bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary vascular response to normoxia and KCa channel activity is developmentally regulated
Authors: Rhodes Michael T. , Porter Valerie A. , Saqueton Connie B. , Herron Jean M. , Resnik Ernesto R. , Cornfield David N.
Published: December 2017

To address developmental regulation of pulmonary vascular O2 sensing, we tested the hypotheses that 1) fetal but not adult pulmonary artery smooth muscle cells (PASMCs) can directly sense an acute increase in O2, 2) Ca2+-sensitive K+(KCa) channel activity decreases with maturation, and 3) PASMC KCa channel expression decreases with maturation. We used fluorescence microscopy to confirm that fetal but not adult PASMCs are able to sense an acute increase in O2tension. Acute normoxia induced a 22 ± 2% decrease in cytosolic Ca2+ concentration ([Ca2+]i) in fetal PASMCs and no change in [Ca2+]i in adult PASMCs ( P < 0.01). The effects of K+channel antagonists were studied on fetal and adult PASMC [Ca2+]i. Iberiotoxin (10−9 M) caused PASMC [Ca2+]i to increase by 694 ± 22% in the fetus and caused no change in adult PASMCs. KCa channel expression and mRNA levels in distal pulmonary arteries from fetal and adult sheep were examined. Both KCachannel protein and mRNA expression in the distal pulmonary vasculature decreased with maturation. We conclude that maturation-dependent changes in PASMC O2 sensing render the fetal PASMCs uniquely sensitive to an acute increase in O2 tension at a biologically critical time point.
Mechanisms of TNF-α stimulation of amiloride-sensitive sodium transport across alveolar epithelium
Authors: Fukuda Norimasa , Jayr Christian , Lazrak Ahmed , Wang Yibing , Lucas Rudolf , Matalon Sadis , Matthay Michael A.
Published: December 2017

Because tumor necrosis factor (TNF)-α can upregulate alveolar fluid clearance (AFC) in pneumonia or septic peritonitis, the mechanisms responsible for the TNF-α-mediated increase in epithelial fluid transport were studied. In rats, 5 μg of TNF-α in the alveolar instillate increased AFC by 67%. This increase was inhibited by amiloride but not by propranolol. We also tested a triple-mutant TNF-α that is deficient in the lectinlike tip portion of the molecule responsible for its membrane conductance effect; the mutant also has decreased binding affinity to both TNF-α receptors. The triple-mutant TNF-α did not increase AFC. Perfusion of human A549 cells, patched in the whole cell mode, with TNF-α (120 ng/ml) resulted in a sustained increase in Na+currents from 82 ± 9 to 549 ± 146 pA ( P < 0.005; n = 6). The TNF-α-elicited Na+current was inhibited by amiloride, and there was no change when A549 cells were perfused with the triple-mutant TNF-α or after preincubation with blocking antibodies to the two TNF-α receptors before perfusion with TNF-α. In conclusion, although TNF- α can initiate acute inflammation and edema formation in the lung, TNF-α can also increase AFC by an amiloride-sensitive, cAMP-independent mechanism that enhances the resolution of alveolar edema in pathological conditions by either binding to its receptors or activating Na+channels by means of its lectinlike domain.
Gas 6 promotes Axl-mediated survival in pulmonary endothelial cells
Authors: Healy Aileen M. , Schwartz John J. , Zhu Xiahui , Herrick Brian E. , Varnum Brian , Farber Harrison W.
Published: December 2017

We examined Gas 6-Axl interactions in human pulmonary artery endothelial cells (HPAEC) and in Axl-transduced HPAEC to test Gas 6 function during endothelial cell survival. We identified the 5.0-kb Axl, 4.2-kb Rse, and 2.6-kb Gas 6 mRNAs in HPAEC. Immunoprecipitation and Western blotting confirmed the presence of these proteins. Gas 6 is present in cell-associated and secreted fractions of growth-arrested HPAEC, independent of cell density. In addition, the Axl receptor is constitutively phosphorylated in growth-arrested cultures, and exogenous Gas 6 enhanced Axl phosphorylation threefold. Gas 6 added to growth-arrested HPAEC resulted in a significant increase in cell number (1.5 nM Gas 6 increased cell number 35%). Flow cytometry revealed that Gas 6 treatment resulted in 28% fewer apoptosing cells. Transduction of a full-length Axl cDNA into HPAEC resulted in 54% fewer apoptosing cells after Gas 6 treatment. Collectively, the data demonstrate antiapoptotic activities for Gas 6 in HPAEC and suggest that Gas 6 signaling may be relevant to endothelial cell survival in the quiescent environment of the vessel wall.
Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels
Authors: Coppock Elizabeth A. , Martens Jeffrey R. , Tamkun Michael M.
Published: December 2017

