bluuebunny/crossref_metadata_2025 huggingface dataset:
p38 MAP kinase regulates IL-1β responses in cultured airway smooth muscle cells
Authors: Laporte Johanne D. , Moore Paul E. , Lahiri Thomas , Schwartzman Igor N. , Panettieri Reynold A. , Shore Stephanie A.
Published: December 2017

We have previously reported that interleukin (IL)-1β causes β-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1β (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 μM) decreased IL-1β-induced COX-2 by 70 ± 7% ( P < 0.01). SB-203580 had no effect on PGE2release in control cells but caused a significant (70–80%) reduction in PGE2release in IL-1β-treated cells. IL-1β increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-κB, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-κB activation. The NF-κB inhibitor MG-132 did not alter IL-1β-induced COX-2 expression, indicating that NF-κB activation is not required for IL-1β-induced COX-2 expression in HASM cells. IL-1β attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1β induces COX-2 expression, PGE2release, and β-adrenergic hyporesponsiveness.
PKC, p42/p44 MAPK, and p38 MAPK are required for HGF-induced proliferation of H441 cells
Authors: Awasthi Vibhudutta , King Richard J.
Published: December 2017

In this paper, we studied the signaling pathway used by hepatocyte growth factor/scatter factor (HGF) to stimulate mitosis. We show, using H441 cells, that 1) HGF activates membrane-associated protein kinase C (PKC); the activity is transient and peaks within 30 min; 2) HGF activates p42/p44 and p38 mitogen-activated protein kinases (MAPKs); maximum activity in both is within 10 min; and 3) the activation of neither p38 nor p42/p44 MAPK is dependent on PKC, indicating that HGF uses separate and nonintersecting pathways to activate these two classes of kinase. However, phorbol 12-myristate 13-acetate also activates both MAPKs as well as PKC, but this activation is abolished in cells pretreated with the PKC inhibitor GF-109203X. HGF was found to significantly increase [3H]thymidine incorporation within 5 h; peak thymidine incorporation was observed at 16 h. However, when cells were pretreated with inhibitors of p42/p44 (PD-98059), p38 (SB-203580), or PKC (GF-109203X, Gö-6983, or myristoylated inhibitor peptide19–27), HGF-induced thymidine uptake was diminished in a dose-dependent manner. Taken together, these results demonstrate that HGF activates PKC and both MAPKs simultaneously through parallel pathways and that the activation of the MAPKs does not depend on PKC. However, p38 and p42/p44 MAPKs and PKC may all be essential for HGF-induced proliferation of H441 cells.
Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development
Authors: De Paepe Monique E. , Rubin Lewis P. , Jude Craig , Lesieur-Brooks Anne M. , Mills David R. , Luks Francois I.
Published: December 2017

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25–31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 ± 0.34% on gestational day 27 to 3.34 ± 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.
Reactive oxygen species in cell signaling
Authors: Thannickal Victor J. , Fanburg Barry L.
Published: December 2017

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of “oxidative stress” is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.
Heme oxygenase: colors of defense against cellular stress
Authors: Otterbein Leo E. , Choi Augustine M. K.
Published: December 2017

The discovery of the gaseous molecule nitric oxide in 1987 unraveled investigations on its functional role in the pathogenesis of a wide spectrum of biological and pathological processes. At that time, the novel concept that an endogenous production of a gaseous substance such as nitric oxide can impart such diverse and potent cellular effects proved to be very fruitful in enhancing our understanding of many disease processes including lung disorders. Interestingly, we have known for a longer period of time that there exists another gaseous molecule that is also generated endogenously; the heme oxygenase (HO) enzyme system generates the majority if not all of the endogenously produced carbon monoxide. This enzyme system also liberates two other by-products, bilirubin and ferritin, each possessing important biological functions and helping to define the uniqueness of the HO enzyme system. In recent years, interest in HO has emerged in numerous disciplines including the central nervous system, cardiovascular physiology, renal and hepatic systems, and transplantation. We review the functional role of HO in lung biology and its real potential application to lung diseases.
Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes
Authors: Isowa Noritaka , Keshavjee Shaf H. , Liu Mingyao
Published: December 2017

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 μM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1–10 μM) and nocodazole (1–100 μM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.
Autocrine regulation of interleukin-8 production in human monocytes
Authors: Browning Darren D. , Diehl Wade C. , Hsu Matthew H. , Schraufstatter Ingrid U. , Ye Richard D.
Published: December 2017

Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-α (MGSA/GROα), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROα was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.
Visualization of labile zinc and its role in apoptosis of primary airway epithelial cells and cell lines
Authors: Truong-Tran Ai Q. , Ruffin Richard E. , Zalewski Peter D.
Published: December 2017

The respiratory epithelium is vulnerable to noxious substances, resulting in the shedding of cells and decreased protection. Zinc (Zn), an antioxidant and cytoprotectant, can suppress apoptosis in a variety of cells. Here we used the novel Zn-specific fluorophore Zinquin to visualize and quantify labile intracellular Zn in respiratory epithelial cells. Zinquin fluorescence in isolated ciliated tracheobronchial epithelial cells and intact epithelium from sheep and pigs revealed an intense fluorescence in the apical and mitochondria-rich cytoplasm below the cilia. Zinquin fluorescence was quenched by the Zn chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and increased by the Zn ionophore pyrithione. We also assessed whether changes in intracellular labile Zn would influence susceptibility of these cells to apoptosis by hydrogen peroxide. Our results confirm that Zn deficiency enhanced hydrogen peroxide-induced caspase activation from 1.24 ± 0.12 to 2.58 ± 0.53 units · μg protein−1· h−1( P ≤ 0.05); Zn supplementation suppressed these effects. These findings are consistent with the hypothesis that Zn protects upper respiratory epithelial cells and may have implications for human asthma where there is hypozincemia and epithelial damage.
Bcl-2 in LPS- and allergen-induced hyperplastic mucous cells in airway epithelia of Brown Norway rats
Authors: Tesfaigzi Yohannes , Fischer Mark J. , Martin Andrea J. , Seagrave Jeanclare
Published: December 2017

Environmental toxins, infection, and allergens lead to a transient mucous cell hyperplasia (MCH) in airway epithelia; however, the mechanisms for reducing mucous cell numbers during recovery are largely unknown. This study investigated Bcl-2 expression in mucous cells induced by a neutrophilic or eosinophilic inflammatory response. Brown Norway rats intratracheally instilled with lipopolysaccharide (LPS) showed an inflammatory response characterized primarily by neutrophils. Secreted mucin was increased fourfold at 1 day, and the number of mucous cells was increased fivefold 2, 3, and 4 days post-LPS instillation compared with those in noninstilled rats. None of the mucous cells in non- or saline-instilled control animals expressed Bcl-2, whereas 20–30% of mucous cells were Bcl-2 positive 1 and 2 days post-LPS instillation. Brown Norway rats immunized and challenged with ovalbumin (OVA) for 2, 4, and 6 days showed an inflammatory response characterized primarily by eosinophils. Secreted mucin increased fivefold, and mucous cell number increased fivefold after 4 and 6 days of OVA exposure compared with water-immunized control rats challenged with OVA aerosols. Approximately 10–25% of mucous cells were Bcl-2 positive in OVA-immunized and -challenged rats. These data demonstrate Bcl-2 expression in hyperplastic mucous cells of Brown Norway rats regardless of the type of inflammatory response and indicate that apoptotic mechanisms may be involved in the resolution of MCHs.
Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca2+handling
Authors: Darby Peter J. , Kwan C. Y. , Daniel Edwin E.
Published: December 2017

To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca2+-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca2+. We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca2+channels and Ca2+binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca2+pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca2+pump, or the type 1 inositol 1,4,5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca2+pump to the cell periphery, whereas the SR Ca2+pump was located deeper in the cell. The presence of L-type Ca2+channels, the PM Ca2+pump, and the Ca2+bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca2+handling.
Prostaglandin E2-induced interleukin-6 release by a human airway epithelial cell line
Authors: Tavakoli S. , Cowan M. J. , Benfield T. , Logun C. , Shelhamer J. H.
Published: December 2017

