bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide participates in early events associated with NNMU-induced acute lung injury in rats
Authors: Cruz Wilhelm S. , Corbett John A. , Longmore William J. , Moxley Michael A.
Published: December 2017

In this study, the biochemical mechanisms by which N-nitroso- N-methylurethane (NNMU) induces acute lung injury are examined. Polymorphonuclear neutrophil infiltration into the lungs first appears in the bronchoalveolar lavage (BAL) fluid 24 h after NNMU injection (10.58 ± 3.00% of total cells; P < 0.05 vs. control animals). However, NNMU-induced elevation of the alveolar-arterial O2 difference requires 72 h to develop. Daily intraperitoneal injections of the inducible nitric oxide (⋅ NO) synthase (iNOS)-selective inhibitor aminoguanidine (AG) initiated 24 h after NNMU administration improve the survival of NNMU-treated animals. However, AG administration initiated 48 or 72 h after NNMU injection does not significantly improve the survival of NNMU-treated animals. These results suggest that ⋅ NO participates in events that occur early in NNMU-induced acute lung injury. BAL cells isolated from rats 24 and 48 h after NNMU injection produce elevated ⋅ NO and express iNOS during a 24-h ex vivo culture. AG attenuates ⋅ NO production but does not affect iNOS expression, whereas actinomycin D prevents iNOS expression and attenuates ⋅ NO production by BAL cells during this ex vivo culture. These results suggest that NNMU-derived BAL cells can stimulate iNOS expression and ⋅ NO production during culture. In 48-h NNMU-exposed rats, iNOS expression is elevated in homogenates of whole lavaged lungs but not in BAL cells derived from the same lung. These findings suggest that the pathogenic mechanism by which NNMU induces acute lung injury involves BAL cell stimulation of iNOS expression and ⋅ NO production in lung tissue.
Role of neutrophils and α1-antitrypsin in coal- and silica-induced connective tissue breakdown
Authors: Zay K. , Loo S. , Xie C. , Devine D. V. , Wright J. , Churg A.
Published: December 2017

Mineral dusts produce emphysema, and administration of dust to rats results in the rapid appearance of desmosine and hydroxyproline in lavage fluid, confirming that dusts directly induce connective tissue breakdown. To examine the role of neutrophils and α1-antitrypsin (α1-AT) in this process, we instilled silica or coal into normal rats or rats that had been pretreated with antiserum against neutrophils. One day after dust exposure, lavage fluid neutrophils and desmosine and hydroxyproline levels were all elevated; treatment with antiserum against neutrophils reduced neutrophils by 75%, desmosine by 40–50%, and hydroxyproline by 25%. By 7 days, lavage fluid neutrophils and desmosine level had decreased, whereas macrophages and hydroxyproline level had increased. By ELISA analysis, lavage fluid α1-AT levels were increased four- to eightfold at both times. On Western blot, some of the α1-AT appeared as degraded fragments, and by HPLC analysis, 5–10% of the methionine residues were oxidized. At both times, lavage fluid exhibited considerably elevated serine elastase inhibitory capacity and also showed elevations in metalloelastase activity. We conclude that, in this model, connective tissue breakdown is initially driven largely by neutrophil-derived proteases and that markedly elevated levels of functional α1-AT do not prevent breakdown, thus providing in vivo support for the concept of quantum proteolysis proposed by Liou and Campbell (T. G. Liou and E. J. Campbell. Biochemistry 34: 16171–16177, 1995). Macrophage-derived proteases may be of increasing importance over time, especially in coal-treated animals.
Identification of glucocorticoid-responsive elements that control transcription of rat glutamine synthetase
Authors: Chandrasekhar S. , Souba Wiley W. , Abcouwer Steve F.
Published: December 2017

Basal expression of glutamine synthetase (GS) is very low in rat lung and muscle and remarkably enhanced by glucocorticoid hormones during trauma and catabolic states. Although this response is believed to be transcriptionally regulated, the genetic elements responsible for tissue-specific glucocorticoid induction of GS expression have not been identified. A rat lung epithelial cell line (L2) and a glucocorticoid receptor-deficient human prostate cancer cell line (PC3), together with GS reporter gene constructs, were utilized in gene transfer experiments to identify two regions within the rat genomic clone gGS3 that imparted dexamethasone (Dex) responsiveness to both the homologous GS promoter and the heterologous herpes simplex virus thymidine kinase promoter in glucocorticoid receptor-dependent fashions. One region lies nearly 6 kb upstream of the GS transcription initiation site, and the other lies within the first intron of the GS gene. Dex responsiveness was localized to a 325-bp fragment of the intron region containing a canonical glucocorticoid response element and to a 225-bp fragment of the far-upstream region containing three separate glucocorticoid response element half-sites. The GS promoter exhibited relatively high basal activity that was repressed by inclusion of the far-upstream or the intron glucocorticoid-responsive region. Dex treatment negated this repression. A model is suggested in which the glucocorticoid-receptor unit causes derepression of lung and muscle GS transcription during trauma and catabolic states.
Proline in vasoactive peptides: consequences for peptide hydrolysis in the lung
Authors: Merker Marilyn P. , Audi Said H. , Brantmeier Becky M. , Nithipatikom Kasem , Goldman Robert S. , Roerig David L. , Dawson Christopher A.
Published: December 2017