The hypoxia-induced membrane depolarization and subsequent constriction of small resistance pulmonary arteries occurs, in part, via inhibition of vascular smooth muscle cell voltage-gated K+(KV) channels open at the resting membrane potential. Pulmonary arterial smooth muscle cell KV channel expression, antibody-based dissection of the pulmonary arterial smooth muscle cell K+ current, and the O2 sensitivity of cloned KV channels expressed in heterologous expression systems have all been examined to identify the molecular components of the pulmonary arterial O2-sensitive KV current. Likely components include Kv2.1/Kv9.3 and Kv1.2/Kv1.5 heteromeric channels and the Kv3.1b α-subunit. Although the mechanism of KV channel inhibition by hypoxia is unknown, it appears that KV α-subunits do not sense O2 directly. Rather, they are most likely inhibited through interaction with an unidentified O2 sensor and/or β-subunit. This review summarizes the role of KV channels in hypoxic pulmonary vasoconstriction, the recent progress toward the identification of KV channel subunits involved in this response, and the possible mechanisms of KV channel regulation by hypoxia.
Carbon monoxide attenuates aeroallergen-induced inflammation in mice
Authors: Chapman Jeffrey T. , Otterbein Leo E. , Elias Jack A. , Choi Augustine M. K.
Published: December 2017

Carbon monoxide (CO) generated by catalysis of heme by heme oxygenase is increased in the exhaled air of asthmatic patients. Based on recent studies demonstrating that asthma is an inflammatory disease associated with increased oxidants and that CO confers cytoprotection in oxidant-induced lung injury and inflammation, we sought to better understand the functional role of CO in asthma by using an aeroallergen model. Mice were sensitized to ovalbumin, challenged with aerosolized ovalbumin, and maintained in either CO (250 parts/million) or room air for 48 h. The differential effects of CO on bronchoalveolar lavage (BAL) fluid cell types were observed, with a marked attenuation of BAL fluid eosinophils in the CO-treated animals at 24 and 48 h. A marked reduction of the proinflammatory cytokine interleukin-5 was observed in the CO-treated mice, with no significant changes for other proinflammatory cytokines. These differential effects of CO were also observed with leukotrienes (LTs) and prostaglandins in that CO significantly decreased BAL fluid PGE2, and LTB4but exerted negligible effect on thromboxane B2or LTC4/D4/E4. Our data suggest a putative immunoregulatory role for CO in aeroallergen-induced inflammation in mice.
Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice
Authors: Velsor Leonard W. , van Heeckeren Anna , Day Brian J.
Published: December 2017

Recent studies suggest that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and when defective creates an antioxidant imbalance. To test whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue from CFTR knockout (CFTR-KO) and wild-type (WT) mice were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and γ-glutamyltransferase. In the CFTR-KO mice, the ELF concentration of GSH was decreased (51%) compared with that in WT mice. The concentration of GSH in the lung tissue of CFTR-KO mice, however, was not significantly different from that in WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice (48 and 28%, respectively). Tissue lipid and DNA oxidation were evaluated by measurement of thiobarbituric acid-reactive substances and 8-hydroxy-2′-deoxyguanosine, respectively. The levels of thiobarbituric acid-reactive substances and 8-hydroxy-2′-deoxyguanosine in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice. These data support our hypothesis that a mutation in the CFTR gene can affect the antioxidant defenses in the lung and may contribute to the exaggerated inflammatory response observed in CF.
Sodium 4-phenylbutyrate downregulates HSC70expression by facilitating mRNA degradation
Authors: Rubenstein Ronald C. , Lyons Bridget M.
Published: December 2017