Human airway epithelial cell release of interleukin (IL)-6 in response to lipid mediators was studied in an airway cell line (BEAS-2B). Prostaglandin (PG) E2(10−7M) treatment caused an increase in IL-6 release at 2, 4, 8, and 24 h. IL-6 release into the culture medium at 24 h was 3,396 ± 306 vs. 1,051 ± 154 pg/ml (PGE2-treated cells vs. control cells). PGE2(10−7to 10−10M) induced a dose-related increase in IL-6 release at 24 h. PGF2α(10−6M) treatment caused a similar effect to that of PGE2(10−7M). PGE2analogs with relative selectivity for PGE2receptor subtypes were studied. Sulprostone, a selective agonist for the EP-3 receptor subtype had no effect on IL-6 release. 11-Deoxy-16,16-dimethyl-PGE2, an EP-2/4 agonist, and 17-phenyl trinor PGE2, an agonist selective for the EP-1 > EP-3 receptor subtype (10−6to 10−8M), caused dose-dependent increases in IL-6 release. 8-Bromo-cAMP treatment resulted in dose-related increases in IL-6 release. RT-PCR of BEAS-2B cell mRNA demonstrated mRNA for EP-1, EP-2, and EP-4 receptors. After PGE2treatment, increases in IL-6 mRNA were noted at 4 and 18 h. Therefore, PGE2increases airway epithelial cell IL-6 production and release.
Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes
Authors: Stevens Paul A. , Wissel Heide , Zastrow Stefan , Sieger Daniela , Zimmer Klaus-Peter
Published: December 2017

Surfactant protein (SP) A and SP-A-mediated lipid uptake by isolated type II cells were investigated with biochemical and morphological methods. Inhibition of coated-pit function by potassium depletion severely reduced both SP-A and SP-A-mediated lipid internalization. Lipid uptake in the absence of SP-A was not affected. With confocal laser scanning microscopy and immunoelectron microscopy, SP-A and lipid predominantly (60%) colocalized in intracellular vesicles carrying early endosomal markers (EEA1) 5 min after endocytosis but were negative for the late endosomal or lysosomal marker LAMP-1. As estimated by subcellular fractionation, at this time point, 23% of the internalized SP-A and 45% of internalized lipid were localized within light (<0.38 M sucrose) fractions, which contain lamellar bodies and are positive for EEA1. The remaining label was predominantly found within EEA1-positive and plasma membrane-containing subfractions (≥1 M sucrose). We suggest that in isolated type II cells in vitro, SP-A and lipid are taken up together via the coated-pit pathway and that at early time points, both components reside in the same early endosomal compartment.
Ionizing radiation enhances matrix metalloproteinase-2 production in human lung epithelial cells
Authors: Araya Jun , Maruyama Muneharu , Sassa Kazuhiko , Fujita Tadashi , Hayashi Ryuji , Matsui Shoko , Kashii Tatsuhiko , Yamashita Naohiro , Sugiyama Eiji , Kobayashi Masashi
Published: December 2017

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.
Overexpression of tumor necrosis factor-α produces an increase in lung volumes and pulmonary hypertension
Authors: Fujita Masaki , Shannon John M. , Irvin Charles G. , Fagan Karen A. , Cool Carlyne , Augustin Andrei , Mason Robert J.
Published: December 2017

Tumor necrosis factor (TNF)-α is a key proinflammatory cytokine that is thought to be important in the development of pulmonary fibrosis, whereas its role in pulmonary emphysema has not been as thoroughly documented. In the present study, TNF-α was overexpressed in alveolar type II cells under the control of the human surfactant protein C promoter. In this report, we further characterized the pulmonary abnormalities and provided a physiological assessment of these mice. Histopathology of the lungs revealed chronic inflammation, severe alveolar air space enlargement and septal destruction, and bronchiolitis. However, pulmonary fibrosis was very limited and only seen in the subpleural, peribronchiolar, and perivascular regions. Physiological assessment showed an increase in lung volumes and a decrease in elastic recoil characteristic of emphysema; there was no evidence of restrictive lung disease characteristic of pulmonary fibrosis. In addition, the mice raised in ambient conditions in Denver developed pulmonary hypertension. Gelatinase activity was increased in the lavage fluid from these lungs. These results suggest that in these mice TNF-α contributed to the development of pulmonary emphysema through chronic lung inflammation and activation of the elastolytic enzymes but by itself was unable to produce significant pulmonary fibrosis.
Effects of PKC isozyme inhibitors on constrictor responses in the feline pulmonary vascular bed
Authors: De Witt Bracken J. , Kaye Alan D. , Ibrahim Ikhlass N. , Bivalacqua Trinity J. , D'Souza Fiona M. , Banister Ronald E. , Arif A. Salam , Nossaman Bobby D.
Published: December 2017

