above the level induced by bleomycin alone. Coadministration of the ANG II receptor antagonist saralasin blocked the DX₆₀₀-induced increase in lung collagen. Moreover, purified recombinant human ACE-2, delivered to mice systemically by osmotic minipump, attenuated bleomycin-induced lung collagen accumulation. Together, these data show that ACE-2 mRNA and activity are severely downregulated in both human and experimental lung fibrosis and suggest that ACE-2 protects against lung fibrogenesis by limiting the local accumulation of the profibrotic peptide ANG II.

Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1

Kang, B. N., Jude, J. A., Panettieri, R. A., Walseth, T. F., Kannan, M. S. — 2008-07-03

The enzymatic activity of CD38, ADP-ribosyl cyclase, synthesizes the calcium mobilizing molecule cyclic ADP-ribose from β-NAD. In human airway smooth muscle (HASM) cells, CD38 expression is augmented by the inflammatory cytokine, TNF-, causing increased intracellular calcium response to agonists. The transcriptional and posttranscriptional regulation of CD38 expression involves signaling through MAPKs and requires activation of NF-B and activator protein-1 (AP-1). The cytokine-augmented CD38 expression is decreased by anti-inflammatory glucocorticoids due to inhibition of NF-B activation and other mechanisms. In this study, we investigated glucocorticoid regulation of CD38 expression in HASM cells through the MKP-1. In HASM cells, dexamethasone and TNF- induced MKP-1 expression (both mRNA and protein) rapidly. Dexamethasone decreased TNF--induced phosphorylation of the major MAPKs, i.e., ERK, p38, and JNK, and decreased the activation of NF-B and AP-1. Dexamethasone also decreased CD38 expression induced by TNF-, and part of this effect was attributable to decreased transcript stability. In cells transfected with MKP-1-specific small interfering RNAs (siRNAs), there was significant attenuation of MKP-1 expression and partial, but nonsignificant, reversal of dexamethasone inhibition of CD38 expression. These results indicate that regulation of CD38 expression in HASM cells by glucocorticoids involves decreased signaling through MAPKs and activation of transcription factors. The glucocorticoid effects on decreased CD38 expression and function result from regulation through transcription and transcript stability.

Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures

Sabnis, A. S., Reilly, C. A., Veranth, J. M., Yost, G. S. — 2008-07-03

Recognition of temperature is a critical element of sensory perception and allows mammals to evaluate both their external environment and internal status. The respiratory epithelium is constantly exposed to the external environment, and prolonged inhalation of cold air is detrimental to human airways. However, the mechanisms responsible for adverse effects elicited by cold air on the human airways are poorly understood. Transient receptor potential melastatin family member 8 (TRPM8) is a well-established cold- and menthol-sensing cation channel. We recently discovered a functional cold- and menthol-sensing variant of the TRPM8 ion channel in human lung epithelial cells. The present study explores the hypothesis that this TRPM8 variant mediates airway cell inflammatory responses elicited by cold air/temperatures. Here, we show that activation of the TRPM8 variant in human lung epithelial cells leads to increased expression of several cytokine and chemokine genes, including IL-1, -1β, -4, -6, -8, and -13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-. Our results provide new insights into mechanisms that potentially control airway inflammation due to inhalation of cold air and suggest a possible role for the TRPM8 variant in the pathophysiology of asthma.

Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice

2008-07-03

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M₃ muscarinic receptor was implicated in the relaxing action of ACh and M₁ muscarinic receptor (M₁AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M₁AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M₁AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M₁AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M₁AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."

The inhaled Rho kinase inhibitor Y-27632 protects against allergen-induced acute bronchoconstriction, airway hyperresponsiveness, and inflammation

Schaafsma, D., Bos, I. S. T., Zuidhof, A. B., Zaagsma, J., Meurs, H. — 2008-07-03