Intracellular trafficking of the ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) is repaired by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA downregulates protein and mRNA expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family) by ∼40–50% and decreases formation of a HSC70-ΔF508 CFTR complex that may be important in the intracellular degradation of ΔF508 CFTR. We examined the potential mechanisms by which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells, 1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70 promoter or of a human HSC70 promoter fragment in luciferase reporter assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays. In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA degradation by ∼40%. The initial rate of35S-HSC70 protein synthesis in 4PBA-treated IB3-1 cells was reduced by ∼40%, consistent with the steady-state mRNA level, whereas its rate of degradation was unaltered by 4PBA. 4PBA also reduced the steady-state accumulation of35S-HSC70 by ∼40%. These data suggest that 4PBA decreases the expression of HSC70 mRNA and protein by inducing cellular adaptations that result in the decreased stability of HSC70 mRNA.
Induction of HSP70 promotes ΔF508 CFTR trafficking
Authors: Choo-Kang Lee R. , Zeitlin Pamela L.
Published: December 2017

The ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) is a temperature-sensitive trafficking mutant that is detected as an immature 160-kDa form (band B) in gel electrophoresis. The goal of this study was to test the hypothesis that HSP70, a member of the 70-kDa heat shock protein family, promotes ΔF508 CFTR processing to the mature 180-kDa form (band C). Both pharmacological and genetic techniques were used to induce HSP70. IB3-1 cells were treated with sodium 4-phenylbutyrate (4PBA) to promote maturation of ΔF508 CFTR to band C. A dose-dependent increase in band C and total cellular HSP70 was observed. Under these conditions, HSP70-CFTR complexes were increased and 70-kDa heat shock cognate protein-CFTR complexes were decreased. Increased ΔF508 CFTR maturation was also seen after transfection with an HSP70 expression plasmid and exposure to glutamine, an inducer of HSP70. With immunofluorescence techniques, the increased appearance of CFTR band C correlated with CFTR distribution beyond the perinuclear regions. These data suggest that induction of HSP70 promotes ΔF508 CFTR maturation and trafficking.
Activation of NF-κB in airway epithelial cells is dependent on CFTR trafficking and Cl− channel function
Authors: Weber Adam J. , Soong Grace , Bryan Ruth , Saba Shahryar , Prince Alice
Published: December 2017

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant ΔF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause “cell stress” and activation of nuclear factor (NF)-κB. G551D mutants also lack Cl− channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-κB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked ΔF508 allele caused a sevenfold activation of NF-κB compared with wild-type CFTR or the G551D mutant ( P < 0.001). NF-κB was also activated in 9/HTEo−/pCep-R cells and in 16HBE/p cftrantisense cell lines, which lack CFTR Cl− channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-κB was associated with elevated interleukin-8 expression. Impaired CFTR Cl− channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-κB in cells with the CFTR mutation.
IL-12 attenuates bleomycin-induced pulmonary fibrosis
Authors: Keane Michael P. , Belperio John A. , Burdick Marie D. , Strieter Robert M.
Published: December 2017

Interleukin (IL)-12 is a potent inducer of interferon (IFN)-γ. We postulated that IL-12 would attenuate bleomycin-induced pulmonary fibrosis. To test this hypothesis, we administered IL-12 or murine serum albumin to bleomycin-treated mice by daily intraperitoneal injection until day 12. Mice treated with IL-12 demonstrated decreased hydroxyproline levels compared with control treated mice. Furthermore, administration of IL-12 led to a time-dependent increase in both lung and bronchoalveolar lavage fluid IFN-γ. The antifibrotic effect of IL-12 could be attenuated with simultaneous administration of neutralizing anti-IFN-γ antibodies. These findings support the notion that IL-12 attenuates bleomycin-induced pulmonary fibrosis via modulation of IFN-γ production.
Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase
Authors: Vivekananda Jeevalatha , Smith Dolphin , King Richard J.
Published: December 2017

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-α and interleukin-1β are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-α, thereby amplifying the sphingomyelinase activation, and TNF-α, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-α may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.
X-ray microanalysis of airway surface liquid collected in cystic fibrosis mice
Authors: Zahm Jean-Marie , Baconnais Sonia , Davidson Donald J. , Webb Sheila , Dorin Julia , Bonnet Noel , Balossier Gérard , Puchelle Edith
Published: December 2017