To examine the hypothesis that trans isomers of bradykinin and [Gly6]bradykinin are preferentially hydrolyzed by lung peptidases, we studied the fractional inactivation of these peptides in the perfused rat lung using a bioassay after a single-pass bolus injection and high-performance liquid chromatography after lung recirculation. In the bioassay studies, when the peptides passed through the lung, 25.6-fold more bradykinin or 7-fold more [Gly6]bradykinin was required to elicit a contraction equivalent to that produced when the peptides did not pass through the lung. In the recirculation studies, hydrolysis progress curves with rapid and slow phases were observed, with a higher fraction of bradykinin than [Gly6]bradykinin hydrolyzed in the rapid phase. Cyclophilin increased the hydrolysis rate during the slow phase for both peptides. Kinetic analysis indicated that the slowly hydrolyzed peptide fraction, presumably the cis fraction, was 0.13 for bradykinin and 0.43 for [Gly6]bradykinin with cis- transisomerization rate constants of 0.074 and 0.049 s−1, respectively, consistent with published nuclear magnetic resonance studies.
Chronic hypoxia augments endothelin-B receptor-mediated vasodilation in isolated perfused rat lungs
Authors: Muramatsu Masashi , Oka Masahiko , Morio Yoshiteru , Soma Sanae , Takahashi Hideki , Fukuchi Yoshinosuke
Published: December 2017

To investigate whether chronic hypoxia affects endothelin-B (ETB) receptor-mediated pulmonary vasodilation, we compared the vasodilator responses to IRL-1620, a selective ETB-receptor agonist, in isolated perfused lungs from normoxic and chronically hypoxic adult male rats. IRL-1620 caused a dose-dependent vasodilation that was greater in the hypertensive lungs than in the normotensive lungs. In normotensive lungs, a nitric oxide (NO) synthase inhibitor, N ω-nitro-l-arginine (l-NNA; 300 μM), and an ATP-sensitive potassium (KATP)-channel inhibitor, glibenclamide (Glib; 10 μM), each reduced the vasodilator response to IRL-1620 (1 nM), but the combination ofl-NNA and Glib inhibited it more effectively than either drug alone. In contrast,l-NNA alone, but not Glib alone, completely blocked IRL-1620-induced vasodilation in hypertensive lungs. The vasodilator response to a KATP-channel opener, NIP-121 (1 μM), but not the response to sodium nitroprusside (1 μM), was enhanced in hypertensive lungs. We also found increased expression of mRNA for the ETB receptor in lung tissue after hypoxic exposure. In addition, semiquantitative immunohistochemistry demonstrated higher expression levels of ETB receptors in the endothelium of distal segments of the pulmonary artery in hypoxic than in normoxic rats. These results suggest that ETB receptor-mediated pulmonary vasodilation is augmented after chronic hypoxic exposure and that release of NO may be the sole mechanism of this vasodilation in hypertensive lungs, whereas both release of NO and activation of KATP channels are involved in normotensive lungs. We speculate that the underlying mechanism responsible for this augmentation may partly be related to upregulation of ETB receptors in the endothelium of pulmonary resistance arteries in hypertensive lungs.
Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements
Authors: Mair Norbert , Haller Thomas , Dietl Paul
Published: December 2017

Measurement of lamellar body (LB) exocytosis at high spatial and temporal resolution was recently enabled by fluorescence of the dye FM 1-43 (FFM1-43). Here, the capabilities of this method were further examined and extended by simultaneous measurement of the cell membrane capacitance ( C m) and laser-scanning confocal microscopy. Step increases in C m were evoked by extracellular ATP (20 μM) or an elevated pipette Ca2+ concentration (≥3 μM). The delay between the first C m step and the increase in FFM1-43 was <1 s, indicating ready access of FM 1-43 to exocytosed LB contents. A specific C m of 0.88 μF/cm2 for the membrane of an exocytosed LB was calculated. Compound exocytosis was occasionally observed. Decreases in C m, indicative of transient fusion or endocytosis, did not occur within 20 min of stimulation. Exocytosis was stimulated by 160 μM guanosine 5′- O-(3-thiotriphosphate) in the pipette, but compound exocytosis was unaffected. The comparison of methods revealed that FM 1-43 is ideally suited to measure the onset of exocytosis and amount of secretion. Patch clamp is superior in resolving fusion events with the plasma membrane.
S-nitrosoglutathione enhances neutrophil DNA fragmentation and cell death
Authors: Fortenberry James D. , Owens Marilyn L. , Brown Lou Ann S.
Published: December 2017