Recently, we have shown that allergen-induced airway hyperresponsiveness (AHR) after the early (EAR) and late (LAR) asthmatic reaction in guinea pigs could be reversed acutely by inhalation of the Rho kinase inhibitor Y-27632. The present study addresses the effects of pretreatment with inhaled Y-27632 on the severity of the allergen-induced EAR and LAR, the development of AHR after these reactions, and airway inflammation. Using permanently instrumented and unrestrained ovalbumin (OA)-sensitized guinea pigs, single OA challenge-induced EAR and LAR, expressed as area under the lung function (pleural pressure, P_pl) time-response curve, were measured, and histamine PC₁₀₀ (provocation concentration causing a 100% increase of P_pl) values were assessed 24 h before, and at 6 and 24 h after, the OA challenge (after the EAR and LAR, respectively). Thirty minutes before and 8 h after OA challenge, saline or Y-27632 (5 mM) was nebulized. After the last PC₁₀₀ value, bronchoalveolar lavage (BAL) was performed, and the inflammatory cell profile was determined. It was demonstrated that inhalation of Y-27632 before allergen challenge markedly reduced the immediate allergen-induced peak rise in P_pl, without significantly reducing the overall EAR and LAR. Also, pretreatment with Y-27632 considerably protected against the development of AHR after the EAR and fully prevented AHR after the LAR. These effects could not be explained by a direct effect of Y-27632 on the histamine responsiveness, because of the short duration of the acute bronchoprotection of Y-27632 (<90 min). In addition, Y-27632 reduced the number of total inflammatory cells, eosinophils, macrophages, and neutrophils recovered from the BAL. Altogether, inhaled Y-27632 protects against acute allergen-induced bronchoconstriction, development of AHR after the EAR and LAR, and airway inflammation in an established guinea pig model of allergic asthma.

Induction of IL-8 by Mycoplasma pneumoniae membrane in BEAS-2B cells

2008-07-03

Mycoplasma pne umoniae is an extracellular pathogen, residing on mucosal surfaces of the respiratory and genital tracts. The lack of cell walls in mycoplasmas facilitates the direct contact of the bacterial membrane with the host cell. The cell membrane of mycoplasma is the major inducer of the host pathogenic response. Airway diseases caused by M. pneumoniae include bronchiolitis, bronchitis, and rarely bronchiectasis. In such disorders, neutrophil infiltration of the airways predominates. More recently, M. pneumoniae has been implicated in the pathogenesis of asthma. Epithelial cells play an important role in recruiting inflammatory cells into the airways. Since M. pneumoniae infection of human epithelial cells induces expression of IL-8—a potent activator of neutrophils—we investigated the signaling and transcriptional mechanisms by which mycoplasma membrane induces expression of this chemokine. In BEAS-2B human bronchial epithelial cells, mycoplasma membrane fraction (MMF) increased IL-8 mRNA and protein production. Activation of the transcriptional elements activating protein-1, nuclear factor-interleukin-6, and particularly NF-B are essential for optimal IL-8 production by MMF. The mitogen-activated protein kinases individually played a modest role in MMF-induced IL-8 production. Toll-like receptor-2 did not play a significant role in MMF-induction of IL-8. Antibiotics with microbicidal activity against M. pneumoniae are also known to have anti-inflammatory effects. Whereas clarithromycin, azithromycin, and moxifloxacin individually were able to inhibit TNF--induction of IL-8, each failed to inhibit MMF-induction of IL-8.

Ciliated epithelial cell lifespan in the mouse trachea and lung

Rawlins, E. L., Hogan, B. L. M. — 2008-07-03

The steady-state turnover of epithelial cells in the lung and trachea is highly relevant to investigators who are studying endogenous stem cells, manipulating gene expression in vivo, or using viral vectors for gene therapy. However, the average lifetime of different airway epithelial cell types has not previously been assessed using currently available genetic techniques. Here, we use Cre/loxP genetic technology to indelibly label a random fraction of ciliated cells throughout the airways of a cohort of mice and follow them in vivo for up to 18 mo. We demonstrate that ciliated airway epithelial cells are a terminally differentiated population. Moreover, their average half-life of 6 mo in the trachea and 17 mo in the lung is much longer than previously available estimates, with significant numbers of labeled cells still present after 18 mo.