The airway surface liquid (ASL) that lines the airway surface epithelium plays a major role in airway antibacterial defense and mucociliary transport efficiency, two key factors in cystic fibrosis (CF) disease. A major difficulty is to collect ASL in native conditions without stimulation or alteration of the underlying airway epithelium. Using a cryoprobe specifically adapted to collect native ASL from the tracheal mouse surface, we analyzed by X-ray microanalysis the complete ASL and plasma ion content in Cftrtm1Hgu/Cftrtm1Hgu mice compared with that in control littermates. ASL ion content from eight Cftrtm1Hgu/Cftrtm1Hgu mice and eight control littermates did not appear significantly different. The mean (±SE) concentrations were 2,352 ± 367 and 2,058 ± 401 mmol/kg dry weight for Na, 1,659 ± 272 and 1,448 ± 281 mmol/kg dry weight for Cl, 357 ± 57 and 337 ± 38 mmol/kg dry weight for S, 1,066 ± 220 and 787 ± 182 mmol/kg dry weight for K, 400 ± 82 and 301 ± 58 mmol/kg dry weight for Ca, 105 ± 31 and 105 ± 20 mmol/kg dry weight for Mg, 33 ± 15 and 29 ± 9 mmol/kg dry weight for P in non-CF and CF mice, respectively. This cryotechnique appears to be a promising technique for analyzing the complete elemental composition of native ASL in CF and non-CF tissues.
Recombinant human Fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits
Authors: Matute-Bello Gustavo , Liles W. Conrad , Frevert Charles W. , Nakamura Morio , Ballman Kim , Vathanaprida Charie , Kiener Peter A. , Martin Thomas R.
Published: December 2017

This study investigated whether recombinant human soluble Fas ligand (rh-sFasL) induces apoptosis of primary type II pneumocytes in vitro and lung injury in vivo. Type II cells isolated from normal rabbit lung expressed Fas on their surface and became apoptotic after an 18-h incubation with rh-sFasL. Fas expression in normal rabbit lungs was localized by immunohistochemistry to alveolar and airway epithelia and alveolar macrophages. The administration of 10 μg of rh-sFasL into the right lungs of rabbits resulted 24 h later in both significantly more bronchoalveolar lavage fluid total protein and significantly more tissue changes compared with those in the left lungs, which received rh-sFasL plus Fas:Ig (a fusion protein that binds and blocks sFasL). Tissue changes included thickening of the alveolar walls, neutrophilic infiltrates, apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive) cells in the alveolar walls, and increased expression of interleukin-8 by alveolar macrophages (as determined by immunohistochemistry). We conclude that the alveolar epithelium of normal rabbits expresses Fas and that sFasL induces lung injury and inflammation in rabbits.
Anti-neutrophil chemokine preserves alveolar development in hyperoxia-exposed newborn rats
Authors: Auten Richard L. , Mason S. Nicholas , Tanaka David T. , Welty-Wolf Karen , Whorton Mary H.
Published: December 2017

Inflammation may contribute to lung injury and impaired alveolar development in bronchopulmonary dysplasia. We treated hyperoxia-exposed newborn rats with antibodies to the neutrophil chemokine cytokine-induced neutrophil chemoattractant-1 (CINC-1) during 95% O2exposure to reduce adverse effects of hyperoxia-induced inflammation on lung development. Rats were exposed at birth to air, 95% O2, or 95% O2+ anti-CINC-1 (injected on days 3 and 4). Bromodeoxyuridine (BrdU) was injected 6 h before death. Anti-CINC-1 treatment improved weight gain but not survival at day 8. Anti-CINC-1 reduced bronchoalveolar lavage neutrophils at day 8 to levels equal to air controls. Total detectable lung CINC-1 was reduced to air control levels. Lung compliance was improved by anti-CINC-1, achieving air control levels in the 10-μg anti-CINC-1 group. Anti-CINC-1 preserved proliferating cell nuclear antigen expression in airway epithelium despite 95% O2exposure. BrdU incorporation was depressed by hyperoxia but preserved by anti-CINC-1 to levels similar to air control. Alveolar volume and surface density were decreased by hyperoxia but preserved by anti-CINC-1 to levels equal to air control. Blockade of neutrophil influx in newborns may avert early lung injury and avoid alveolar developmental arrest that contributes to bronchopulmonary dysplasia.
Strain dependence of airway hyperresponsiveness reflects differences in eosinophil localization in the lung
Authors: Takeda K. , Haczku A. , Lee J. J. , Irvin C. G. , Gelfand E. W.
Published: December 2017