Enhancing the clearance of neutrophils by enhancing apoptotic cell death and macrophage recognition may be beneficial in acute lung injury. Exogenous nitric oxide gas depresses neutrophil oxidative functions and accelerates cell death (A. H. Daher, J. D. Fortenberry, M. L. Owens, and L. A. Brown. Am. J. Respir. Cell Mol. Biol. 16: 407–412, 1997). We hypothesized that S-nitrosoglutathione (GSNO), a physiologically relevant nitric oxide donor, could also enhance neutrophil DNA fragmentation. Neutrophils were incubated for 2–24 h in the absence and presence of GSNO (dose range 0.1–5 mM) and evaluated for cell death by a fluorescent viability/cytotoxicity assay. Neutrophil DNA fragmentation was assessed by cell death detection ELISA and by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling assay. Neutrophil oxidative function was also determined. Incubation with GSNO increased cell death at 2, 4, and 24 h. GSNO incubation for 24 h significantly increased DNA fragmentation in a dose-dependent fashion at 0.5 (median 126% of control value; P = 0.002) and 5 mM (185% of control value; P = 0.002) by terminal deoxynucleotidyltransferase-mediated fluorescence-labeled dUTP nick end labeling and at 0.5 mM by ELISA (164% of control value; P = 0.03). The apoptosis-to-total cell death ratio increased with increasing GSNO concentration ( P < 0.05). Effects were mitigated by coincubation with superoxide dismutase. Five millimolar GSNO decreased overall superoxide generation and O2consumption but not when adjusted for dead neutrophils. GSNO significantly enhances cell death and neutrophil DNA fragmentation in a dose-dependent fashion.
Surfactant protein A enhances the binding and deacylation ofE. coliLPS by alveolar macrophages
Authors: Stamme Cordula , Wright Jo Rae
Published: December 2017

Surfactant protein (SP) A and SP-D are involved in multiple immunomodulatory functions of innate host defense partly via their interaction with alveolar macrophages (AMs). In addition, both SP-A and SP-D bind to bacterial lipopolysaccharide (LPS). To investigate the functional significance of this interaction, we first tested the ability of SP-A and SP-D to enhance the binding of tritium-labeled Escherichia coli LPS to AMs. In contrast to SP-D, SP-A enhanced the binding of LPS by AMs in a time-, temperature-, and concentration-dependent manner. Coincubation with surfactant-like lipids did not affect the SP-A-mediated enhancement of LPS binding. At SP-A-to-LPS molar ratios of 1:2–1:3, the LPS binding by AMs reached 270% of control values. Second, we investigated the role of SP-A in regulating the degradation of LPS by AMs. In the presence of SP-A, deacylation of LPS by AMs increased by ∼2.3-fold. Pretreatment of AMs with phosphatidylinositol-specific phospholipase C had no effect on the SP-A-enhanced LPS binding but did reduce the amount of serum-enhanced LPS binding by 50%, suggesting that a cell surface molecule distinct from CD14 mediates the effect of SP-A. Together the results for the first time provide direct evidence that SP-A enhances LPS binding and degradation by AMs.
Diesel exhaust particles are taken up by human airway epithelial cells in vitro and alter cytokine production
Authors: Boland Sonja , Baeza-Squiban Armelle , Fournier Thierry , Houcine Odile , Gendron Marie-Claude , Chévrier Michèle , Jouvenot Gilles , Coste André , Aubier Michel , Marano Francelyne
Published: December 2017

The involvement of diesel exhaust particles (DEPs) in respiratory diseases was evaluated by studying their effects on two in vitro models of human airway epithelial cells. The cytotoxicity of DEPs, their phagocytosis, and the resulting immune response were investigated in a human bronchial epithelial cell line (16HBE14o−) as well as in human nasal epithelial cells in primary culture. DEP exposure induced a time- and dose-dependent membrane damage. Transmission electron microscopy showed that DEPs underwent endocytosis by epithelial cells and translocated through the epithelial cell sheet. Flow cytometric measurements allowed establishment of the time and dose dependency of this phagocytosis and its nonspecificity with different particles (DEPs, carbon black, and latex particles). DEPs also induced a time-dependent increase in interleukin-8, granulocyte-macrophage colony-stimulating factor, and interleukin-1β release. This inflammatory response occurred later than phagocytosis, and its extent seems to depend on the content of adsorbed organic compounds because carbon black had no effect on cytokine release. Furthermore, exhaust gas posttreatments, which diminished the adsorbed organic compounds, reduced the DEP-induced increase in granulocyte-macrophage colony-stimulating factor release. These results suggest that DEPs could 1) be phagocytosed by airway epithelial cells and 2) induce a specific inflammatory response.
Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages
Authors: Wright Jo Rae , Zlogar Daniel F. , Taylor Julie C. , Zlogar Thomas M. , Restrepo Clara I.
Published: December 2017

Surfactant protein (SP) A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species. Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls. In addition, purified preparations of SPs often contain endotoxin. The goals of this study were 1) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP. Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-β-d-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels. Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin. There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages. SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production. These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination.
Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils
Authors: Costello Richard W. , Evans Christopher M. , Yost Bethany L. , Belmonte Kristen E. , Gleich Gerald J. , Jacoby David B. , Fryer Allison D.
Published: December 2017

