bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells
Authors: Petrache Irina , Choi Mary E. , Otterbein Leo E. , Chin Beek Yoke , Mantell Lin L. , Horowitz Stuart , Choi Augustine M. K.
Published: December 2017

We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH2-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.
Inducible nitric oxide synthase in the lung and exhaled nitric oxide after hyperoxia
Authors: Cucchiaro Giovanni , Tatum Arthur H. , Brown Michael C. , Camporesi Enrico M. , Daucher John W. , Hakim Tawfic S.
Published: December 2017

The effect of hyperoxia on nitric oxide (NO) production in intact animals is unknown. We described the effects of hyperoxia on inducible nitric oxide synthase (iNOS) expression and NO production in the lungs of rats exposed to high concentrations of oxygen. Animals were placed in sealed Plexiglas chambers and were exposed to either 85% oxygen (hyperoxic group) or 21% oxygen (negative control group). Animals were anesthetized after 24 and 72 h of exposure and were ventilated via a tracheotomy. We measured NO production in exhaled air (ENO) by chemiluminescence. The lungs were then harvested and processed for detection of iNOS by immunohistochemistry and Western blotting analysis. The same experiments were repeated in animals exposed to hyperoxia for 72 h after they were infused with l-arginine. We used rats that were injected intraperitoneally with Escherichia coli lipopolysaccharide to induce septic shock as a positive control group. Hyperoxia and septic shock induced expression of iNOS in the lung. However, ENOwas elevated only in septic shock rats but was normal in the hyperoxic group. Exogenous infusion of l-arginine after hyperoxia did not increase ENO. To exclude the possibility that in the hyperoxic group NO was scavenged by oxygen radicals to form peroxynitrite, lungs were studied by immunohistochemistry for the detection of nitrotyrosine. Nitrotyrosine was found in septic shock animals but not in the hyperoxic group, further suggesting that NO is not synthesized in rats exposed to hyperoxia. We conclude that hyperoxia induces iNOS expression in the lung without an increase in NO concentration in the exhaled air.
Actin reorganization in airway smooth muscle cells involves Gqand Gi-2activation of Rho
Authors: Hirshman Carol A. , Emala Charles W.
Published: December 2017

Extracellular stimuli induce cytoskeleton reorganization (stress-fiber formation) in cells and Ca2+sensitization in intact smooth muscle preparations by activating signaling pathways that involve Rho proteins, a subfamily of the Ras superfamily of monomeric G proteins. In airway smooth muscle, the agonists responsible for cytoskeletal reorganization via actin polymerization are poorly understood. Carbachol-, lysophosphatidic acid (LPA)-, and endothelin-1-induced increases in filamentous actin staining are indicative of actin reorganization (filamentous-to-globular actin ratios of 2.4 ± 0.3 in control cells, 6.7 ± 0.8 with carbachol, 7.2 ± 0.8 with LPA, and 7.4 ± 0.9 with endothelin-1; P < 0.001; n = 14 experiments). Although the effect of all agonists was blocked by C3 exoenzyme (inactivator of Rho), only carbachol was blocked by pertussis toxin. Although carbachol-induced actin reorganization was blocked in cells pretreated with antisense oligonucleotides directed against Gαi-2alone, LPA- and endothelin-1-induced actin reorganization were only blocked when both Gαi-2and Gqα were depleted. These data indicate that in human airway smooth muscle cells, carbachol induces actin reorganization via a Gαi-2pathway, whereas LPA or endothelin-1 induce actin reorganization via either a Gαi-2or a Gqα pathway.
Mechanical force-induced signal transduction in lung cells
Authors: Liu Mingyao , Tanswell A. Keith , Post Martin
Published: December 2017

The lung is a unique organ in that it is exposed to physical forces derived from breathing, blood flow, and surface tension throughout life. Over the past decade, significant progress has been made at the cellular and molecular levels regarding the mechanisms by which physical forces affect lung morphogenesis, function, and metabolism. With the use of newly developed devices, mechanical forces have been applied to a variety of lung cells including fetal lung cells, adult alveolar epithelial cells, fibroblasts, airway epithelial and smooth muscle cells, pulmonary endothelial and smooth muscle cells, and mesothelial cells. These studies have led to new insights into how cells sense mechanical stimulation, transmit signals intra- and intercellularly, and regulate gene expression at the transcriptional and posttranscriptional levels. These advances have significantly increased our understanding of the process of mechanotransduction in lung cells. Further investigation in this exciting research field will facilitate our understanding of pulmonary physiology and pathophysiology at the cellular and molecular levels.
CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands
Authors: Ballard Stephen T. , Trout Laura , Bebök Zsuzsa , Sorscher E. J. , Crews Angela
Published: December 2017

Previous studies demonstrated that ACh-induced liquid secretion by porcine bronchi is driven by active Cl−and H[Formula: see text] secretion. The present study was undertaken to determine whether this process was localized to submucosal glands and mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). When excised, cannulated, and treated with ACh, porcine bronchi secreted 15.6 ± 0.6 μl ⋅ cm−2⋅ h−1. Removal of the surface epithelium did not significantly affect the rate of secretion, indicating that the source of the liquid was the submucosal glands. Pretreatment with diphenylamine-2-carboxylate, a relatively nonselective Cl−-channel blocker, significantly reduced liquid secretion by 86%, whereas pretreatment with DIDS, which inhibits a variety of Cl−channels but not CFTR, had no effect. When bronchi were pretreated with glibenclamide or 5-nitro-2-(3-phenylpropylamino)benzoic acid (both inhibitors of CFTR), the rate of ACh-induced liquid secretion was significantly reduced by 39 and 91%, respectively, compared with controls. Agents that blocked liquid secretion also caused disproportionate reductions in H[Formula: see text] secretion. Polyclonal antibodies to the CFTR bound preferentially to submucosal gland ducts and the surface epithelium, suggesting that this channel was localized to these sites. These data suggest that ACh-induced gland liquid secretion by porcine bronchi is driven by active secretion of both Cl−and H[Formula: see text] and is mediated by the CFTR.
α-Adrenergic blockade restores normal fluid transport capacity of alveolar epithelium after hemorrhagic shock
Authors: Laffon M. , Lu L. N. , Modelska K. , Matthay M. A. , Pittet J. F.
Published: December 2017