Measurement of extravascular lung water in patients with pulmonary edema

Matthay, M. A. — 2008-06-02

Indicator dilution measurements of extravascular lung water: basic assumptions and observations

Effros, R. M., Pornsuriyasak, P., Porszasz, J., Casaburi, R. — 2008-06-02

Since they were introduced more than five decades ago, a variety of single-pass indicator, thermal, and osmotic dilution approaches have been developed for detecting and measuring excess fluid in the lungs. This brief review discusses why studies of the extravascular lung water (EVLW) continue to intrigue physiologists and clinicians and the likelihood that they will become sufficiently reliable for more widespread use. Emphasis is placed on the basic assumptions that underlie these measurements and limitations imposed by the nature of the data that are collected. A distinction is made between approaches that are based on compartmental models of solute and water exchange and those that represent extensions of more conventional washout procedures, which have been utilized extensively for measurements of gas volumes in the lungs. Although the compartmental approach has been used to simplify indicator dilution studies by eliminating the need for a vascular indicator, it is based on assumptions that may not be realistic. Early recirculation inevitably limits the period in which observations can be made and impairs detection of those portions of the lungs with decreased perfusion. These general principles are also used to develop a new method of analyzing osmotic transient studies. A short account is given of EVLW observations that have been made in animals and humans. Both the sensitivity and specificity of EVLW measurements in humans are uncertain, and the normal clinical range of EVLW remains in doubt.

Does adiponectin play a role in pulmonary emphysema?

Wert, S. E. — 2008-06-02

Alveolar macrophage activation and an emphysema-like phenotype in adiponectin-deficient mice

2008-06-02

Adiponectin is an adipocyte-derived collectin that acts on a wide range of tissues including liver, brain, heart, and vascular endothelium. To date, little is known about the actions of adiponectin in the lung. Herein, we demonstrate that adiponectin is present in lung lining fluid and that adiponectin deficiency leads to increases in proinflammatory mediators and an emphysema-like phenotype in the mouse lung. Alveolar macrophages from adiponectin-deficient mice spontaneously display increased production of tumor necrosis factor- (TNF-) and matrix metalloproteinase (MMP-12) activity. Consistent with these observations, we found that pretreatment of alveolar macrophages with adiponectin leads to TNF- and MMP-12 suppression. Together, our findings show that adiponectin leads to macrophage suppression in the lung and suggest that adiponectin-deficient states may contribute to the pathogenesis of inflammatory lung conditions such as emphysema.

Intratracheal perfluorocarbons diminish LPS-induced increase in systemic TNF-{alpha}

2008-06-02

Perfluorocarbons (PFC) reduce the production of various inflammatory cytokines, including TNF-. The anti-inflammatory effect is not entirely understood. If anti-inflammatory properties are caused by a mechanical barrier, PFC in the alveoli should have no effect on the inflammatory response to intravenous LPS administration. To test that hypothesis, rats (n = 31) were administered LPS intravenously and were either spontaneously breathing (Spont), conventionally ventilated (CMV), or receiving partial liquid ventilation (PLV). Serum concentration of TNF- was measured. The pulmonary expressions of TNF- and TNF- receptor 1 protein and of TNF- and ICAM-1 mRNA were determined. LPS caused a significant (P < 0.001) increase in serum TNF-. Serum TNF- concentration was similar in LPS/Spont (525 ± 180 pg/ml) and LPS/CMV (504 ± 154 pg/ml) but was significantly (P < 0.001) lower in animals of the LPS/PLV group (274 ± 101 pg/ml). Immunohistochemical data on TNF- protein expression showed a LPS-induced increase in TNF- and TNF- receptor 1 expression that was diminished by partial liquid ventilation. PCR measurements revealed a lower expression of TNF- and ICAM-1 mRNA in LPS/PLV than in LPS/CMV or LPS/Spont animals. Semiquantitative histological evaluation revealed only minor alveolar inflammation with no significant differences between the groups. Low serum TNF- concentration in PFC-treated animals is most likely explained by a decreased production of TNF- in the lung.

Toll-like receptor 2 is upregulated by hog confinement dust in an IL-6-dependent manner in the airway epithelium