Different strains of mice exhibit different degrees of airway hyperresponsiveness after sensitization to and airway challenge with ovalbumin. Antibody responses in BALB/c mice far exceeded those in C57BL/6 mice; in contrast, although responsiveness to methacholine was much higher in the BALB/c mice, the number of eosinophils in the bronchoalveolar lavage fluid was higher in C57BL/6 animals. Sensitized and challenged BALB/c mice developed increases in lung resistance and decreases in dynamic compliance after methacholine or 5-hydroxytryptamine inhalation. C57BL/6 mice only exhibited significant levels of responsiveness when dynamic compliance was monitored in response to inhaled 5-hydroxytryptamine. Eosinophils accumulated in the peribronchial and peripheral lung tissue in BALB/c mice but were distributed diffusely in the peripheral lung tissue of C57BL/6 mice. Thus, in addition to differences in antibody responses and cholinergic agonist reactivity, differences in the responses of large and small airways may reflect the selective distribution of eosinophils in lung tissue.
Migration and gelatinases in cultured fetal, adult, and hyperoxic alveolar epithelial cells
Authors: Buckley S. , Driscoll B. , Shi W. , Anderson K. , Warburton D.
Published: December 2017

Alveolar epithelial cell (AEC) migration mediated by matrix metalloproteinases (MMPs) is required for lung development and repair after injury such as hyperoxia. Of specific interest in lung remodeling are the gelatinases, which are upregulated in AEC after hyperoxia. We correlated migration and gelatinase production in AEC cultured from fetal, adult, and hyperoxic rats. Fetal AEC (19–20 days) had higher MMP-2 and MMP-9 gelatinase expression than adult AEC, with fivefold higher MMP-9 activity, and were migratory through gelatin, responding to epidermal growth factor, keratinocyte growth factor, and fibroblast growth factor-10. MMP-2 and MMP-9 expression and migratory activity could be detected from the time of plating. In contrast, adult AEC migrated and expressed MMP-2 and MMP-9 proteins only after 48 h of culture. AEC from hyperoxic rats were significantly more migratory through gelatin than control adult AEC, with significantly higher MMP-9 activity. Inhibition of MMPs with doxycycline reduced the migration of AEC from hyperoxic rats to the level of control adult AEC. Fibronectin-cultured “hyperoxic” AEC acquired a temporary capacity for migration similar to the A549 lung cancer cell line, which is both highly migratory and invasive and is derived from the AEC type 2 lineage. These data suggest that MMP activity is associated with a migratory phenotype in fetal, hyperoxic, and transformed AEC in vitro, and we speculate that MMPs may play a key mechanistic role in AEC migration in vivo during lung development and repair.
Shear stress regulation of endothelial NOS in fetal pulmonary arterial endothelial cells involves PKC
Authors: Wedgwood Stephen , Bekker Janine M. , Black Stephen M.
Published: December 2017

We have shown that increased pulmonary blood flow at birth increases the activity and expression of endothelial nitric oxide (NO) synthase (eNOS). However, the signal transduction pathway regulating this process is unclear. Because protein kinase C (PKC) has been shown to be activated in response to shear stress, we undertook a study to examine its role in mediating shear stress effects on eNOS. Initial experiments demonstrated that PKC activity increased in response to shear stress. NO production in response to shear stress was found to be biphasic, with an increase in NO release up to 1 h, a plateau phase until 4 h, and another increase between 4 and 8 h. PKC inhibition reduced the initial rise in NO release by 50% and the second increase by 70%. eNOS mRNA and protein levels were also increased in response to shear stress, whereas PKC inhibition prevented this increase. The stimulation of PKC activity with phorbol ester increased eNOS gene expression without increasing NO release. These results suggest that PKC may play different roles in shear stress-mediated release of NO and increased eNOS gene expression.
Gene suppression by tristetraprolin and release by the p38 pathway
Authors: Zhu Wei , Brauchle Maria A. , Di Padova Franco , Gram Hermann , New Liguo , Ono Koh , Downey Jocelyn S. , Han Jiahuai
Published: December 2017

Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-α and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.
Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line
Authors: Hoshino Yuma , Mio Tadashi , Nagai Sonoko , Miki Hiroyuki , Ito Isao , Izumi Takateru
Published: December 2017