Activation of β-adrenergic receptors in the lung is an important mechanism that can prevent alveolar flooding after brief but severe hemorrhagic shock. However, a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the normal upregulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Because hemorrhage increases proinflammatory cytokine expression in the lung partly through the activation of α-adrenergic receptors, the objective of this study was to determine whether α-adrenergic blockade would restore the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock. Hemorrhagic shock was associated with a significant increase of interleukin-1β (IL-1β) concentration in the lung and a failure of the alveolar epithelium to respond to β-adrenergic agonists, with the upregulation of vectorial fluid transport despite intra-alveolar administration of exogenous catecholamines. In contrast, catecholamine-mediated upregulation of alveolar liquid clearance was restored by pretreatment with phentolamine, an α-adrenergic-receptor antagonist. Phentolamine pretreatment also significantly attenuated the shock-mediated increase of IL-1β concentration in the lung. Additional experiments demonstrated that the inhibition of IL-1β binding to its receptor by the administration of IL-1-receptor antagonist restored the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock. In summary, the results of these studies indicate that the activation of α-adrenergic receptors after hemorrhagic shock prevents the β-adrenergic-dependent upregulation of alveolar fluid clearance by modulating the severity of the pulmonary inflammatory response.
Soluble guanylate cyclase gene expression and localization in rat lung after exposure to hypoxia
Authors: Li Dechun , Zhou Nan , Johns Roger A.
Published: December 2017

The nitric oxide (NO)-cGMP signal transduction pathway plays an important role in the regulation of pulmonary vascular tone and resistance in pulmonary hypertension. A number of studies have demonstrated that endothelial (e) and inducible nitric oxide synthases (NOS) are upregulated in hypoxia-exposed rat lung. These changes in NOS expression have been found to correlate with the process of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension, and remodeling is increased in the absence of eNOS. In this study, we examined the expression and localization of soluble guanylate cyclase (sGC), the primary receptor for NO, in hypoxia- and normoxia-treated rat lungs. Male Sprague-Dawley rats were exposed to hypoxia (10% O2, normobaric) or normoxia for 1, 3, 5, and 21 days. The lungs were used for Western analysis of sGC protein, sGC enzyme activity, immunohistochemistry using antiserum against sGC α1- and β1-subunits, and nonradioactive in situ hybridization (NRISH) using a digoxigenin-labeled sGC α1-subunit cRNA probe. Western blot analysis revealed a more than twofold increase of sGC protein α1-subunit in rat lungs exposed to 3, 5, and 21 days of hypoxia, correlating well with sGC enzyme activity. Immunohistochemistry and NRISH demonstrated increased expression of sGC in the smooth muscle cells of the pulmonary arteries and arterioles in the hypoxic rat lungs when compared with normoxic controls. Based on our results, the upregulation of sGC may play an important role in the regulation of smooth muscle tone and pressure in the pulmonary circulation during chronic hypoxia.
Developmental shift in the relative percentages of lung fibroblast subsets: role of apoptosis postseptation
Authors: Awonusonu Feyisola , Srinivasan Suseela , Strange Jennifer , Al-Jumaily Walid , Bruce Margaret C.
Published: December 2017

We have used the lipophilic, fluorescent dye Nile red and flow cytometry to identify and isolate two rat lung fibroblast subsets, lipid-containing interstitial cells (LICs) and non-LICs (NLICs) and to quantitate developmental changes in the relative percentages of these subsets. A significant decrease was observed in the percentage of LICs (from 79.0 ± 3.8% on postnatal day 4 to 28.6 ± 4.2% on day 30; P < 0.0001). To determine whether one or both subsets undergo apoptosis postseptation, fibroblasts from 16- to 18-day rats were treated with BODIPY-conjugated dUTP to label DNA strand breaks, which were then quantitated by flow cytometry. Apoptotic cells were judged to be predominantly LICs based on flow cytometric estimates of cell size and granularity and on light-microscopic colocalization of intracellular lipid and Hoechst-positive apoptotic bodies. Cell proliferation was compared in LICs and NLICs with both an in vitro [3H]thymidine incorporation assay and cell cycle analysis of propidium iodide-stained cells. Results of both assays indicated that on days 4– 5, LICs proliferated more rapidly than NLICs. Tropoelastin and fibronectin mRNA expression, evaluated by RT-PCR, indicated that although tropoelastin mRNA levels did not differ, fibronectin mRNA levels were approximately ninefold greater in LICs. These results demonstrate the feasibility of a flow cytometric assay for the analysis of size, granularity, and intracellular lipid content of neonatal rat lung fibroblast subsets. Subsets differed substantially with respect to proliferative capacity, fibronectin mRNA expression, and incidence of apoptosis postseptation. Together with the observed changes in relative percentages of fibroblast subsets with age, these data suggest that the ratio of LICs to NLICs could be a critical determinant of fibroblast function during lung development.
Threshold calcium levels for lamellar body exocytosis in type II pneumocytes
Authors: Haller Thomas , Auktor Klaus , Frick Manfred , Mair Norbert , Dietl Paul
Published: December 2017

Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca2+concentration ([Ca2+]i). In fura 2-loaded cells, [Ca2+]iwas selectively elevated by flash photolysis of a cell-permeant caged Ca2+compound ( o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca2+influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579–1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca2+]ielevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca2+]iand thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca2+. This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca2+signal below ∼300 nmol/l. Our results suggest a highly Ca2+-sensitive step in LB exocytosis.
Glucocorticoids ablate IL-1β-induced β-adrenergic hyporesponsiveness in human airway smooth muscle cells
Authors: Moore Paul E. , Laporte Johanne D. , Gonzalez Sonia , Moller Winfried , Heyder Joachim , Panettieri Reynold A. , Shore Stephanie A.
Published: December 2017

We have previously reported that interleukin (IL)-1β decreases responsiveness of cultured human airway smooth muscle (HASM) cells to β-agonists. The purpose of this study was to determine whether glucocorticoids inhibit this IL-1β effect. Dexamethasone (Dex; 10−6M) had no effect on concentration-related decreases in cell stiffness in response to isoproterenol (Iso) in control cells as measured by magnetic twisting cytometry but prevented the decreased responsiveness to Iso observed in IL-1β (20 ng/ml)-treated cells. In addition, Dex had no effect on Iso-stimulated cAMP formation in control cells but prevented the IL-1β-induced reduction in Iso-stimulated cAMP formation. Similar effects on cell stiffness and cAMP responses were seen after pretreatment with the glucocorticoid fluticasone proprionate (FP). Dex and FP also prevented IL-1β-induced hyporesponsiveness to PGE2stimulation. In contrast, neither IL-1β nor glucocorticoids had any effect on cell stiffness responses to dibutyryl cAMP. We have previously reported that the IL-1β effect on β-adrenergic responsiveness is mediated through cyclooxygenase-2 expression and prostanoid formation. Consistent with these observations, IL-1β-induced cyclooxygenase-2 expression was virtually abolished by FP at concentrations of 10−10M and greater, with a resultant decrease in PGE2formation. However, Dex did not inhibit IL-1β-induced nuclear translocation of nuclear factor-κB or activator protein-1 in HASM cells. In summary, our results indicate that, in HASM cells, glucocorticoids alone do not alter responses to β-agonists but do inhibit IL-1β-induced β-adrenergic hyporesponsiveness. Glucocorticoids mediate this effect by inhibiting prostanoid formation but without altering nuclear factor-κB or activator protein-1 translocation.
CCSP deficiency does not alter surfactant homeostasis during adenoviral infection
Authors: Ikegami Machiko , Harrod Kevin S. , Whitsett Jeffrey A. , Jobe Alan H.
Published: December 2017

Clara cell secretory protein (CCSP) deficiency in mice is associated with increased susceptibility to pulmonary inflammation after hyperoxia or viral infection. Because adenoviral exposure perturbs pulmonary surfactant homeostasis in vivo, we hypothesized that CCSP deficiency would influence surfactant metabolism after pulmonary infection. Alveolar and total lung saturated phosphatidylcholine pool sizes were similar in CCSP-deficient [CCSP(−/−)] and wild-type [CCSP(+/+)] mice before and 7 days after intratracheal administration of adenovirus. Radiolabeled choline and palmitate incorporation into saturated phosphatidylcholine was similar, and there was no alteration by previous infection 7 days before the incorporation measurements. Furthermore, CCSP deficiency did not influence clearance of [14C]dipalmitoylphosphatidylcholine and125I-labeled recombinant surfactant protein C. Increased persistence of alveolar capillary leak was observed in CCSP(−/−) mice after adenoviral infection. Surfactant lipid homeostasis was not influenced by CCSP before or after administration of adenovirus to the lung. Persistence of alveolar capillary leak in CCSP(−/−) mice after adenovirus provides further evidence for the role of CCSP in the regulation of pulmonary inflammation.
Histamine alters cadherin-mediated sites of endothelial adhesion
Authors: Winter Michael C. , Kamath Anant M. , Ries Dana R. , Shasby Sandra S. , Chen Yih-Tai , Shasby D. Michael
Published: December 2017

We tested the hypothesis that histamine alters the focal apposition of endothelial cells by acting on sites of cadherin-mediated cell-cell adhesion. Focal apposition was measured as the impedance of a cell-covered electrode, which was partitioned into a cell-matrix resistance, a cell-cell resistance, and membrane capacitance. Histamine causes an immediate, short-lived decrease in the impedance of an electrode covered with human umbilical vein endothelial (HUVE) cells. ECV304 cells are a line of spontaneously transformed HUVE cells that do not express the endothelial cadherin, cadherin-5. Histamine increased ECV304 cell calcium to 600 nM. Histamine did not increase myosin light chain phosphorylation of control or transfected ECV304 cells. ECV304 cells transfected with either E-cadherin or cadherin-5 on a dexamethasone-responsive plasmid (pLKneo) increased their cell-cell resistance when stimulated with dexamethasone, whereas ECV304 cells transfected with pLKneo-lacZ did not. Histamine did not affect the impedance of ECV304 cells transfected with pLKneo-lacZ. In contrast, histamine decreased the cell-cell resistance of ECV304 cells transfected with either pLKneo-E-cadherin or pLKneo-cadherin-5. From these data, we conclude that histamine acts on sites of cadherin-mediated cell-cell apposition.
Superoxide anion impairs Ca2+mobilization in cultured human nasal epithelial cells
Authors: Koyama Tetsuya , Oike Masahiro , Komiyama Sohtaro , Ito Yushi
Published: December 2017