2008-06-02

Hog confinement workers are at high risk to develop chronic bronchitis as a result of their exposure to organic dust. Chronic bronchitis is characterized by inflammatory changes of the airway epithelium. A key mediator in inflammation is Toll-like receptor 2 (TLR2). We investigated the role of TLR2 in pulmonary inflammation induced by hog confinement dust. Normal human bronchial epithelial cells (NHBE) were grown in culture and exposed to hog confinement dust extract. Hog confinement dust upregulated airway epithelial cell TLR2 mRNA in a concentration- and time-dependent manner using real-time PCR. There was a similar increase in TLR2 protein at 48 h as shown by Western blot. TLR2 was upregulated on the surface of airway epithelial cells as shown by flow cytometry. A similar upregulation of pulmonary TLR2 mRNA and protein was shown in a murine model of hog confinement dust exposure. Hog confinement dust is known to stimulate epithelial cells to produce IL-6. To determine whether TLR2 expression was being regulated by IL-6, the production of IL-6 was blocked using an IL-6-neutralizing antibody. This resulted in attenuation of the dust-induced upregulation of TLR2. To further demonstrate the importance of IL-6 in the regulation of TLR2, NHBE were directly stimulated with recombinant human IL-6. IL-6 alone was able to upregulate TLR2 in airway epithelial cells. Hog confinement dust upregulates TLR2 in the airway epithelium through an IL-6-dependent mechanism.

Prolonged heterologous {beta}2-adrenoceptor desensitization promotes proasthmatic airway smooth muscle function via PKA/ERK1/2-mediated phosphodiesterase-4 induction

Hu, A., Nino, G., Grunstein, J. S., Fatma, S., Grunstein, M. M. — 2008-06-02

β2-Adrenergic receptor (β2AR) agonists acutely relieve bronchoconstriction via cAMP-mediated relaxation of airway smooth muscle (ASM). Airway constrictor responsiveness may be significantly heightened, however, following protracted exposure to these agents, presumably reflecting the effects of β2AR desensitization in ASM accompanying prolonged cAMP signaling. Because cAMP phosphodiesterase (PDE) activity can significantly modulate ASM contractility, we investigated the mechanism regulating PDE expression and its potential role in mediating changes in agonist-induced constrictor and relaxation responsiveness in ASM following its heterologous β2AR desensitization by prolonged exposure to cAMP-elevating agents. Isolated rabbit ASM tissues and cultured human ASM cells treated for 24 h with the receptor- or nonreceptor-coupled cAMP-stimulating agent, prostaglandin E₂ (PGE₂) or forskolin, respectively, exhibited constrictor hyperresponsiveness to acetylcholine and impaired β2AR-mediated relaxation and cAMP accumulation. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity, reflective of increased transcription of the PDE4D5 isoform, and were prevented by pretreatment of the ASM with a PDE4 inhibitor. Extended studies using gene silencing and pharmacological approaches to inhibit specific intracellular signaling molecules demonstrated that the mechanism underlying PGE₂-induced transcriptional upregulation of PDE4D5 involves PKA-dependent activation of G_i protein signaling via the β-subunits, the latter eliciting downstream activation of ERK1/2 and its consequent induction of PDE4D5 transcription. Collectively, these findings identify that β2AR desensitization in ASM following prolonged exposure to cAMP-elevating agents is associated with proasthmatic-like changes in ASM responsiveness that are mediated by upregulated PDE4 expression induced by activated cross talk between the PKA and ERK1/2 signaling pathways.

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium

Koff, J. L., Shao, M. X. G., Ueki, I. F., Nadel, J. A. — 2008-06-02

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF--converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)- is the EGFR ligand in this signaling cascade by using TGF- neutralizing antibody and by showing that epithelial production of TGF- occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1-> ROS-> TACE-> TGF--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Quement, C. L., Guenon, I., Gillon, J.-Y., Lagente, V., Boichot, E. — 2008-06-02

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.

Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model

2008-06-02

Sphingosine 1-phosphate (S1P) produced by sphingosine kinase (SPHK) is implicated in acute immunoresponses, however, mechanisms of SPHK/S1P signaling in the pathogenesis of bronchial asthma are poorly understood. In this study, we hypothesized that SPHK inhibition could ameliorate lung inflammation in ovalbumin (OVA)-challenged mouse lungs. Six- to eight-week-old C57BL/6J mice were sensitized and exposed to OVA for 3 consecutive days. Twenty-four hours later, mice lungs and bronchoalveolar lavage (BAL) fluid were analyzed. For an inhibitory effect, either of the two different SPHK inhibitors, N,N-dimethylsphingosine (DMS) or SPHK inhibitor [SK-I; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole], was nebulized for 30 min before OVA inhalation. OVA inhalation caused S1P release into BAL fluid and high expression of SPHK1 around bronchial epithelial walls and inflammatory areas. DMS or SK-I inhalation resulted in a decrease in S1P amounts in BAL fluid to basal levels, accompanied by decreased eosinophil infiltration and peroxidase activity. The extent of inhibition caused by DMS inhalation was higher than that caused by SK-I. Like T helper 2 (Th2) cytokine release, OVA inhalation-induced increase in eotaxin expression was significantly suppressed by DMS pretreatment both at protein level in BAL fluid and at mRNA level in lung homogenates. Moreover, bronchial hyperresponsiveness to inhaled methacholine and goblet cell hyperplasia were improved by SPHK inhibitors. These data suggest that the inhibition of SPHK affected acute eosinophilic inflammation induced in antigen-challenged mouse model and that targeting SPHK may provide a novel therapeutic tool to treat bronchial asthma.