Injury of the alveolar epithelium by cigarette smoke is presumed to be an important process in the pathogenesis of smoking-related pulmonary diseases. We investigated the cytotoxic effects of cigarette smoke extract (CSE) on an alveolar type II cell-derived cell line (A549). CSE caused apoptosis at concentrations of 5% or less and necrosis at 10% or more. When CSE was exposed to air before application to A549 cells, the cytotoxic effects were attenuated. CSE caused cell death without direct contact with the cells. Acrolein and hydrogen peroxide, two major volatile factors in cigarette smoke, caused cell death in a similar manner. Aldehyde dehydrogenase, a scavenger of aldehydes, and N-acetylcysteine, a scavenger of oxidants and aldehydes, completely inhibited CSE-induced apoptosis. CSE and acrolein increased intracellular oxidant activity. In conclusion, apoptosis of alveolar epithelial cells may be one of the mechanisms of lung injury induced by cigarette smoking. This cytotoxic effect might be due to an interaction between aldehydes and oxidants present in CSE or formed in CSE-exposed cells.
Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions
Authors: Hickman-Davis Judy M. , Fang Ferric C. , Nathan Carl , Shepherd Virginia L. , Voelker Dennis R. , Wright Jo Rae
Published: December 2017

Surfactant protein (SP) A and SP-D are members of the collectin superfamily. They are widely distributed within the lung, are capable of antigen recognition, and can discern self versus nonself. SPs recognize bacteria, fungi, and viruses by binding mannose and N-acetylglucosamine residues on microbial cell walls. SP-A has been shown to stimulate the respiratory burst as well as nitric oxide synthase expression by alveolar macrophages. Although nitric oxide (NO·) is a well-recognized microbicidal product of macrophages, the mechanism(s) by which NO· contributes to host defense remains undefined. The purpose of this symposium was to present current research pertaining to the specific role of SPs and reactive oxygen-nitrogen species in innate immunity. The symposium focused on the mechanisms of NO·-mediated toxicity for bacterial, human, and animal models of SP-A- and NO·-mediated pathogen killing, microbial defense mechanisms against reactive oxygen-nitrogen species, specific examples and signaling pathways involved in the SP-A-mediated killing of pulmonary pathogens, the structure and binding of SP-A and SP-D to bacterial targets, and the immunoregulatory functions of SP-A.
Serine proteases increase oxidative stress in lung cells
Authors: Aoshiba Kazutetsu , Yasuda Kimihiko , Yasui Shuji , Tamaoki Jun , Nagai Atsushi
Published: December 2017

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1–100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2′-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H2O2, and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.
Surfactant proteins A and D in Eustachian tube epithelium
Authors: Paananen Reija , Sormunen Raija , Glumoff Virpi , van Eijk Martin , Hallman Mikko
Published: December 2017

Surfactant protein (SP) A and SP-D are collectins that have roles in host defense. The Eustachian tube (ET) maintains the patency between the upper airways and the middle ear. Dysfunction of local mucosal immunity in ET may predispose infants to recurrent otitis media. We recently described preliminary evidence of the expression of SP-A and SP-D in the ET. Our present aim was to establish the sites of SP-A and SP-D expression within the epithelium of the ET in vivo. With in situ hybridization, electron microscopy, and immunoelectron microscopy, the cells responsible for SP-A and SP-D expression and storage were identified. SP-A expression was localized within the ET epithelium, and the protein was found in the electron-dense granules of microvillar epithelial cells. Being concentrated in the epithelial lining, only a few cells revealed intracellular SP-D, and it was not associated with granules. The SP-A and SP-D immunoreactivities in ET lavage fluid, as shown by Western blot analyses, were similar to those in bronchoalveolar lavage fluid. We propose that there are specialized cells in the ET epithelium expressing and secreting SP-A and SP-D. SP-A and SP-D may be important for antibody-independent protection of the middle ear against infections.
Persistent mucin glycoprotein alterations in equine recurrent airway obstruction
Authors: Jefcoat A. M. , Hotchkiss J. A. , Gerber V. , Harkema J. R. , Basbaum C. B. , Robinson N. E.
Published: December 2017

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (α-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of α-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.
Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease?
Authors: Finkelstein Erik I. , Nardini Mirella , van der Vliet Albert
Published: December 2017