M2muscarinic receptors limit acetylcholine release from the pulmonary parasympathetic nerves. M2receptors are dysfunctional in antigen-challenged guinea pigs, causing increased vagally mediated bronchoconstriction. Dysfunction of these M2receptors is due to eosinophil major basic protein, which is an antagonist for M2receptors. Histamine-induced bronchoconstriction is composed of a vagal reflex in addition to its direct effect on airway smooth muscle. Because hyperreactivity to histamine is seen in antigen-challenged animals, we hypothesized that hyperreactivity to histamine may be due to increased vagally mediated bronchoconstriction caused by dysfunction of M2receptors. In anesthetized, antigen-challenged guinea pigs, histamine-induced bronchoconstriction was greater than that in control guinea pigs. After vagotomy or atropine treatment, the response to histamine in antigen-challenged animals was the same as that in control animals. In antigen-challenged animals, blockade of eosinophil influx into the airways or neutralization of eosinophil major basic protein prevented the development of hyperreactivity to histamine. Thus hyperreactivity to histamine in antigen-challenged guinea pigs is vagally mediated and dependent on eosinophil major basic protein.
Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs
Authors: Calvet Jean-Henri , Planus Emmanuelle , Rouet Patricia , Pezet Sophie , Levame Micheline , Lafuma Chantal , Harf Alain , D’Ortho Marie-Pia
Published: December 2017

Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity ( r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 × 10−5 M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment.
Effects of eosinophil granule major basic protein on phosphatidylcholine secretion in rat type II pneumocytes
Authors: Okumura Manabu , Kai Hirofumi , Shinozawa Shinya , Isohama Yoichiro , Miyata Takeshi
Published: December 2017

Eosinophils are involved in inflammatory diseases such as asthma. We previously reported that activated eosinophils increased the phosphatidylcholine (PC) secretion in primary cultures of rat type II pneumocytes. Increased PC secretion was confirmed to be partly mediated by superoxide anions released from activated eosinophils. However, the influence of eosinophil granule proteins on PC secretion is unknown at present. In this study, we determined whether eosinophil major basic protein (MBP) influences PC secretion. MBP dose dependently increased the PC secretion in rat type II pneumocytes without producing any cell damage. The MBP-induced increase in PC secretion was significantly reduced by preadministration of either H-7, a protein kinase inhibitor, or 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, a chelator of intracellular Ca2+, but not by H-89, a protein kinase inhibitor. Our results suggest that the MBP-induced increase in PC secretion may provide mechanical stability and protect against lung atelectasis.
Cyclosporin A protects lung function from hyperoxic damage
Authors: Matthew E. , Pun R. , Simonich M. , Iwamoto H. , Dedman J.
Published: December 2017

Cyclosporin A (CsA), an inhibitor of protein phosphatase 2B (calcineurin), has been shown to play a role in exocytosis and neutrophil mobility. Hyperoxia (>95% oxygen for 72 h) causes lung injury and reduces lung compliance. This model is indicative of deficiencies in surfactant and elicits a vigorous immune response leading to further damage. We examined the effects of CsA on surfactant-secreting lung alveolar type II cells. CsA enhances ATP-stimulated increases in whole cell capacitance in the presence of 2 mM extracellular Ca2+. This measurement corresponds with increases in exocytosis. Because of its effect on the immune system and exocytosis from type II cells, CsA was examined for its protective effects against hyperoxia-induced lung damage in mice. We found that CsA (50 mg ⋅ kg−1⋅ day−1) attenuated hyperoxia-induced reductions in lung compliance when administered before or during 72 h of >95% oxygen ( P < 0.05). CsA (10 mg ⋅ kg−1⋅ day−1) also had a protective effect against hyperoxia-induced changes in neutrophil infiltration, capillary congestion, edema, and hyaline membrane formation. Wet lung weight-to-dry lung weight ratios did not show any significant changes after hyperoxia or hyperoxia plus CsA ( P < 0.05). CsA may be useful to treat patients undergoing prolonged high-oxygen therapy and possibly other lung injuries.
Development of intrinsic tone in isolated pulmonary arterioles
Authors: Liu Qiang , Sylvester J. T.
Published: December 2017

In isolated porcine pulmonary arterioles with endothelium, intraluminal diameter measured at a transmural pressure of 20 mmHg decreased spontaneously from 233 ± 11 to 171 ± 12 μm in 135 min. This intrinsic constriction was not prevented by indomethacin, tetraethylammonium, or superoxide dismutase. Indomethacin plus NG-nitro-l-arginine methyl ester caused initial constriction and BQ-123 or BQ-123 plus BQ-788 caused initial dilation, but these treatments did not prevent subsequent progressive constriction. In pulmonary arterioles with endothelium exposed to calcium-free conditions and pulmonary arterioles without endothelium, the intraluminal diameter measured at a transmural pressure of 20 mmHg was constant at 239 ± 16 and 174 ± 7 μm, respectively. Thus the spontaneous development of tone in isolated pulmonary arterioles required extracellular calcium and resulted from 1) time-independent smooth muscle contraction caused by mechanisms intrinsic to smooth muscle and 2) time-dependent contraction caused by decreasing activity of endothelium-derived relaxing factors other than nitric oxide, vasodilator prostaglandins, and hyperpolarizing factors acting on calcium-dependent potassium channels or increasing activity of endothelium-derived contracting factors other than endothelin-1, vasoconstrictor prostaglandins, and superoxide anions. Further investigation is indicated to identify these unknown mechanisms and determine their role in pulmonary vasoreactivity.
Mechanisms regulating cAMP-mediated growth of bovine neonatal pulmonary artery smooth muscle cells
Authors: Guldemeester Alexandra , Stenmark Kurt R. , Brough George H. , Stevens Troy
Published: December 2017