RAGE: developmental expression and positive feedback regulation by Egr-1 during cigarette smoke exposure in pulmonary epithelial cells

2008-06-02

The receptor for advanced glycation end-products (RAGE) is a member of the immunoglobin superfamily of multiligand receptors. Following ligand binding, mechanisms associated with host defense, tissue remodeling, and inflammation are activated. RAGE is highly expressed in pulmonary epithelium transitioning from alveolar type (AT) II to ATI cells and is upregulated in the presence of ligand; however, the regulation and function of RAGE during development are less clear. Herein, immunohistochemistry demonstrated a temporal-spatial pattern of RAGE expression in pulmonary epithelial cells from embryonic day 17.5 to postnatal day 10. Cotransfection experiments revealed that the mouse RAGE promoter was activated by early growth response gene 1 (Egr-1) and inhibited by thyroid transcription factor-1 (TTF-1) via interaction with specific regulatory elements. A rat ATI cell line (R3/1) with endogenous RAGE expression also differentially regulated RAGE when transfected with TTF-1 or Egr-1. Because Egr-1 is markedly induced in pulmonary epithelial cells exposed to cigarette smoke extract (CSE; Reynolds PR, Hoidal JR. Am J Respir Cell Mol Biol 35: 314–319, 2006.), we sought to investigate RAGE induction by CSE. Employing RT-PCR and Western blotting, RAGE and common ligands (amphoterin and S100A12) were upregulated in epithelial (R3/1 and A549) and macrophage (RAW) cell lines following exposure to CSE. Immunostaining for RAGE in cells similarly exposed and in lungs from mice exposed to cigarette smoke for 6 mo revealed elevated RAGE expression in pulmonary epithelium. After the addition of glyoxylated BSA, an advanced glycation end-product that binds RAGE, real-time RT-PCR detected a 200-fold increase in Egr-1. These results indicate that Egr-1 regulates RAGE expression during development and the likelihood of positive feedback involving Egr-1 and RAGE in cigarette smoke-related disease.

Expression of the proapoptotic protein Bax is reduced in bronchial mucous cells of asthmatic subjects

2008-06-02

The present studies were designed to determine whether our findings in mice showing that the Bcl-2-associated protein X (Bax), which plays a role in the resolution of allergen-induced mucous cell metaplasia, can be applied to asthma in humans. Immunostaining of autopsy tissues from mild and severe asthmatic subjects showed a significant reduction in the percentage of Bax-positive mucous cells compared with those from nonasthmatic controls. To exclude the possibility that postmortem changes may have affected Bax expression, Bax mRNA levels in airway epithelial cells obtained from nonsmoking asthmatic subjects were compared with those from nonasthmatic controls. Because the number of cells obtained by bronchial brushings is limited, we developed a robust preamplification procedure of cDNA before quantitative real-time PCR to allow detection of 100 gene targets from limited sample size, even when it was prepared from partially degraded RNA. cDNA was prepared by reverse transcription from RNA isolated from bronchial epithelial cells obtained by bronchial brushings from well-characterized subjects without lung disease and from subjects with mild asthma. Quantitative analysis showed that Bax mRNA levels were significantly reduced in samples obtained from asthma patients compared with nonasthma controls. Furthermore, Bax mRNA levels were reduced when primary airway epithelial cells from 10 individuals were treated in culture with the T helper 2 cytokine IL-13. These studies show that Bax expression is reduced in airway epithelial cells of even mild asthmatic subjects and suggest that restoring Bax expression may provide a clinical approach for restoring the normal numbers of epithelial cells and reduced mucous hypersecretion in asthma.

