bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of endotoxin-induced lung inflammation by interleukin-10 gene transfer in mice
Authors: Dokka Sujatha , Malanga Carl J. , Shi Xianglin , Chen Fei , Castranova Vincent , Rojanasakul Yon
Published: December 2017

Interleukin (IL)-10 is an anti-inflammatory cytokine that has great potential for use in the treatment of inflammatory and immune illnesses. In this study, gene transfer was used to induce IL-10 transgene expression in murine lungs for treatment of endotoxin-induced lung inflammation. Gene transfer was performed with a cytomegalovirus (CMV)-IL-10 plasmid with the aid of the liposomal agents LipofectAMINE and N-[1-(2,3-dioleoyl)propyl]- N, N, N-trimethylammonium methylsulfate (DOTAP). Administration of the endotoxin caused a marked increase in lung inflammation as indicated by increased tumor necrosis factor (TNF)-α release and neutrophil count. Pretreatment of the mice with IL-10 plasmid with and without LipofectAMINE had no inhibitory effect on lung inflammation and IL-10 transgene expression. LipofectAMINE by itself induced lung inflammation, an effect that was not observed with DOTAP. IL-10 plasmid when codelivered with DOTAP expressed biologically active IL-10 protein and caused a reduction in endotoxin-induced inflammation. Transgene expression was observed as early as 3 h after administration, peaked at 12 h, and declined thereafter. We conclude that IL-10 gene transfer is a feasible approach for the treatment of lung inflammation.
L-type Ca2+channels, resting [Ca2+]i, and ET-1-induced responses in chronically hypoxic pulmonary myocytes
Authors: Shimoda Larissa A. , Sham James S. K. , Shimoda Tenille H. , Sylvester J. T.
Published: December 2017

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca2+channels contributes to 1) maintenance of resting intracellular Ca2+concentration ([Ca2+]i), 2) increased [Ca2+]iin response to ET-1 (10−8M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca2+]iin PASMCs from intrapulmonary arteries of rats exposed to 10% O2for 21 days was 293.9 ± 25.2 nM (vs. 153.6 ± 28.7 nM in normoxia). Resting [Ca2+]iwas decreased after extracellular Ca2+removal but not with nifedipine (10−6M), an L-type Ca2+channel antagonist. After CH, the ET-1-induced increase in [Ca2+]iwas reduced and was abolished after extracellular Ca2+removal or nifedipine. Removal of extracellular Ca2+reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca2+]iin PASMCs from chronically hypoxic rats does not require activation of L-type Ca2+channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca2+]i.
Phagocytosis of particulate air pollutants by human alveolar macrophages stimulates the bone marrow
Authors: Mukae Hiroshi , Hogg James C. , English Dean , Vincent Renaud , Van Eeden Stephan F.
Published: December 2017

Epidemiologic studies have shown an association between the level of ambient particulate matter < 10 μm (PM10) and cardiopulmonary mortality. We have shown that exposure of rabbits to PM10stimulates the bone marrow. In this study, we determined whether human alveolar macrophages (AMs) that phagocytose atmospheric PM10produce mediators capable of stimulating the bone marrow. AMs incubated with PM10for 24 h produced tumor necrosis factor-α in a dose-dependent manner (86.8 ± 53.29 pg/ml with medium alone; 1,087.2 ± 257.3 pg/ml with 0.1 mg/ml of PM10; P < 0.02). Instillation of the supernatants from AMs incubated with 0.1 mg/ml of PM10into the lungs of rabbits ( n = 6) increased circulating polymorphonuclear leukocyte (PMN) and band cell counts as well as shortened the PMN transit time through the bone marrow (87.9 ± 3.3 h) compared with unstimulated human AMs (104.9 ± 2.4 h; P < 0.01; n = 5 rabbits). The supernatants from rabbit AMs incubated with 0.1 mg/ml of PM10( n = 4 rabbits) caused a similar shortening in the PMN transit time through the bone marrow (91.5 ± 1.6 h) compared with human AMs. We conclude that mediators released from AMs after phagocytosis of PM10induce a systemic inflammatory response that includes stimulation of the bone marrow.
Surfactant components modulate fibroblast apoptosis and type I collagen and collagenase-1 expression
Authors: De Lara Luis Vázquez , Becerril Carina , Montaño Martha , Ramos Carlos , Maldonado Vilma , Meléndez Jorge , Phelps David S. , Pardo Annie , Selman Moisés
Published: December 2017