We examined the effects of superoxide anion ([Formula: see text]) on the intracellular Ca2+concentration in cultured human nasal epithelial cells. The cells were exposed to[Formula: see text] by pretreatment with xanthine (X) and xanthine oxidase (XO); control cells were treated with X alone. When Ca2+-containing Krebs solution was reperfused in the thapsigargin-treated, store-depleted cells, reapplication-induced intracellular Ca2+concentration elevation was significantly smaller in X/XO-treated cells than in the control cells, suggesting that [Formula: see text] impairs Ca2+release-activated Ca2+entry (CRAC). Bath application of ATP induced a steep Ca2+transient in both control and X/XO-treated cells. However, the concentration-response curve of the ATP-induced Ca2+transient was shifted to a higher concentration in X/XO-treated cells. The impairments of CRAC and ATP-induced Ca2+transient induced by X/XO were reversed by superoxide dismutase. Furthermore, all these X/XO-induced effects were also observed in cells pretreated with pyrogallol, also an [Formula: see text] donor. These results indicate that [Formula: see text] impairs at least two mechanisms involved in Ca2+mobilization in human nasal epithelial cells, i.e., CRAC and ATP-induced Ca2+release.
Surfactant protein C in fetal and ventilated preterm rabbit lungs
Authors: Ross Gary F. , Ikegami Machiko , Steinhilber Wolfram , Jobe Alan H.
Published: December 2017

The developing lung contains surfactant protein (SP) C mRNA levels comparable to term values before mature type II cells and alveolar surfactant lipids are detectable. Estimates of the amount of mature SP-C in the alveolar lavages of preterm lungs are not available. We used an antibody to a recombinant human SP-C to measure the amount of SP-C in alveolar lavages of preterm fetal rabbits, ventilated preterm rabbits, and term rabbits. The amounts of SP-C were compared with the amounts of saturated phosphatidylcholine (Sat PC). Median Sat PC amounts increased about 680-fold, and median SP-C values increased by over 5,000-fold in alveolar washes from 27 days gestation to term. There was no increase in Sat PC or SP-C with ventilation at 27 and 28 days gestation, but ventilation increased both Sat PC and SP-C at 29 days gestation. The molar percent of SP-C relative to Sat PC also increased with gestational age and with ventilation at 29 days gestation. proSP-C was abundant in a membrane fraction from lung tissue at 27 and 28 days gestation when minimal mature SP-C was detected in alveolar washes. At 29 days and at term, proSP-C decreased in membrane fractions. The preterm lung that is surfactant lipid deficient is also severely deficient in mature SP-C.
Quantitative trait locus mapping of airway responsiveness to chromosomes 6 and 7 in inbred mice
Authors: De Sanctis G. T. , Singer J. B. , Jiao A. , Yandava C. N. , Lee Y. H. , Haynes T. C. , Lander E. S. , Beier D. R. , Drazen J. M.
Published: December 2017

Quantitative trait locus (QTL) mapping was used to identify chromosomal regions contributing to airway hyperresponsiveness in mice. Airway responsiveness to methacholine was measured in A/J and C3H/HeJ parental strains as well as in progeny derived from crosses between these strains. QTL mapping of backcross [(A/J × C3H/HeJ) × C3H/HeJ] progeny ( n = 137–227 informative mice for markers tested) revealed two significant linkages to loci on chromosomes 6 and 7. The QTL on chromosome 6 confirms the previous report by others of a linkage in this region in the same genetic backgrounds; the second QTL, on chromosome 7, represents a novel locus. In addition, we obtained suggestive evidence for linkage (logarithm of odds ratio = 1.7) on chromosome 17, which lies in the same region previously identified in a cross between A/J and C57BL/6J mice. Airway responsiveness in a cross between A/J and C3H/HeJ mice is under the control of at least two major genetic loci, with evidence for a third locus that has been previously implicated in an A/J and C57BL/6J cross; this indicates that multiple genetic factors control the expression of this phenotype.
NADPH and heme redox modulate pulmonary artery relaxation and guanylate cyclase activation by NO
Authors: Gupte Sachin A. , Rupawalla Tasneem , Phillibert Donald , Wolin Michael S.
Published: December 2017

The hemoprotein oxidant ferricyanide (FeCN) converts the iron of the heme on soluble guanylate cyclase (sGC) from Fe2+to Fe3+, which prevents nitric oxide (NO) from binding the heme and stimulating sGC activity. This study uses FeCN to examine whether modulation of the redox status of the heme on sGC influences the relaxation of endothelium-removed bovine pulmonary arteries (BPA) to NO. Pretreatment of the homogenate of BPA with 50 μM FeCN resulted in a loss of stimulation of sGC activity by the NO donor 10 μM S-nitroso- N-acetylpenicillamine (SNAP). In the FeCN-treated homogenate reconcentrated to the enzyme levels in BPA, 100 μM NADPH restored NO stimulation of sGC, and this effect of NADPH was prevented by an inhibitor of flavoprotein electron transport, 1 μM diphenyliodonium (DPI). In BPA the relaxation to SNAP was not altered by FeCN, inhibitors of NADPH generation by the pentose phosphate pathway [250 μM 6-aminonicotinamide (6-AN) and 100 μM epiandrosterone (Epi)], or 1 μM DPI. However, the combination of FeCN with 6-AN, Epi, or DPI inhibited ( P < 0.05) relaxation to SNAP without significantly altering the relaxation of BPA to forskolin. The inhibitory effects of 1 μM 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (a probe that appears to convert NO-heme of sGC to its Fe3+-heme form) on relaxation to SNAP were also enhanced by DPI. These observations suggest that a flavoprotein containing NADPH oxidoreductase may influence cGMP-mediated relaxation of BPA to NO by maintaining the heme of sGC in its Fe2+oxidation state.
Neutrophils enhance clearance of necrotic epithelial cells in ozone-induced lung injury in rhesus monkeys
Authors: Hyde Dallas M. , Miller Lisa A. , McDonald Ruth J. , Stovall Mary Y. , Wong Viviana , Pinkerton Kent E. , Wegner Craig D. , Rothlein Robert , Plopper Charles G.
Published: December 2017