GSK-3/{beta}-catenin signaling axis in airway smooth muscle: role in mitogenic signaling

2008-06-02

β-Catenin plays a dual role in cellular signaling by stabilizing cadherin-mediated cell-cell contact and by regulating gene transcription associated with cell cycle progression. Nonetheless, its presence and function in airway smooth muscle have not been determined. We hypothesized a central role for β-catenin in mitogenic signaling in airway smooth muscle in response to growth factor stimulation. Immunocytochemical and biochemical analysis revealed that human airway smooth muscle cells indeed express abundant β-catenin, which was localized primarily to the plasma membrane in quiescent cells. Treatment of airway smooth muscle cells with PDGF or FBS induced sustained phosphorylation of glycogen synthase kinase-3 (GSK-3), a negative regulator in its unphosphorylated form that promotes β-catenin degradation. GSK-3 phosphorylation was also increased in airway smooth muscle cells with a proliferative phenotype compared with quiescent airway smooth muscle cells with a mature phenotype. Parallel with the increase in GSK-3 phosphorylation, growth factor treatment induced an increased expression and nuclear presence of β-catenin and activated promitogenic signaling in airway smooth muscle, including the phosphorylation of retinoblastoma protein, DNA synthesis ([³H]thymidine incorporation), and cell proliferation. Importantly, small interfering RNA knockdown of β-catenin strongly reduced retinoblastoma protein phosphorylation, [³H]thymidine incorporation, and cell proliferation induced by PDGF and FBS. Collectively, these data reveal the existence of a GSK-3/β-catenin signaling axis in airway smooth muscle that is regulated by growth factors and of central importance to mitogenic signaling.

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection

2008-06-02

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.

The human zinc transporter SLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lung epithelia

Besecker, B., Bao, S., Bohacova, B., Papp, A., Sadee, W., Knoell, D. L. — 2008-06-02

Zinc is an essential micronutrient and cytoprotectant involved in the host response to inflammatory stress. We tested whether zinc transporters, the critical regulators that maintain intracellular zinc concentrations, play a role in cell survival, particularly in lung epithelia, during inflammation. Initially, mRNA transcripts were quantitatively measured by RT-PCR for all known human zinc transporters, including 14 importers (SLC39A_1–14) and 10 exporters (SLC30A_1–10), in primary human lung epithelia obtained from multiple human donors and BEAS-2B cell cultures under baseline and TNF--stimulated conditions. While many zinc transporters were constitutively expressed, only SLC39A8 (Zip8) mRNA was strongly induced by TNF-. Endogenous Zip8 protein was not routinely detected under baseline conditions. In sharp contrast, TNF- induced the expression of a glycosylated protein that translocated to the plasma membrane and mitochondria. Increased Zip8 expression resulted in an increase in intracellular zinc content and coincided with cell survival in the presence of TNF-. Inhibition of Zip8 expression using a short interfering RNA probe reduced cellular zinc content and impaired mitochondrial function in response to TNF-, resulting in loss of cell viability. These data are the first to characterize human Zip8 and remarkably demonstrate that upregulation of Zip8 is sufficient to protect lung epithelia against TNF--induced cytotoxicity. We conclude that Zip8 is unique, relative to other Zip proteins, by functioning as an essential zinc importer at the onset of inflammation, thereby facilitating cytoprotection within the lung.

Impact of the Fc{gamma}II-receptor on quartz uptake and inflammatory response by alveolar macrophages

2008-06-02

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcII-receptor (FcII-R). Fluorescence immunocytochemistry, demonstrating FcII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcII-R in DQ12 phagocytosis. FcII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcII-R. Consistent with the uptake results, FcII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF- release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.