During lung injury, fibroblasts migrate into the alveolar spaces where they can be exposed to pulmonary surfactant. We examined the effects of Survanta and surfactant protein A (SP-A) on fibroblast growth and apoptosis and on type I collagen, collagenase-1, and tissue inhibitor of metalloproteinase (TIMP)-1 expression. Lung fibroblasts were treated with 100, 500, and 1,000 μg/ml of Survanta; 10, 50, and 100 μg/ml of SP-A; and 500 μg/ml of Survanta plus 50 μg/ml of SP-A. Growth rate was evaluated by a formazan-based chromogenic assay, apoptosis was evaluated by DNA end labeling and ELISA, and collagen, collagenase-1, and TIMP-1 were evaluated by Northern blotting. Survanta provoked fibroblast apoptosis, induced collagenase-1 expression, and decreased type I collagen affecting mRNA stability ∼10-fold as assessed with the use of actinomycin D. Collagen synthesis and collagenase activity paralleled the gene expression results. SP-A increased collagen expression ∼2-fold and had no effect on collagenase-1, TIMP-1, or growth rate. When fibroblasts were exposed to a combination of Survanta plus SP-A, the effects of Survanta were partially reversed. These findings suggest that surfactant lipids may protect against intraluminal fibrogenesis by inducing fibroblast apoptosis and decreasing collagen accumulation.
TGF-β1 and fibroblast growth factor-1 modify fibroblast growth factor-2 production in type II cells
Authors: Li Cheng-Ming , Khosla Jody , Pagan Ines , Hoyle Paul , Sannes Philip L.
Published: December 2017

Fibroblast growth factor (FGF)-2, which stimulates DNA synthesis by type II cells in the lung, has been shown to be regulated by transforming growth factor (TGF)-β1, an important inflammatory cytokine, in vascular epithelium. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-β1 or FGF-1, which also stimulates DNA synthesis by type II cells. Isolated rat type II cells were exposed to 0–40 ng/ml of TGF-β1 or 0–500 ng/ml of FGF-1 in serum-free medium for 1–5 days. With a specific immunoassay, significant increases of FGF-2 protein in type II cell lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment with 8 ng/ml of TGF-β1. Similarly, transcripts for FGF-2 were dramatically increased above those in control cells with TGF-β1 or FGF-1, as were those for FGF receptor-1. These results demonstrate important regulatory links between FGF-2 and both TGF-β1 and FGF-1 in the alveolar epithelium that could contribute to the regulation of normal cell turnover, development, and the repair processes after injury in the lung.
Modulation of allergic inflammation in mice deficient in TNF receptors
Authors: Rudmann Daniel G. , Moore Mark W. , Tepper Jeffery S. , Aldrich Melinda C. , Pfeiffer Juergen W. , Hogenesch Harm , Tumas Daniel B.
Published: December 2017

Tumor necrosis factor-α (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(−/−) mice] in a murine model of allergic inflammation and found that TNFR(−/−) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(−/−) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(−/−) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(−/−) mouse.
Temporal/spatial expression of nuclear receptor coactivators in the mouse lung
Authors: Naltner Angela , Wert Susan , Whitsett Jeffrey A. , Yan Cong
Published: December 2017

Our laboratory has previously demonstrated that retinoic acid nuclear receptor, thyroid transcription factor-1 (TTF-1), and nuclear receptor coactivators such as cAMP response element binding protein (CREB) binding protein (CBP)/p300 and steroid receptor coactivator-1 (SRC-1) form an enhanceosome on the 5′-enhancer region of the human surfactant protein B gene. Immunohistochemistry was used to identify cells that coexpressed CBP/p300, SRC-1, retinoid X receptor, and TTF-1 in the developing and mature lung. CBP/p300 and SRC-1 were expressed in the adult mouse lung, CBP and p300 being present in both alveolar type I and type II epithelial cells and SRC-1 and TTF-1 being restricted to type II epithelial cells. CBP/p300, SRC-1, and TTF-1 were readily detected in the nuclei of developing respiratory epithelial tubules in fetal mice from embryonic days 10 to 18.CBP/p300 and SRC-1 were also detected in developing mesenchymal cells. These coactivators were coexpressed with TTF-1 and SP-B in human pulmonary adenocarcinoma cells (H441 cells) in vitro. Interaction assays with a two-hybrid reporter analysis demonstrated direct interactions among TTF-1, SRC-1, and CBP/p300 in H441 cells. These findings support a role for retinoic acid receptor and nuclear receptor coactivators in the regulation of SP-B gene expression in the respiratory epithelium.
Neutrophil recruitment and increased permeability during acute lung injury induced by lipopolysaccharide
Authors: Chignard M. , Balloy V.
Published: December 2017