To test the hypothesis that neutrophil influx is important for the removal of necrotic airway epithelial cells, rhesus monkeys were treated with a function-blocking monoclonal antibody (MAb) against CD18 followed by exposure to ozone or filtered air. CD18 MAb-treated, ozone-exposed monkeys showed a significant inhibition of neutrophil emigration and an accumulation of necrotic airway epithelial cells. In a subsequent experiment, monkeys were given CD18 MAb or an isotype control immunoglobulin before ozone or filtered-air exposure. Complement 5a was instilled into lobes of the right lung at the end of the exposure. Lavage neutrophils were significantly elevated in the right lobes compared with those in the contralateral left lobes; consequently, there were significantly fewer necrotic cells in the airways of the right lung, whereas large aggregations of necrotic cells were observed in the contralateral airways of the left lung. These data indicate that neutrophil influx in ozone-induced injury in primates is CD18 dependent and that neutrophils contribute to the repair of airway epithelium by removal of injured epithelial cells.
Resistance of hypotransferrinemic mice to hyperoxia-induced lung injury
Authors: Yang Funmei , Coalson Jacqueline J. , Bobb Heather H. , Carter Jacqueline D. , Banu Jameela , Ghio Andrew J.
Published: December 2017

Oxidative stress plays a central role in the pathogenesis of acute and chronic pulmonary diseases. Safe sequestration of iron, which participates in the formation of the hydroxyl radical, is crucial in the lung's defense. We used a mouse line defective in the major iron transport protein transferrin to investigate the effect of aberrant iron metabolism on the lung's defense against oxidative injury. The tolerance to hyperoxic lung injury was greater in the hypotransferrinemic than in wild-type mice as documented by histopathology and biochemical indexes for lung damage. There was no increase in the levels of intracellular antioxidants, inflammatory cytokines, and heme oxygenase-1 in the hypotransferrinemic mouse lung compared with those in wild-type mice. However, there were elevated expressions of ferritin and lactoferrin in the lung of hypotransferrinemic mice, especially in the alveolar macrophages. Our results suggest that pulmonary lactoferrin and ferritin protect animals against oxidative stress, most likely via their capacity to sequester iron, and that alveolar macrophages are the key participants in iron detoxification in the lower respiratory tract.
Exacerbation of bleomycin-induced lung injury in mice by amifostine
Authors: Ortiz Luis A. , Lasky Joseph A. , Safah Hana , Reyes Medel , Miller Alan , Lungarella Giuseppe , Friedman Mitchell
Published: December 2017

Bleomycin (BLM) induces lung injury and fibrosis in the murine lung and enhances tumor necrosis factor (TNF)-α and collagen mRNA expression in the murine lung. Amifostine is a cytoprotective agent that protects normal tissues from the cytotoxic effects of chemo- and radiation therapy. We investigated the effect of amifostine in BLM-induced lung injury in mice. Mice received intraperitoneal amifostine (200 mg/kg) 30 min before and/or 1, 3, and 7 days after an intratracheal injection of saline or BLM (4 U/kg). The animals were killed 14 days after BLM exposure, and their lungs were studied for TNF-α and collagen mRNA expression, hydroxyproline content, and histopathology. Light microscopy demonstrated that amifostine exacerbated the BLM-induced lung injury in mice. Increased TNF-α mRNA expression as a result of BLM exposure was not modulated by amifostine treatment. In contrast, amifostine treatment enhanced the BLM-induced expression of α1(I) procollagen mRNA in the lung. Similarly, mice treated with amifostine before BLM exposure accumulated significantly higher amounts of hydroxyproline (111 ± 5 μg/lung) than BLM-treated animals (90 ± 6 μg/lung). These data suggest that amifostine treatment exacerbates BLM-induced lung injury in mice.
The potential of various lipopolysaccharides to release IL-8 and G-CSF
Authors: Koyama Sekiya , Sato Etsuro , Nomura Hiroshi , Kubo Keishi , Miura Masakazu , Yamashita Tetsuji , Nagai Sonoko , Izumi Takateru
Published: December 2017

Lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa is less cytotoxic than that from Escherichia coli. But P. aeruginosa induces a prominent sustained lung inflammation as in cystic fibrosis and diffuse panbronchiolotis. The present study examined the potential for several LPSs obtained from E. coli and P. aeruginosa to release neutrophil chemotactic activity (NCA) from lung cells. LPSs differently stimulated A549 cells, BEAS-2B cells, and lung fibroblasts to release NCA [ P. aeruginosa > E. coli 0127:B8 (Difco) > E. coli 055:B5 (Sigma) > E. coli 026:B6 (Sigma)]. E. coli 0127:B8 (Sigma) and 0111:B4 (Sigma) did not stimulate these cells. NCA was chemotactic by checkerboard analysis. Molecular-sieve column chromatography revealed three chemotactic peaks. The release of NCA was inhibited by cycloheximide and lipoxygenase inhibitors. Experiments with blocking antibodies suggested that much of the NCA was secondary to the release of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF). Thus we examined the concentrations of IL-8 and G-CSF and found that the potency of the various LPSs to stimulate NCA closely paralleled the potency in releasing IL-8 and G-CSF. But a difference among LPSs to stimulate A549 cells was observed. Finally, the release of IL-6 showed similar results. These data suggest that P. aeruginosa LPS may stimulate lung cells to release more NCA than E. coli LPSs, leading to sustained lung inflammation.
Cloning and expression of guinea pig TIMP-2. Expression in normal and hyperoxic lung injury
Authors: Meléndez Jorge , Maldonado Vilma , Bingle Collin D. , Selman Moisés , Pardo Annie
Published: December 2017

Tissue inhibitors of metalloproteinases (TIMPs) play a key regulatory role in extracellular matrix remodeling. By screening a lung library with a human TIMP-2 cDNA probe, we have isolated the cDNA corresponding to guinea pig TIMP-2. The 3.5-kb cDNA presents an open reading frame that predicts a protein of 220 amino acids showing 97.2, 96.8, 97.2, and 77.3% overall identity with human, mouse, rat, and chicken TIMP-2, respectively. Guinea pig TIMP-2 cDNA was expressed in CHO-K1 cells, showing a protein with the expected molecular weight and activity. Northern blot analysis revealed TIMP-2 expression in brain, kidney, intestine, spleen, heart, and lung. Transforming growth factor-β downregulated TIMP-2 mRNA in guinea pig lung fibroblasts, whereas a variety of other stimuli showed no effect. In normal and hyperoxia-exposed lungs, TIMP-2 mRNA was mainly localized in alveolar macrophages and epithelial cells. No quantitative differences were found by Northern blot. These results confirm that TIMP-2 is highly conserved in mammals and largely expressed in lungs.
In situ localization and regulation of thromboxane A2synthase in normal and LPS-primed lungs
Authors: Ermert L. , Ermert M. , Duncker H.-R. , Grimminger F. , Seeger W.
Published: December 2017

Thromboxane (Tx) A2synthase catalyzes the conversion of prostaglandin H2to the unstable metabolite TxA2, which is a potent mediator of vasoconstriction and bronchoconstriction. The cellular localization of TxA2synthase was examined by immunohistochemistry and in situ hybridization in human and rat lung tissues. Bronchial epithelial cells, bronchial smooth muscle cells, peribronchial nerve fibers, single cells of bronchus-associated lymphoid tissue, single cells located in the alveolar septum, and alveolar macrophages exhibited positive immunostaining for TxA2synthase protein in lung tissue of both species. In addition, vascular smooth muscle cells of muscular and partially muscular vessels displayed strong (rat) and moderate (human) immunostaining for TxA2synthase. In situ hybridization performed in the rat lungs demonstrated TxA2synthase mRNA localization in accordance with the immunostaining pattern. Perfusing isolated rat lungs with endotoxin for 1 and 2 h resulted in a marked increase in TxA2synthase protein staining intensity in most cell types as measured by quantitative image analysis, whereas the in situ hybridization signal was unchanged. We conclude that the pulmonary distribution of TxA2synthase displays close similarity between rat and human lung tissues and matches well with the previously described immunolocalization of cyclooxygenase-1 and cyclooxygenase-2 in this tissue. Endotoxin challenge is suggested to cause a rapid upregulation of TxA2synthase at the posttranscriptional level. These data provide a morphological basis for the understanding of the role of TxA2in the regulation of lung bronchial and vascular tone and in immunologic events.
cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B
Authors: Pietschmann Sylvia M. , Pison Ulrich
Published: December 2017

Pulmonary surfactant promotes alveolar stability by lowering the surface tension at the air-liquid interface in the peripheral air spaces. The three surfactant proteins SP-A, SP-B, and SP-C contribute to dynamic surface properties involved during respiration. We have cloned and sequenced the complete cDNAs for ovine SP-A and SP-C and two distinct forms of ovine SP-B cDNAs. The nucleotide sequence of ovine SP-A cDNA consists of 1,901 bp and encodes a protein of 248 amino acids. Ovine SP-C cDNA contains 809 bp, predicting a protein of 190 amino acids. Ovine SP-B is encoded by two mRNA species, which differ by a 69-bp in-frame deletion in the region coding for the active airway protein. The larger SP-B cDNA comprises 1,660 bp, encoding a putative protein of 374 amino acids. With the sequences reported, a more complete analysis of surfactant regulation and the determination of their physiological function in vivo will be enabled.
Inhaled carbon monoxide does not cause pulmonary vasodilation in the late-gestation fetal lamb
Authors: Grover Theresa R. , Rairigh Robyn L. , Zenge Jeanne P. , Abman Steven H. , Kinsella John P.
Published: December 2017