The effects of Gö-6976, a Ca2+-dependent protein kinase C (PKC) isozyme inhibitor, and rottlerin, a PKC-δ isozyme/calmodulin (CaM)-dependent kinase III inhibitor, on responses to vasopressor agents were investigated in the feline pulmonary vascular bed. Injections of angiotensin II, norepinephrine (NE), serotonin, BAY K 8644, and U-46619 into the lobar arterial constant blood flow perfusion circuit caused increases in pressure. Gö-6976 reduced responses to angiotensin II; however, it did not alter responses to serotonin, NE, or U-46619, whereas Gö-6976 enhanced BAY K 8644 responses. Rottlerin reduced responses to angiotensin II and NE, did not alter responses to serotonin or U-46619, and enhanced responses to BAY K 8644. Immunohistochemistry of feline pulmonary arterial smooth muscle cells demonstrated localization of PKC-α and -δ isozymes in response to phorbol 12-myristate 13-acetate and angiotensin II. Localization of PKC-α and -δ isozymes decreased with administration of Gö-6976 and rottlerin, respectively. These data suggest that activation of Ca2+-dependent PKC isozymes and Ca2+-independent PKC-δ isozyme/CaM-dependent kinase III mediate angiotensin II responses. These data further suggest that Ca2+-independent PKC-δ isozyme/CaM-dependent kinase III mediate responses to NE. A rottlerin- or Gö-6976-sensitive mechanism is not involved in mediating responses to serotonin and U-46619, but these PKC isozyme inhibitors enhanced BAY K 8644 responses in the feline pulmonary vascular bed.
Connexin expression by alveolar epithelial cells is regulated by extracellular matrix
Authors: Guo Yihe , Martinez-Williams Cara , Yellowley Clare E. , Donahue Henry J. , Rannels D. Eugene
Published: December 2017

Extracellular matrix (ECM) proteins promote attachment, spreading, and differentiation of cultured alveolar type II epithelial cells. The present studies address the hypothesis that the ECM also regulates expression and function of gap junction proteins, connexins, in this cell population. Expression of cellular fibronectin and connexin (Cx) 43 increase in parallel during early type II cell culture as Cx26 expression declines. Gap junction intercellular communication is established over the same interval. Cells plated on a preformed, type II cell-derived, fibronectin-rich ECM demonstrate accelerated formation of gap junction plaques and elevated gap junction intercellular communication. These effects are blocked by antibodies against fibronectin, which cause redistribution of Cx43 protein from the plasma membrane to the cytoplasm. Conversely, cells cultured on a laminin-rich ECM, Matrigel, express low levels of Cx43 but high levels of Cx26, reflecting both transcriptional and translational regulation. Cx26 and Cx43 thus demonstrate reciprocal regulation by ECM constituents.
Intercellular Ca2+signaling in alveolar epithelial cells through gap junctions and by extracellular ATP
Authors: Isakson Brant E. , Evans W. Howard , Boitano Scott
Published: December 2017

Inter- and extracellular-mediated changes in intracellular Ca2+concentration ([Ca2+]i) can ensure coordinated tissue function in the lung. Cultured rat alveolar epithelial cells (AECs) have been shown to respond to secretagogues with increases in [Ca2+]iand have been shown to be gap junctionally coupled. However, communication of [Ca2+]ichanges in AECs is not well defined. Monolayers of AECs were mechanically perturbed and monitored for [Ca2+]ichanges. Perturbation of AECs was administered by a glass probe to either mechanically stimulate or mechanically wound individual cells. Both approaches induced a change in [Ca2+]iin the stimulated cell that was propagated to neighboring cells (Ca2+waves). A connexin mimetic peptide shown to uncouple gap junctions eliminated Ca2+waves in mechanically stimulated cells but had no effect on mechanically wounded cells. In contrast, apyrase, an enzyme that effectively removes ATP from the extracellular milieu, had no effect on mechanically stimulated cells but severely restricted mechanically wounded Ca2+wave propagation. We conclude that AECs have the ability to communicate coordinated Ca2+changes using both gap junctions and extracellular ATP.
Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep
Authors: Bachurski Cindy J. , Ross Gary F. , Ikegami Machiko , Kramer Boris W. , Jobe Alan H.
Published: December 2017

Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.
Toll-like receptor 4 mediates ozone-induced murine lung hyperpermeability via inducible nitric oxide synthase
Authors: Kleeberger Steven R. , Reddy Sekhar P. M. , Zhang Liu-Yi , Cho Hye-Youn , Jedlicka Anne E.
Published: December 2017

We tested the hypotheses that 1) inducible nitric oxide synthase (iNOS) mediates ozone (O3)-induced lung hyperpermeability and 2) mRNA levels of the gene for iNOS ( Nos2) are modulated by Toll-like receptor 4 ( Tlr4) during O3exposure. Pretreatment of O3-susceptible C57BL/6J mice with a specific inhibitor of total NOS ( NG-monomethyl-l-arginine) significantly decreased the mean lavageable protein concentration (a marker of lung permeability) induced by O3(0.3 parts/million for 72 h) compared with vehicle control mice. Furthermore, lavageable protein in C57BL/B6 mice with targeted disruption of Nos2 [ Nos2(−/−)] was 50% less than the protein in wild-type [ Nos2(+/+)] mice after O3. To determine whether Tlr4 modulates Nos2 mRNA levels, we studied C3H/HeJ (HeJ) and C3H/HeOuJ mice that differ only at a missense mutation in Tlr4 that confers resistance to O3-induced lung hyperpermeability in the HeJ strain. Nos2 and Tlr4 mRNA levels were significantly reduced and correlated in resistant HeJ mice after O3relative to those in susceptible C3H/HeOuJ mice. Together, the results are consistent with an important role for iNOS in O3-induced lung hyperpermeability and suggest that Nos2 mRNA levels are mediated through Tlr4.
Chloride channel function is linked to epithelium-dependent airway relaxation
Authors: Fortner Christopher N. , Lorenz John N. , Paul Richard J.
Published: December 2017

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl−secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl−channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl−channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl−in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl−secretion using Cl−-free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl−channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl−channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl−channel other than CFTR is active in the SP response. This work introduces a possible role for Cl−pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.
IL-10 gene knockout attenuates allergen-induced airway hyperresponsiveness in C57BL/6 mice
Authors: Justice J. Paul , Shibata Y. , Sur S. , Mustafa J. , Fan M. , Van Scott M. R.
Published: December 2017

Intratracheal administration of interleukin-10 (IL-10) has been reported to inhibit allergic inflammation but augment airway hyperresponsiveness (AHR). In the present study, airway and smooth muscle responsiveness to methacholine (MCh) were compared in wild-type (WT) and IL-10-deficient (IL-10-KO) mice to investigate the role of endogenous IL-10 in AHR development. Naive WT and IL-10-KO mice exhibited similar dose-dependent increases in airway resistance (Raw) to intravenous MCh. Sensitization and challenge with ragweed (RW) induced a twofold increase in responsiveness to intravenous MCh in WT mice, but hyperresponsiveness was not observed in similarly treated IL-10-KO mice. Likewise, tracheal rings from RW-sensitized and -challenged WT mice exhibited a fourfold greater responsiveness to MCh than IL-10-KO tracheal preparations. Measurements of airway constriction by whole body plethysmography further supported the Raw and tracheal ring data (i.e., AHR was not observed in the absence of IL-10). Interestingly, factors previously implicated in the development of AHR, including IL-4, IL-5, IL-13, IgA, IgG1, IgE, eosinophilia, and lymphocyte recruitment to the airways, were upregulated in the IL-10-KO mice. Treatment with recombinant murine IL-10 at the time of allergen challenge reduced the magnitude of inflammation but reinstated AHR development in IL-10-KO mice. Adoptive transfer of mononuclear splenocytes to IL-10-sufficient severe combined immunodeficient mice indicated that lymphocytes were an important source of the IL-10 impacting AHR development. These results provide evidence that IL-10 expression promotes the development of allergen-induced smooth muscle hyperresponsiveness.
l-Arginine attenuates lipopolysaccharide-induced lung chemokine production
Authors: Calkins Casey M. , Bensard Denis D. , Heimbach Julie K. , Meng Xianzhong , Shames Brian D. , Pulido Edward J. , McIntyre Robert C.
Published: December 2017