Neonatal pulmonary artery smooth muscle cells (PASMCs) exhibit enhanced growth capacity and increased growth responses to mitogenic stimuli compared with adult PASMCs. Because intracellular signals mediating enhanced growth responses in neonatal PASMCs are incompletely understood, we questioned whether 1) Gqagonists increase cAMP content and 2) increased cAMP is proproliferative. Endothelin-1 and angiotensin II increased both cAMP content and proliferation in neonatal but not in adult PASMCs. Inhibition of protein kinase C and protein kinase A activity nearly eliminated the endothelin-1- and angiotensin II-induced growth of neonatal PASMCs. Moreover, cAMP increased proliferation in neonatal but not in adult cells. Protein kinase C-stimulated adenylyl cyclase was expressed in both cell types, suggesting that insensitivity to stimulation of cAMP in adult cells was not due to decreased enzyme expression. Our data collectively indicate that protein kinase C stimulation of cAMP is a critical signal mediating proliferation of neonatal PASMCs that is absent in adult PASMCs and therefore may contribute to the unique proproliferative phenotype of these neonatal cells.
Respiratory epithelial cells demonstrate lactoferrin receptors that increase after metal exposure
Authors: Ghio Andrew J. , Carter Jacqueline D. , Dailey Lisa A. , Devlin Robert B. , Samet James M.
Published: December 2017

Human airway epithelial cells can increase expression of both lactoferrin and ferritin after exposure to catalytically active metal. These proteins transport and store metal, with coordination sites fully complexed, and therefore can diminish the oxidative stress. The intracellular transport of lactoferrin results in a transfer of complexed metal to ferritin, where it is stored in a less reactive form. This effort to control the injurious properties of metals would be facilitated by lactoferrin receptors (LfRs) on airway epithelial cells. We tested the hypotheses that 1) LfRs exist on respiratory epithelial cells and 2) exposure to both an air pollution particle, which has abundant concentrations of metals, and individual metal salts increase the expression of LfRs. Before exposure to either the particle or metals, incubation of BEAS-2B cells with varying concentrations of125I-labeled lactoferrin demonstrated lactoferrin binding that was saturable. Measurement of125I-lactoferrin binding after the inclusion of 100 μg/ml of oil fly ash in the incubation medium demonstrated increased binding within 5 min of exposure, which reached a maximal value at 45 min. Inclusion of 1.0 mM deferoxamine in the incubation of BEAS-2B cells with 100 μg/ml of oil fly ash decreased lactoferrin binding. Comparable to the particle, exposure of BEAS-2B cells to either 1.0 mM vanadyl sulfate or 1.0 mM iron (III) sulfate, but not to nickel sulfate, for 45 min elevated LfR activity. We conclude that LfRs on respiratory epithelial cells increased after exposure to metal. LfRs could participate in decreasing the oxidative stress presented to the lower respiratory tract by complexing catalytically active metals.
Transcriptional activation and protein binding by two regions of the rat surfactant protein A promoter
Authors: Rosenberg Elizabeth , Li Feng , Smith Candyce I. , Reisher Samuel R. , Feinstein Sheldon I.
Published: December 2017

Surfactant protein A (SP-A) is expressed in lung alveolar type II cells and bronchiolar Clara cells. We have identified two active regions in the promoter of the rat SP-A gene by deletion analysis of a plasmid containing 163 bp before the start of transcription (−163 bp), linked to a reporter gene. Constructs were transfected into lung cell lines derived from each of the cell types that produces SP-A. We found a novel region of promoter activity at ∼90 bp before the transcriptional start (SP-A−90). Mutation of four nucleotides in SP-A−90that are highly conserved among species (−92 to −89 bp) decreased expression of the SP-A construct by ∼50% in both cell lines. Electrophoretic mobility shift analysis showed specific binding to SP-A−90by nuclear proteins from the cell lines, as well as from rat lung and liver. The electrophoretic mobility of the bands shifted by lung nuclear proteins changed late in fetal development. Although in the Clara cell line no reduction of promoter activity was seen on deletion of the region upstream of SP-A−90, in the type II cell line, deletion of residues −163 to −133 did reduce activity by ∼50%. This region contains a recognition element for thyroid transcription factor-1 (TTF-1). Endogenous TTF-1 binding activity was substantially higher in the type II cell line than in the Clara cell line, but cotransfection of a TTF-1 expression plasmid enhanced expression of the SP-A construct better in the Clara cell line than in the type II cell line. These results suggest that the recognition element for TTF-1 has varying activity in the lung cell lines of different origin due to the availability of TTF-1.
Stretch induces cytokine release by alveolar epithelial cells in vitro
Authors: Vlahakis Nicholas E. , Schroeder Mark A. , Limper Andrew H. , Hubmayr Rolf D.
Published: December 2017