As observed with nitric oxide (NO), carbon monoxide (CO) binds and may activate soluble guanylate cyclase and increase cGMP levels in smooth muscle cells in vitro. Because inhaled NO (INO) causes potent and sustained pulmonary vasodilation, we hypothesized that inhaled CO (ICO) may have similar effects on the perinatal lung. To determine whether ICOcan lower pulmonary vascular resistance (PVR) during the perinatal period, we studied the effects of ICOon late-gestation fetal lambs. Catheters were placed in the main pulmonary artery, left pulmonary artery (LPA), aorta, and left atrium to measure pressure. An ultrasonic flow transducer was placed on the LPA to measure blood flow to the left lung. After baseline measurements, fetal lambs were mechanically ventilated with a hypoxic gas mixture (inspired O2fraction < 0.10) to maintain a constant fetal arterial [Formula: see text]. After 60 min (baseline), the lambs were treated with ICO[5–2,500 parts/million (ppm)]. Comparisons were made with INO(5 and 20 ppm) and combined INO(5 ppm) and ICO(100 and 2,500 ppm). We found that ICOdid not alter left lung blood flow or PVR at any of the study doses. In contrast, low-dose INOdecreased PVR by 47% ( P < 0.005). The combination of INOand ICOdid not enhance the vasodilator response to INO. To determine whether endogenous CO contributes to vascular tone in the fetal lung, zinc protoporphyrin IX, an inhibitor of heme oxygenase, was infused into the LPA in three lambs. Zinc protoporphyrin IX had no effect on baseline PVR, aortic pressure, or the pressure gradient across the ductus arteriosus. We conclude that ICOdoes not cause vasodilation in the near-term ovine transitional circulation, and endogenous CO does not contribute significantly to baseline pulmonary vascular tone or ductus arteriosus tone in the late-gestation ovine fetus.
A method for measuring the oxygen consumption of intact cell monolayers
Authors: Mamchaoui Kamel , Saumon Georges
Published: December 2017

This report describes an open-air method for measuring the O2 consumption (Q˙o 2) of intact monolayers of cultured cells. This method is based on Fick's second law of diffusion. It requires only a micromanipulator and a miniature O2 electrode to measure the[Formula: see text] gradient in the culture medium in the well. It was compared with the conventional oxygraph chamber method. Both methods gave the same value forQ˙o 2 in freshly isolated rat type II cells: 166 ± 15.3 nmol ⋅ h−1 ⋅ 106cells−1 for the open-air method and 151 ± 11.6 nmol ⋅ h−1 ⋅ 106cells−1 for the oxygraph chamber method ( n = 11 experiments). But the open-air method gave significantly larger values for Q˙o 2 in cells cultured for 2 days (236 ± 8.8 nmol ⋅ h−1 ⋅ 106cells−1) than the oxygraph method (71 ± 15.2 nmol ⋅ h−1 ⋅ 106cells−1; P < 0.001; n = 12 experiments). This suggests that the way cells are detached from their substratum to be placed in the oxygraph chamber affects theirQ˙o 2. The open-air method may be useful for studies on the metabolic properties of monolayers because the cells do not risk being damaged.
Increased CD44 expression in human bronchial epithelial repair after damage or plating at low cell densities
Authors: Leir Shih-Hsing , Baker Janice E. , Holgate Stephen T. , Lackie Peter M.
Published: December 2017

We have investigated the effect of mechanical damage, cell density, and cell-derived soluble mediators on CD44 expression in a model of bronchial epithelial repair. CD44 (all isoforms) and variant-containing isoforms (CD44v3, CD44v6, and CD44v9) were identified with flow cytometry and immunocytochemistry with image analysis. After mechanical damage, CD44 expression increased up to 500 μm from the wound edge and for up to 48 h in two human bronchial epithelium-derived cell lines, 16HBE14o− and NCI-H292. CD44 expression was unchanged by interferon-γ and increased by <50% by tumor necrosis factor-α. To exclude other soluble factors, a Vaseline spacer was used to temporarily divide petri dishes, with cells at high density on one side and those at low density on the other. After the spacer was removed, the cells at low cell density growing in the shared medium expressed up to fourfold higher CD44, although cell proliferation was unchanged. Thus increased CD44 expression at low cell density was not mediated by soluble factors and may reflect functional involvement in cell motility, dedifferentiation, or altered cell-substrate adhesion in epithelial repair.
Autocrine cytokine signaling mediates effects of rhinovirus on airway responsiveness
Authors: Grunstein Michael M. , Hakonarson Hakon , Maskeri Neil , Chuang Sing
Published: December 2017

The airway responses to allergen exposure in allergic asthma are qualitatively similar to those elicited by specific viral respiratory pathogens, most notably rhinovirus (RV), suggesting that the altered airway responsiveness seen in allergic asthma and that elicited by viral respiratory tract infection may share a common underlying mechanism. To the extent that T helper cell type 2 (Th2) cytokines have been implicated in the pathogenesis of allergic asthma, this study examined the potential role(s) of Th2-type cytokines in mediating pro-asthmatic-like changes in airway smooth muscle (ASM) responsiveness after inoculation of naive ASM with human RV. Isolated rabbit ASM tissues and cultured human ASM cells were exposed to RV (serotype 16) for 24 h in the absence and presence of monoclonal blocking antibodies (MAbs) or antagonists directed against either the Th2-type cytokines interleukin (IL)-4 and IL-5, intercellular adhesion molecule (ICAM)-1 (the endogenous host receptor for most RVs), or the pleiotropic proinflammatory cytokine IL-1β. Relative to control (vehicle-treated) tissues, RV-exposed ASM exhibited significantly enhanced isometric contractility to acetylcholine and impaired relaxation to isoproterenol. These pro-asthmatic-like changes in ASM responsiveness were ablated by pretreating the RV-exposed tissues with either IL-5-receptor-α blocking antibody or human recombinant IL-1-receptor antagonist, whereas IL-4 neutralizing antibody had no effect. Extended studies further demonstrated that inoculation of ASM cells with RV elicited 1) an increased mRNA expression and release of IL-5 protein, which was inhibited in the presence of anti-ICAM-1 MAb, and 2) an enhanced release of IL-1β protein, which was inhibited in the presence of IL-5 receptor-α antibody. Collectively, these observations provide new evidence demonstrating that RV-induced changes in ASM responsiveness are largely attributed to ICAM-1-dependent activation of a cooperative autocrine signaling mechanism involving upregulated IL-5-mediated release of IL-1β by the RV-exposed ASM itself.
Endotoxin priming of the cyclooxygenase-2-thromboxane axis in isolated rat lungs
Authors: Ermert M. , Merkle M. , Mootz R. , Grimminger F. , Seeger W. , Ermert L.
Published: December 2017