Cigarette smoking is known to contribute to inflammatory diseases of the respiratory tract by promoting recruitment of inflammatory-immune cells such as neutrophils and perhaps by altering neutrophil functional properties. We investigated whether acrolein, a toxic unsaturated aldehyde found in cigarette smoke, could directly affect neutrophil function. Exposure of freshly isolated human neutrophils to acrolein markedly inhibited spontaneous neutrophil apoptosis as indicated by loss of membrane asymmetry and DNA fragmentation and induced increased neutrophil production of the chemokine interleukin-8 (IL-8). Acrolein (1–50 μM) was found to induce marked activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs), and inhibition of p38 MAPK activation by SB-203580 prevented acrolein-induced IL-8 release. However, inhibition of either ERK or p38 MAPK did not affect acrolein-dependent inhibition of apoptosis. Acrolein exposure prevented the activation of caspase-3, a crucial step in the execution of neutrophil apoptosis, presumably by direct inhibition of the enzyme. Our results indicate that acrolein may contribute to smoke-induced inflammatory processes in the lung by increasing neutrophil recruitment and reducing neutrophil clearance by apoptosis.
Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs
Authors: MacRitchie Amy N. , Albertine Kurt H. , Sun Jiancheng , Lei Paul S. , Jensen Suzanne C. , Freestone Allen A. , Clair Philip M. , Dahl Mar Janna , Godfrey Emily A. , Carlton David P. , Bland Richard D.
Published: December 2017

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, α-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.
Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter
Authors: Sun Guobin , Crissman Kay , Norwood Joel , Richards Judy , Slade Ralph , Hatch Gary E.
Published: December 2017

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains ∼10% by weight of soluble transition metals, was added (0–200 μg/ml) to BALF or sELF and exposed to 20%18O2 (24°C, 4 h). Oxygen incorporation was quantified as excess 18O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.
Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V
Authors: Xu Dingbang , Isaacs Cary , Hall Ian P. , Emala Charles W.
Published: December 2017

Adenylyl cyclases are a nine-member family of differentially regulated enzymes responsible for the synthesis of cAMP. cAMP is an important second messenger that contributes to the regulation of airway smooth muscle tone. However, little is known regarding the expression and regulation of adenylyl cyclase isoforms in airway smooth muscle cells. Nondegenerate specific primers were designed for all nine known isoforms of human adenylyl cyclase. RT-PCR experiments were performed using total RNA extracted from whole human brain (positive control), whole rat brain (negative control), whole human trachea, human airway smooth muscle, and primary cultures of human airway smooth muscle cells. Seven of the nine known isoforms of adenylyl cyclase (isoforms I, III–VII, and IX) were expressed at the mRNA level in both human airway smooth muscle and primary cultures of human airway smooth muscle cells. Immunoblot and adenylyl cyclase functional assay indicated that isoform V is likely among the functionally predominant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that multiple isoforms of adenylyl cyclase enzymes are coexpressed in human airway smooth muscle cells and that isoform V is among the functionally important isoforms.
Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture
Authors: Zhu Y. K. , Liu X. D. , Sköld C. M. , Umino T. , Wang H. J. , Spurzem J. R. , Kohyama T. , Ertl R. F. , Rennard S. I.
Published: December 2017

Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-α, interleukin-1β, and interferon-γ), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% ( P < 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% ( P < 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% ( P < 0.01). α1-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema.
Neutrophil maturation and activation determine anatomic site of clearance from circulation
Authors: Suratt Benjamin T. , Young Scott K. , Lieber Jonathan , Nick Jerry A. , Henson Peter M. , Worthen G. Scott
Published: December 2017