Mechanical ventilation can injure the lung, causing edema and alveolar inflammation. Interleukin-8 (IL-8) plays an important role in this inflammatory response. We postulated that cyclic cell stretch upregulates the production and release of IL-8 by human alveolar epithelium in the absence of structural cell damage or paracrine stimulation. To test this hypothesis, alveolar epithelial cells (A549 cells) were cultured on a deformable silicoelastic membrane. When stretched by 30% for up to 48 h, the cells released 49 ± 34% more IL-8 ( P < 0.001) than static controls. Smaller deformations (20% stretch) produced no consistent increase in IL-8. Stretch of 4 h duration increased IL-8 gene transcription fourfold above baseline. Stretch had no effect on cell proliferation, cell viability as assessed by51Cr release assay, or the release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α. We conclude that deformation per se can trigger inflammatory signaling and that alveolar epithelial cells may be active participants in the alveolitis associated with ventilator-induced lung injury.
Airway epithelial tight junctions and binding and cytotoxicity ofPseudomonas aeruginosa
Authors: Lee Alfred , Chow Dar , Haus Brian , Tseng Wanru , Evans David , Fleiszig Suzanne , Chandy Grischa , Machen Terry
Published: December 2017

The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosato apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells. Binding of noncytotoxic P. aeruginosa strain O1 was assessed with P. aeruginosa that expressed green fluorescent protein. Binding of cytotoxic P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters with small (0.45-μm) or large (2.0-μm) pores. High transepithelial resistance ( Rt) Calu-3 and cultured bovine tracheal monolayers ( Rt> 1,000 Ω ⋅ cm2) bound P. aeruginosa very infrequently (<1 P. aeruginosa/100 cells) at the apical membrane, but P. aeruginosabound frequently to cells near “free edges” at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes. Wounded high Rtepithelia showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P. aeruginosa, and dead and living cells near the wound bound P. aeruginosa similarly. Compared with high Rtepithelia, low RtCFT1 ( Rt= 100–200 Ω ⋅ cm2) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P. aeruginosa binding throughout the monolayer. Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low Rtthan in high Rtairway epithelia. Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide. P. aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low RtCFT1 cells.
IL-4 induces ICAM-1 expression in human bronchial epithelial cells and potentiates TNF-α
Authors: Striz Ilja , Mio Tadashi , Adachi Yuichi , Heires Peggy , Robbins Richard A. , Spurzem John R. , Illig Mary J. , Romberger Debra J. , Rennard Stephen I.
Published: December 2017

Interleukin (IL)-4 is thought to contribute to the Th2 type of immune response and hence the development of allergic reactions such as asthma. In asthmatic patients, the airway epithelium expresses increased amounts of the cell surface adhesion molecule intercellular adhesion molecule (ICAM)-1 (CD54). One cytokine capable of inducing ICAM-1 in airway epithelial cells, tumor necrosis factor-α (TNF-α), is present in asthma. This study evaluated if IL-4 either alone or together with TNF-α costimulation might modulate CD54 expression by human bronchial epithelial cells (HBECs). CD54 positivity increased in response to IL-4 (16 ± 2% positive vs. 3 ± 1%, P < 0.01); greater induction of CD54 resulted from TNF-α (45 ± 2%, P < 0.001). Costimulation with TNF-α plus IL-4 further augmented expression (56 ± 1%, P < 0.05). Immunoperoxidase results were confirmed by flow cytometry. RT-PCR revealed no increase in ICAM-1 mRNA expression under control conditions or after stimulation with IL-4 alone. TNF-α increased IL-4 mRNA, and IL-4 potentiated this. Functionally, IL-4 augmented the adhesion of THP-1 monocyte/macrophage cells to monolayers of HBECs both alone and in the presence of TNF-α. We conclude that 1) IL-4 augments epithelial cell ICAM-1 expression, 2) IL-4 potentiates the adhesion of THP-1 monocyte/macrophage cells to epithelial cells, and 3) modulation of epithelial cell ICAM-1 expression by IL-4 may play a role in the immunopathology of bronchial asthma.
Effects of depletion of neutrophils or macrophages on development of cigarette smoke-induced emphysema
Authors: Ofulue A. Felix , Ko Mary
Published: December 2017