Enhanced prostanoid generation has been implicated in vascular abnormalities occurring during endotoxemia and sepsis, and the lung is particularly prone to such events. Prostanoids are generated from arachidonic acid (AA) via cyclooxygenase (COX)-1 or -2, both isoenzymes recently demonstrated to be expressed in different lung cell types. Upregulation of COX may underlie the phenomenon that endotoxin [lipopolysaccharide (LPS)]-exposed lungs show markedly enhanced vasoconstrictor responses to secondarily applied stimuli (priming). Isolated rat lungs were perfused with a physiological salt buffer solution in the absence and presence of 1.5% rat plasma and exposed to different concentrations of LPS (1,000 or 10,000 ng/ml) during a 2-h priming period. No change in physiological variables was noted during this period, although enhanced baseline liberation of both thromboxane (Tx) A2and PGI2as well as of tumor necrosis factor (TNF)-α was evident compared with that in control lungs in the absence of LPS. LPS priming caused a significant elevation in AA-induced pulmonary arterial pressure, ventilation pressure, and lung weight gain. Concomitant increased levels of TxA2were found in the buffer perfusate. All changes were largely suppressed by three selective, structurally unrelated COX-2 inhibitors (NS-398, DUP-697, and SC-236) in both buffer- and buffer-plasma-perfused lungs. Anti-TNF-α neutralizing antibodies were ineffective under conditions of buffer perfusion. In the presence of plasma components, manyfold augmented TNF-α generation was noted, and anti-TNF-α antibodies significantly suppressed the increase in ventilation pressure but not in the vascular pressor response and lung edema formation. We conclude that the propensity of LPS-primed lungs to respond with enhanced vasoconstriction, edema formation, and bronchoconstriction to a secondarily applied stimulus proceeds nearly exclusively via COX-2 and increased Tx formation, with TNF-α generation being involved in the change in bronchomotor reactivity in the presence of plasma constituents. In context with recent immunohistological investigations, LPS-induced upregulation of the COX-2-thromboxane synthase axis in vascular and bronchial smooth muscle cells is suggested to underlie these events.
Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells
Authors: Su Yunchao , Block Edward R.
Published: December 2017

Pulmonary artery endothelial cells (PAEC) were exposed to normoxia or hypoxia (0% O2-95% N2-5% CO2) in the presence and absence of calpain inhibitor I or calpeptin, after which endothelial nitric oxide synthase (eNOS) activity and protein content were assayed. Exposure to hypoxia decreased eNOS activity but not eNOS protein content. Both calpain inhibitor I and calpeptin prevented the hypoxic decrease of eNOS activity. Incubation of calpain with total membrane preparations of PAEC caused dose-dependent decreases in eNOS activity independent of changes in eNOS protein content. Exposure of PAEC to hypoxia also caused time-dependent decreases of heat shock protein 90 (HSP90) that were prevented by calpain inhibitor I and calpeptin. Moreover, the HSP90 content in anti-eNOS antibody-induced immunoprecipitates from hypoxic PAEC lysates was reduced, and repletion of HSP90 reversed the decrease of eNOS activity in these immunoprecipitates. Incubation of PAEC with a specific inhibitor of HSP90 (geldanamycin) mimicked the hypoxic decrease of eNOS activity. These results indicate that the hypoxia-induced reduction in eNOS activity in PAEC is due to a decrease in HSP90 caused by calpain activation.
Experimental silicosis: a shift to a preferential IFN-γ-based Th1 response in thoracic lymph nodes
Authors: Garn Holger , Friedetzky Anke , Kirchner Andrea , Jäger Ruth , Gemsa Diethard
Published: December 2017

In chronic silicosis, mechanisms leading to lymphocyte activation are still poorly understood, although it is well known that not only the lung but also the draining lymph nodes are affected. In the present study, we investigated T-cell activation by analysis of cytokine expression in the enlarged thoracic lymph nodes of rats 2 mo after an 8-day silica aerosol exposure. In the case of helper T cell (Th) type 1 cytokines, we found a significant increase in interferon (IFN)-γ mRNA expression, whereas interleukin (IL)-2 expression remained unchanged. In contrast, gene transcription for the Th2-type cytokines IL-4 and IL-10 was diminished. In addition, with use of an in vitro lymphocyte-macrophage coculture system, an enhanced IFN-γ and a reduced IL-10 release were shown with cells from silicotic animals. With regard to IFN-γ-inducing cytokines, we observed enhanced IL-12 mRNA levels in vivo, whereas IL-18 gene expression was slightly decreased. These data indicate that a persistent shift toward an IFN-γ-dominated type 1 (Th1/cytotoxic T cell type 1) T-cell reaction pattern occurred within the thoracic lymph nodes of silicotic animals. Thus a mutual activation of lymphocytes and macrophages may maintain the chronic inflammatory changes that characterize silicosis.