The long-term disposition of circulating neutrophils and the site of disappearance from circulation remain unclear. We investigated neutrophil localization in mice using111In-labeled murine peripheral blood neutrophils, mature bone marrow neutrophils, and peritoneal exudate neutrophils to track in vivo localization of these different cell populations. Infused peripheral neutrophils were found to localize equally between liver and marrow sites by 4 h (31.2 ± 1.9 vs. 31.9 ± 1.8%), whereas exudate neutrophils predominantly localized to liver (42.0 ± 1.1%) and marrow-derived neutrophils to the marrow (65.9 ± 6.6%) where they were found to localize predominantly in the hematopoietic cords. Stimulation of marrow neutrophils before infusion caused a shift in localization from marrow to liver, and subsequent induction of an inflammatory site after infusion and marrow sequestration led to remobilization of infused marrow neutrophils but not of peripheral neutrophils. These results indicate that the marrow participates in removing neutrophils from circulation, with evidence supporting both storage and perhaps disposal functions. Furthermore, models for circulating neutrophil homeostasis should consider that the site of retention is governed by the maturation and activation states of the cell.
Role of cell surface glycosylation in mediating repair of human airway epithelial cell monolayers
Authors: Dorscheid Delbert R. , Wojcik Kimberly R. , Yule Kelly , White Steven R.
Published: December 2017

Our laboratory recently demonstrated the pattern of cell surface glycosylation of nonsecretory central airway epithelium (Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, and White SR. Histochem J 31: 145–151, 1999), but the role of glycosylation in airway epithelial cell migration and repair is unknown. We examined the functional role of cell surface carbohydrates in wound repair after mechanical injury of 1HAEo−human airway epithelial and primary bronchial epithelial monolayers. Wound repair stimulated by epidermal growth factor was substantially attenuated by 10−7M tunicamycin (TM), an N-glycosylation inhibitor, but not by the inhibitors deoxymannojirimycin or castanospermine. Wound repair of 1HAEo−and primary airway epithelial cells was blocked completely by removal of cell surface terminal fucose residues by α-fucosidase. Cell adhesion to collagen matrix was prevented by TM but was only reduced ∼20% from control values with prior α-fucosidase treatment. Cell migration in Blind Well chambers stimulated by epidermal growth factor was blocked by pretreatment with TM but α-fucosidase pretreatment produced no difference from control values. These data suggest that cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration and that N-glycosylation with terminal fucosylation plays a role in the complex process of repair by coordination of certain cell-cell functions.
Transcriptional mechanisms of acute lung injury
Authors: Fan Jie , Ye Richard D. , Malik Asrar B.
Published: December 2017

Acute lung injury occurs as a result of a cascade of cellular events initiated by either infectious or noninfectious inflammatory stimuli. An elevated level of proinflammatory mediators combined with a decreased expression of anti-inflammatory molecules is a critical component of lung inflammation. Expression of proinflammatory genes is regulated by transcriptional mechanisms. Nuclear factor-κB (NF-κB) is one critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of acute lung injury. Activation and regulation of NF-κB are tightly controlled by a complicated signaling cascade. In acute lung injury caused by infection of bacteria, Toll-like receptors play a central role in initiating the innate immune system and activating NF-κB. Anti-inflammatory cytokines such as interleukin-10 and interleukin-13 have been shown to suppress inflammatory processes through inhibiting NF-κB activation. NF-κB can interact with other transcription factors, and these interactions thereby lead to greater transcriptional selectivity. Modification of transcription is likely to be a logical therapeutic target for acute lung injury.
sGC and PDE5 are elevated in lambs with increased pulmonary blood flow and pulmonary hypertension
Authors: Black Stephen M. , Sanchez Lucienne S. , Mata-Greenwood Eugenia , Bekker Janine M. , Steinhorn Robin H. , Fineman Jeffrey R.
Published: December 2017

Utilizing aortopulmonary vascular graft placement, we established a lamb model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. We previously demonstrated that endothelial nitric oxide synthase (eNOS) is increased in lambs at age 4 wk. However, these lambs display a selective impairment of endothelium-dependent pulmonary vasodilation that is suggestive of a derangement downstream of NO release. Thus our objective was to characterize potential alterations in the expression and activity of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5) induced by increased pulmonary blood flow and pulmonary hypertension. Late-gestational fetal lambs ( n = 10) underwent in utero placement of an aortopulmonary vascular graft (shunt). Western blotting analysis on lung tissue from 4-wk-old shunted lambs and age-matched controls showed that protein for both subunits of sGC was increased in shunted lamb lungs compared with age-matched controls. Similarly, cGMP levels were increased in shunted lamb lungs compared with age-matched controls. However, PDE5 expression and activity were also increased in shunted lambs. Thus although cGMP generation was increased, concomitant upregulation of PDE5 expression and activity may have (at least partially) limited and accounted for the impairment of endothelium-dependent pulmonary vasodilation in shunted lambs.