The aim of this study was to ascertain the putative roles of neutrophils or macrophages in the pathogenesis of cigarette smoking-induced emphysema on the basis of effects of anti-neutrophil (anti-PMN) antibody or anti-monocyte/macrophage (anti-MoMac) antibody on the development of emphysema in cigarette smoke-exposed rats. Rats were treated with rabbit anti-PMN or anti-MoMac antibody and exposed 7 days/wk for 2 mo to cigarette smoke inhalation; rats treated with nonimmunized rabbit IgG (control antibody) and exposed to cigarette smoke or normal room air served as controls. Antibody treatments began 24 h before the start of smoke or air exposure and was continued with 1 treatment/wk. Total and differential cell counts in bronchoalveolar lavage fluid and collagenase-dissociated lung and determinations of the elastinolytic activity of lung neutrophils or macrophages in [3H]elastin-coated wells indicated specific suppression of neutrophil accumulation and neutrophil-related elastinolytic burden in the lungs of the anti-PMN antibody-treated smoke-exposed rats, in contrast to specific suppression of macrophage accumulation and macrophage-related elastinolytic burden in the lungs of the anti-MoMac antibody-treated smoke-exposed rats. Cigarette smoke exposure-induced lung elastin breakdown (quantitated by immunologic assay of levels of elastin-derived peptides and desmosine in lavage fluid) and emphysema in the lungs (based on morphometric analysis of alveolar mean linear intercepts and alveolar tissue density in fixed lungs) were not prevented in the lungs of anti-PMN antibody-treated smoke-exposed rats but was clearly prevented in lungs of the anti-MoMac antibody-treated smoke-exposed rats. These findings implicate macrophages rather than neutrophils as the critical pathogenic factor in cigarette smoke-induced emphysema.
E-4010, a selective phosphodiesterase 5 inhibitor, attenuates hypoxic pulmonary hypertension in rats
Authors: Hanasato Norihisa , Oka Masahiko , Muramatsu Masashi , Nishino Mayu , Adachi Hideyuki , Fukuchi Yoshinosuke
Published: December 2017

The purpose of this study was to determine whether E-4010, a newly synthesized potent and selective orally active phosphodiesterase (PDE) 5 inhibitor, would prevent the development of chronic hypoxia-induced pulmonary hypertension in rats. In conscious, pulmonary hypertensive rats, a single oral administration of E-4010 (1.0 mg/kg) caused an acute, long-lasting reduction in mean pulmonary arterial pressure (PAP), with no significant effects on systemic arterial pressure, cardiac output, and heart rate. In rats that received food containing 0.01 or 0.1% E-4010 during the 3-wk exposure to hypoxia, mean PAP was significantly decreased (mean PAP 24.0 ± 0.9, 16.2 ± 0.8, and 12.8 ± 0.5 mmHg in rats treated with 0, 0.01, and 0.1% E-4010-containing food, respectively), whereas mean systemic arterial pressure was unchanged and cardiac output was slightly increased compared with chronically hypoxic control rats. Right ventricular hypertrophy, medial wall thickness in pulmonary arteries corresponding to the respiratory and terminal bronchioles, and the degree of muscularization of more distal arteries were less severe in E-4010-treated rats. Long-term treatment with E-4010 caused an increase in cGMP levels in lung tissue and plasma but not in aortic tissue and no significant change in cAMP levels in either lung, aorta, or plasma. These results suggest that long-term oral treatment with E-4010 reduced the increase in PAP, right ventricular hypertrophy, and pulmonary arterial remodeling induced by exposure to chronic hypoxia, probably through increasing cGMP levels in the pulmonary vascular smooth muscle.
Interactions of keratinocyte growth factor with a nitrating species after marrow transplantation in mice
Authors: Haddad Imad Y. , Panoskaltsis-Mortari Angela , Ingbar David H. , Resnik Ernesto R. , Yang Shuxia , Farrell Catherine L. , Lacey David L. , Cornfield David N. , Blazar Bruce R.
Published: December 2017

We reported that allogeneic T cells given to irradiated mice at the time of marrow transplantation stimulated tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and nitric oxide (⋅ NO) production in the lung, and the addition of cyclophosphamide (known to stimulate superoxide production) favored the generation of a nitrating species. Although keratinocyte growth factor (KGF) prevents experimental lung injury by promoting epithelial repair, its effects on the production of inflammatory mediators has not been studied. KGF given before transplantation inhibited the T cell-induced increase in bronchoalveolar lavage fluid protein, TNF-α, IFN-γ, and nitrite levels measured on day 7 after transplantation without modifying cellular infiltration or proinflammatory cytokines and inducible ⋅ NO synthase mRNA. KGF also suppressed ⋅ NO production by alveolar macrophages obtained from mice injected with T cells. In contrast, the same schedule of KGF failed to prevent permeability edema or suppress TNF-α, IFN-γ, and ⋅ NO production in mice injected with both T cells and cyclophosphamide. Because only epithelial cells respond to KGF, these data are consistent with the production of an epithelial cell-derived mediator capable of downregulating macrophage function. However, the presence of a nitrating agent impairs KGF-derived responses.
Syntaxin 1A is transiently expressed in fetal lung mesenchymal cells: potential developmental roles
Authors: Brimhall Bradley B. , Sikorski Kristan A. , Torday John , Shahsafaei Aliakbar , Haley Kathleen J. , Sunday Mary E.
Published: December 2017