Chemokines stimulate the influx of leukocytes into tissues. Their production is regulated by nuclear factor-κB (NF-κB), an inducible transcription factor under the control of inhibitory factor κB-α (IκB-α). We have previously demonstrated that l-arginine (l-Arg) attenuates neutrophil accumulation and pulmonary vascular injury after administration of lipopolysaccharide (LPS). We hypothesized thatl-Arg would attenuate the production of lung chemokines by stabilizing IκB-α and preventing NF-κB DNA binding. We examined the effect of l-Arg on chemokine production, IκB-α degradation, and NF-κB DNA binding in the lung after systemic LPS. To block nitric oxide (NO) production, a NO synthase inhibitor was given before l-Arg. LPS induced the production of chemokine protein and mRNA. l-Arg attenuated the production of chemokine protein and mRNA, prevented the decrease in IκB-α levels, and inhibited NF-κB DNA binding. NO synthase inhibition abolished the effects of l-Arg on all measured parameters. Our results suggest that l-Arg abrogates chemokine protein and mRNA production in rat lung after LPS. This effect is dependent on NO and is mediated by stabilization of IκB-α levels and inhibition of NF-κB DNA binding.
Phosphodiesterase 5 inhibition restores impaired ACh relaxation in hypertensive conduit pulmonary arteries
Authors: Oka Masahiko
Published: December 2017

Responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were compared in large (LPA) and small pulmonary artery (SPA) rings from normoxic and chronically hypoxic (CH) rats. In addition, the effects of a selective phosphodiesterase (PDE) 5 inhibitor, E-4021, on ACh-induced relaxation were evaluated. Chronic hypoxia markedly decreased both ACh- and SNP-induced relaxations in LPA but not in SPA rings. Pretreatment with E-4021 caused a much greater leftward shift of the concentration-response curve for ACh in hypoxic than in normoxic LPA rings, eliminating the difference in response to ACh between these two vessels. These results suggest that cGMP-dependent relaxation is impaired in the proximal but not in the distal pulmonary artery of CH rats and that increased PDE5 activity could be a mechanism responsible for this impaired responsiveness.
NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium
Authors: Chen Jian-Xiong , Berry Leonard C. , Christman Brian W. , Tanner Miles , Myers Paul R. , Meyrick Barbara O.
Published: December 2017

We examined whether nitric oxide (NO) inhibits prostanoid synthesis through actions on cyclooxygenase (COX) gene expression and activity. Bovine pulmonary artery endothelial cells were pretreated for 30 min with the NO donors 1 mM S-nitroso- N-acetylpenicillamine (SNAP), 0.5 mM sodium nitroprusside (SNP), or 0.2 μM spermine NONOate; controls included cells pretreated with either 1 mM N-acetyl-d-penicillamine or the NO synthase (NOS) inhibitor 1 mM NG-nitro-l-arginine methyl ester with and without addition of lipopolysaccharide (LPS; 0.1 μg/ml) for 8 h. COX-1 and COX-2 gene and protein expression were examined by RT-PCR and Western analysis, respectively; prostanoid measurements were made by gas chromatography-mass spectrometry, and COX activity was studied after a 30-min incubation with 30 μM arachidonic acid. LPS induced COX-2 gene and protein expression and caused an increase in COX activity and an eightfold increase in 6-keto-PGF1αrelease. LPS-stimulated COX-2 gene expression was decreased by ∼50% by the NO donors. In contrast, LPS caused a significant reduction in COX-1 gene expression and treatment with NO donors had little effect. SNAP, SNP, and NONOate significantly suppressed LPS-stimulated COX activity and 6-keto-PGF1αrelease. Our data indicate that increased generation of NO attenuates LPS-stimulated COX-2 gene expression and activity, whereas inhibition of endogenous NOS has little effect.
Vascular release of nonheme iron in perfused rabbit lungs
Authors: Huang Yuh-Chin T. , Ghio Andrew J. , Nozik-Grayck Eva , Piantadosi Claude A.
Published: December 2017

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O2) or hypoxic (95% N2) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released ∼0.25 μg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by ∼50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and ∼60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.
Multiple Ca2+-dependent modulators mediate alkalosis-induced vasodilation in newborn piglet lungs
Authors: Vander Heyden Michele A. , Halla Ted R. , Madden Jane A. , Gordon John B.
Published: December 2017