Transgenic expression of matrix metalloproteinase-9 causes adult-onset emphysema in mice associated with the loss of alveolar elastin

2008-06-02

Matrix metalloproteinase (MMP)-9 has been consistently identified in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, its role in the development of the disease remains undefined. Mice that specifically express human MMP-9 in their macrophages were generated, and morphometric, biochemical, and histological analyses were conducted on the transgenic and littermate control mice over 1 yr to determine the effect of macrophage MMP-9 expression on emphysema formation and lung matrix content. Lung morphometry was normal in transgenic mice at 2 mo of age (mean linear intercept = 50 ± 3 littermate mice vs. 51 ± 2 transgenic mice). However, after 12 mo of age, the MMP-9 transgenic mice developed significant air space enlargement (mean linear intercept = 53 ± 3 littermate mice vs. 61 ± 2 MMP-9 transgenic mice; P < 0.04). Lung hydroxyproline content was not significantly different between wild-type and transgenic mice, but MMP-9 did significantly decrease alveolar wall elastin at 1 yr of age (4.9 ± 0.3% area of alveolar wall in the littermate mice vs. 3.3 ± 0.3% area of alveolar wall in the MMP-9 mice; P < 0.004). Thus these results establish a central role for MMP-9 in the pathogenesis of this disease by demonstrating that expression of this protease in macrophages can alter the extracellular matrix and induce progressive air space enlargement in mice.

Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration

2008-06-02

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls (P < 0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.

Telokin expression and the effect of hypoxia on its phosphorylation status in smooth muscle cells from small and large pulmonary arteries

Madden, J. A., Dantuma, M. W., Sorokina, E. A., Weihrauch, D., Kleinman, J. G. — 2008-06-02

Small pulmonary arteries (SPA), <500 µm diameter of the cat, constrict when exposed to hypoxia, whereas larger arteries (large pulmonary arteries; LPA), >800 µm diameter, show little or no response. It is unknown why different contractile responses occur within the same vascular bed, but activator or repressor proteins within the smooth muscle cell (SMC) can modify myosin phosphatase and myosin light chain kinase (MLCK), thereby influencing the phosphorylation state of myosin light chain (MLC) and ultimately, contraction. Telokin, a protein with a sequence identical to the COOH-terminal domain of MLCK, is expressed in smooth muscle where in its phosphorylated state it inhibits myosin phosphatase, binds to unphosphorylated myosin, and helps maintain smooth muscle relaxation. We measured telokin mRNA and telokin protein in smooth muscle from different diameter feline pulmonary arteries and sought to determine whether changes in the phosphorylation status of telokin and MLC occurred during hypoxia. In pulmonary arteries, telokin expression varied inversely with artery diameter, but cerebral arteries showed neither telokin protein nor telokin mRNA. Although telokin and MLC were distributed uniformly throughout the SPA muscle cell cytoplasm, they were not colocalized. During hypoxia, telokin dephosphorylated, and MLC became increasingly phosphorylated in SPA SMC, whereas in LPA SMC there was no change in either telokin or MLC phosphorylation. When LPA SMC were exposed to phenylephrine, MLC phosphorylation increased with no change in telokin phosphorylation. These results suggest that in SPA, phosphorylated telokin may help maintain relaxation under unstimulated conditions, whereas in LPA, telokin's function remains undetermined.

Cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice

2008-06-02

A variety of mouse models have been used to study the pathogenesis of pulmonary emphysema/chronic obstructive pulmonary disease. The effect of cigarette smoke (CS) is believed to be strain dependent, because certain mouse strains are more susceptible or resistant to development of emphysema. However, the molecular basis of susceptibility of mouse strains to effects of CS is not known. We investigated the effect of CS on lungs of most of the commonly used mouse strains to study the molecular mechanism of susceptibility to effects of CS. C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice were exposed to CS for 3 consecutive days, and various parameters of inflammatory and oxidative responses were assessed in lungs of these mice. We found that the C57BL/6J strain was highly susceptible, the A/J, AKR/J, and CD-1 strains were moderately susceptible, and the 129SvJ strain was resistant to lung inflammatory and oxidant responses to CS exposure. The mouse strain that was more susceptible to effects of CS showed augmented lung inflammatory cell influx, activation of NF-B and p38 MAPK, and increased levels of matrix metalloproteinase-9 and NF-B-dependent proinflammatory cytokines compared with resistant mouse strains. Similarly, decreased levels of glutathione were associated with increased levels of lipid peroxidation products in susceptible mouse strains compared with resistant strains. Hence, we identified the susceptible and resistant mouse strains on the basis of the pattern of inflammatory and oxidant responses. Identification of sensitive and resistant mouse strains could be useful for studying the molecular mechanisms of effects of CS on inflammation and pharmacological interventional studies in CS-exposure mouse models.

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A

2008-06-02

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca²⁺ and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca²⁺ activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.