Lung development is a complex process in which epithelial-mesenchymal interactions play a key role. A conserved secretory apparatus, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is essential for exocytosis in many cell types. Syntaxins, located on the terminal plasma membrane (T-SNAREs), are a critical component of the secretosomal complex involved in vesicular docking, fusion, and exocytosis. We analyzed syntaxin 1A mRNA and protein in fetal rat lung ontogeny, demonstrating peak expression on about day 19 of embryonic development, immediately preceding type II pneumocyte differentiation. Syntaxin 1A is predominantly expressed by lipofibroblasts, which are required for bombesin-like peptide-induced surfactant phospholipid synthesis (choline uptake) by isolated type II cells. In organ cultures, anti-syntaxin 1A antibody HPC-1 blocks choline uptake both at baseline and when induced by bombesin-like peptide or dexamethasone. HPC-1 also promotes thymidine uptake in parallel in a dose-dependent fashion. These observations indicate a potential role for syntaxin 1A during fetal lung development, possibly through involvement in secretion of mesenchymal cell-derived factors that induce terminal type II cell differentiation.
ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells
Authors: Celi Alessandro , Cianchetti Silvana , Petruzzelli Stefano , Carnevali Stefano , Baliva Filomena , Giuntini Carlo
Published: December 2017

Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 ± 3 to 49 ± 7% (SE). A significant increase from 17 ± 4 to 39 ± 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin β-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.
MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation
Authors: Orsini Michael J. , Krymskaya Vera P. , Eszterhas Andrew J. , Benovic Jeffrey L. , Panettieri Reynold A. , Penn Raymond B.
Published: December 2017

Asthma is frequently associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness to contractile agents. Although numerous hormones and cytokines have been shown to induce human ASM (HASM) proliferation, the cellular and molecular mechanisms underlying HASM hyperplasia are largely unknown. Here we characterize the roles of the mitogen-activated protein kinase (MAPK) superfamily [p42/p44 MAPK, c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38] in mediating hormone- and cytokine-induced HASM proliferation. Significant enhancement of [3H]thymidine incorporation in HASM cultures was observed only by treatment with agents (epidermal growth factor, platelet-derived growth factor, thrombin, and phorbol 12-myristate 13-acetate) that promoted a strong and sustained activation of p42/p44 MAPK. Significant activation of the JNK/SAPK and p38 pathways was only observed on stimulation with interleukin (IL)-1β and tumor necrosis factor-α, agents that did not appreciably stimulate HASM proliferation. Two different inhibitors of MAPK/extracellular signal-regulated kinase kinase (MEK), PD-98059 and U-0126, inhibited mitogen-induced [3H]thymidine incorporation in a manner consistent with their ability to inhibit p42/p44 activation. Elk-1 and activator protein-1 reporter activation by mitogens was similarly inhibited by inhibition of MEK, suggesting a linkage between p42/p44 activation, transcription factor activation, and HASM proliferation. These findings establish a fundamental role for p42/p44 activation in regulating HASM proliferation and provide insight into species-specific differences observed among studies in ASM mitogenesis.
Monocyte inflammation augments acrolein-induced Muc5ac expression in mouse lung
Authors: Borchers Michael T. , Wesselkamper Scott , Wert Susan E. , Shapiro Steven D. , Leikauf George D.
Published: December 2017

Acrolein, an unsaturated aldehyde found in smog and tobacco smoke, can induce airway hyperreactivity, inflammation, and mucus hypersecretion. To determine whether changes in steady-state mucin gene expression ( Muc2 and Muc5ac) are associated with inflammatory cell accumulation and neutrophil elastase activity, FVB/N mice were exposed to acrolein (3.0 parts/million; 6 h/day, 5 days/wk for 3 wk). The levels of Muc2 and Muc5ac mRNA were determined by RT-PCR, and the presence of Muc5ac protein was detected by immunohistochemistry. Total and differential cell counts were determined from bronchoalveolar lavage (BAL) fluid, and neutrophil elastase activity was measured in the BAL fluid supernatant. Lung Muc5ac mRNA was increased on days 12and 19, and Muc5ac protein was detected in mucous granules and on the surface of the epithelium on day 19. Lung Muc2 mRNA was not detected at measurable levels in either control or exposed mice. Acrolein exposure caused a significant and persistent increase in macrophages and a rapid but transient increase in neutrophils in BAL fluid. Recoverable neutrophil elastase activity was not significantly altered at any time after acrolein exposure. To further examine the role of macrophage accumulation in mucin gene expression, additional strains of mice (including a strain genetically deficient in macrophage metalloelastase) were exposed to acrolein for 3 wk, and Muc5ac mRNA levels and macrophage accumulation were measured. The magnitude of macrophage accumulation coincided with increased Muc5ac mRNA levels, indicating that excessive macrophage accumulation augments acrolein-induced Muc5ac synthesis and secretion after repeated exposure. These findings support a role for chronic monocytic inflammation in the pathogenesis of mucus hypersecretion observed in chronic bronchitis.