We previously found that alkalosis-induced vasodilation was mediated by endothelium-derived nitric oxide (EDNO) in newborn piglet pulmonary artery and vein rings precontracted with the thromboxane mimetic U-46619. In contrast, prostacyclin or K+channel activation contributed to the response in other preparations. This study was undertaken to determine whether EDNO alone also mediates alkalosis-induced pulmonary vasodilation in piglet lungs vasoconstricted with hypoxia and, if not, to identify the mediator(s) involved. Responses to alkalosis were measured during hypoxia under control conditions after blocking nitric oxide synthase ( Nω-nitro-l-arginine), cyclooxygenase (meclofenamate), or both endothelium-derived modulators (Dual); after blocking voltage-dependent (4-aminopyridine), ATP- dependent (glibenclamide), or Ca2+-dependent K+(KCa; tetraethylammonium) K+channels; and after blocking both endothelium-derived modulators and KCachannels (Triple). Vasodilator responses measured after 20 min of alkalosis were blunted in Dual and tetraethylammonium lungs and abolished in Triple lungs. Thus alkalosis-induced vasodilation in hypoxic lungs appeared to be mediated by three Ca2+-dependent modulators: EDNO, prostacyclin, and KCachannels. In addition, a transient, unidentified modulator contributed to the nadir of the vasodilator response measured at 10 min of alkalosis. Future studies are needed to identify factors that contribute to the discordance between isolated vessels and whole lungs.
Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-α receptors
Authors: Cho Hye-Youn , Zhang Liu-Yi , Kleeberger Steven R.
Published: December 2017

This study was designed to investigate the mechanisms through which tumor necrosis factor ( Tnf) modulates ozone (O3)-induced pulmonary injury in susceptible C57BL/6J (B6) mice. B6 [wild-type ( wt)] mice and B6 mice with targeted disruption (knockout) of the genes for the p55 TNF receptor [ TNFR1(−/−)], the p75 TNF receptor [ TNFR2(−/−)], or both receptors [ TNFR1/TNFR2(−/−)] were exposed to 0.3 parts/million O3for 48 h (subacute), and lung responses were determined by bronchoalveolar lavage. All TNFR(−/−) mice had significantly less O3-induced inflammation and epithelial damage but not lung hyperpermeability than wt mice. Compared with air-exposed control mice, O3elicited upregulation of lung TNFR1 and TNFR2 mRNAs in wt mice and downregulated TNFR1 and TNFR2 mRNAs in TNFR2(−/−) and TNFR1(−/−) mice, respectively. Airway hyperreactivity induced by acute O3exposure (2 parts/million for 3 h) was diminished in knockout mice compared with that in wtmice, although lung inflammation and permeability remained elevated. Results suggested a critical role for TNFR signaling in subacute O3-induced pulmonary epithelial injury and inflammation and in acute O3-induced airway hyperreactivity.
Reactive oxygen species stimulate VEGF production from C2C12skeletal myotubes through a PI3K/Akt pathway
Authors: Kosmidou Ioanna , Xagorari Angeliki , Roussos Charis , Papapetropoulos Andreas
Published: December 2017

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, the expression of which increases in skeletal muscle after exercise. Because exercise is also accompanied by increased intramuscular reactive oxygen species (ROS) generation, we tested the hypothesis that ROS stimulate VEGF production from skeletal myotubes. Differentiated C2C12skeletal myotubes exposed to ROS-producing agents exhibited a concentration-dependent increase in VEGF production, whereas undifferentiated myoblasts did not respond to oxidants. Moreover, conditioned medium from ROS-treated myotubes increased the bovine lung microvascular cell proliferation rate. To study the mechanism(s) involved in the stimulation of VEGF production by ROS, myotubes were pretreated with a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY-294002, before being exposed to hydrogen peroxide or pyrogallol. LY-294002 attenuated both Akt phosphorylation and VEGF production. In addition, oxidants increased nuclear factor-κB-dependent promoter activity in transiently transfected myotubes; however, pretreatment with the pharmacological inhibitor of nuclear factor-κB, diethyldithiocarbamate, did not affect the oxidant-stimulated VEGF release. We conclude that ROS induce VEGF release from myotubes via a PI3K/Akt-dependent pathway.
Effect of endothelin antagonism on the production of cytokines in eosinophilic airway inflammation
Authors: Finsnes Finn , Lyberg Torstein , Christensen Geir , Skjønsberg Ole H.
Published: December 2017

Endothelin (ET)-1 has been launched as an important mediator in bronchial asthma, which is an eosinophilic airway inflammation. However, the interplay between ET-1 and other proinflammatory mediators during the development of airway inflammation has not been elucidated. We wanted to study 1) whether the production of ET-1 precedes the production of other proinflammatory mediators and 2) whether ET-1 stimulates the production of these mediators within the airways. These hypotheses were studied during the development of an eosinophilic airway inflammation in rats. The increase in ET-1 mRNA level in lung tissue preceded the increase in mRNA levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-8. Treatment of the animals with the ET receptor antagonist bosentan resulted in a substantial decrease in the concentrations of tumor necrosis factor-α, IL-4, IL-1β, interferon-γ, and ET-1 in bronchoalveolar lavage fluid. In conclusion, the synthesis of ET-1 as measured by increased mRNA level precedes the synthesis of other proinflammatory cytokines of importance for the development of an eosinophilic airway inflammation, and ET antagonism inhibits the production of these mediators within the airways. Whether treatment with ET antagonists will prove beneficial for patients with eosinophilic airway inflammations like bronchial asthma is not yet known.
IL-1β enhances β2-adrenergic receptor expression in human airway epithelial cells by activating PKC
Authors: Bin Wei , Aksoy Mark O. , Yang Yi , Kelsen Steven G.
Published: December 2017

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1β upregulates β2-adrenergic receptor (β2-AR) density and β2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1β-induced β-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on β-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on β-AR density, and 3) IL-1β treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1β (0.2 nM for 18 h) increased β-AR density to 213% of control values ( P < 0.001). PMA (1 μM for 18 h) increased β-AR density to 225% of control values ( P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1β-induced upregulation of β-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included α, βII, ε, μ, ζ, and λ/ι. IL-1β increased PKC-μ immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1β-induced β-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-μ isozyme. We conclude that signal transduction pathways activated by PKC-μ upregulate β2-AR expression in human airway epithelial cells.
A murine model of smoke inhalation
Authors: Matthew E. , Warden G. , Dedman J.
Published: December 2017

The United States has one of the world's largest per capita fire death rates. House fires alone kill >9,000 Americans annually, and smoke inhalation is the leading cause of mortality from structural fires. Animal models are needed to develop therapies to combat this problem. We have developed a murine model of smoke inhalation through the design, construction, and use of a controlled-environment smoke chamber. There is a direct relationship between the quantity of wood combusted and mortality in mice. As with human victims, the primary cause of death from smoke inhalation is an elevated blood carboxyhemoglobin level. Lethal (78%) and sublethal (50%) carboxyhemoglobin levels were obtained in mice subjected to varying amounts of smoke. Mice exposed to wood smoke demonstrated more dramatic pathology than mice exposed to cotton or polyurethane smoke. A CD-1 model of wood smoke exposure was developed, demonstrating type II cell hypertrophy, cytoplasmic blebbing, cytoplasmic vacuolization, sloughing, hemorrhage, edema, macrophage infiltration, and lymphocyte infiltration. The bronchoalveolar lavage fluid of smoke-exposed mice demonstrated a significant increase in total cell counts compared with those in control mice. These findings are comparable to the lung tissue response observed in human victims of smoke inhalation.
Nitric oxide decreases lung liquid production via guanosine 3′,5′-cyclic monophosphate
Authors: Cummings James J. , Wang Huamei
Published: December 2017

We studied the role of cGMP in nitric oxide (NO)-induced changes in lung liquid production ( Jv) in chronically instrumented fetal sheep. Forty-five studies were done in which Jvwas measured by a tracer dilution technique. Left pulmonary arterial flow (Qlpa) was measured by a Doppler flow probe. There were two series of experiments. In the first, we gave 8-bromo-cGMP, a cGMP analog, by either the pulmonary vascular or intraluminal route; in the second, we used agents to inhibit or enhance endogenous cGMP activity. When infused directly into the pulmonary circulation, 8-bromo-cGMP significantly increased Qlpabut had no effect on Jv. Conversely, when instilled into the lung liquid, 8-bromo-cGMP had no effect on Qlpabut significantly reduced Jv. Inhibition of guanylate cyclase activity with methylene blue totally blocked, whereas phosphodiesterase inhibition with Zaprinast significantly enhanced, the effect of instilled NO on Jv. Thus the reduction in lung liquid caused by NO appears to be mediated by cGMP, perhaps through a direct effect on the pulmonary epithelium.