The intranasal administration of lipopolysaccharide (LPS) to mice triggers a huge influx of polymorphonuclear neutrophils (PMNs) into the airway spaces, with a peak at 48 h. The increase in protein concentration, an index of microvascular permeability, displayed a different pattern, i.e., a first increase with a plateau between 3 and 24 h followed by a second increase peaking at 72 h. When mice were depleted of circulating PMNs, the increase in protein concentration was inhibited at 3 h but not at 24 h. The lack of PMN involvement at 24 h was confirmed by 1) in situ activation of exudated PMNs present in the air spaces on intranasal administration of LPS and 2) induction of the migration of PMNs sequestered in lung vessels on intraperitoneal administration of LPS. These findings show that the increase in microvascular permeability during lung inflammation is due to at least two distinct mechanisms, an early one related to the neutrophil influx and a delayed one occurring even under neutropenic conditions.
KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung
Authors: Yano Toshiyuki , Mason Robert J. , Pan Tianli , Deterding Robin R. , Nielsen Larry D. , Shannon John M.
Published: December 2017

Keratinocyte growth factor (KGF, FGF-7) is a potent mitogen for epithelial cells. We instilled recombinant human KGF to determine the effects of KGF on alveolar epithelial cells. Left lungs of adult rats were instilled intrabronchially with KGF (5 mg/kg) or normal saline. KGF instillation resulted in epithelial cell hyperplasia, and the alveolar bromodeoxyuridine (BrdU) labeling index peaked at 35% on day 2 after instillation. The mRNA levels for the surfactant proteins (SPs) SP-A, SP-B, and SP-D were increased in whole lung tissue on days 1 and 2 after KGF treatment and then returned to control levels on days 3–7. SP-C mRNA levels were increased on days 2–5 after KGF instillation. However, all surfactant protein mRNAs were reduced in type II cells isolated from rats instilled with KGF 2 or 3 days before isolation. These observations were confirmed by in situ hybridization. Instillation of KGF also increased the amount of SP-A and SP-D in lavage fluid. Transcripts for CC10, the 10-kDa Clara cell protein, were decreased. KGF increases the mRNA for the surfactant proteins per lung because of type II cell hyperplasia, but the mRNA per cell is slightly diminished as measured in isolated cells or estimated by in situ hybridization.
Telomerase in alveolar epithelial development and repair
Authors: Driscoll Barbara , Buckley Susan , Bui Kim Chi , Anderson Kathryn D. , Warburton David
Published: December 2017

Telomerase expression and activity were examined in the developing lung and in the adult lung during repair after injury. Both whole lung tissue and primary cultures of type 2 alveolar epithelial cells (AEC2) isolated from fetal and adult rodents were analyzed for 1) telomerase expression by immunohistochemistry and 2) telomerase activity with a telomerase repeat amplification protocol. We found that telomerase was expressed in a temporally regulated manner in fetal lung through the late stages of gestation, with peak expression just before birth. Expression persisted for a brief period in neonates, then decreased to nearly undetectable levels by postnatal day 9. Telomerase expression and activity were reinduced in normally quiescent adult lung by in vivo treatment with hyperoxia. In populations of AEC2 isolated from both developing and repairing lungs, telomerase expression and activity showed a strong correlation with the proliferation marker proliferating cell nuclear antigen. It has been suggested that telomerase expression and activity are hallmarks of stem or progenitor cells. Our observations suggest that a telomerase-positive subpopulation is present within the general AEC2 population. Telomerase may act as a marker for the proliferative status of this subpopulation.
Reversibility of increased microvessel permeability in response to VE-cadherin disassembly
Authors: Gao Xiaopei , Kouklis Panos , Xu Ning , Minshall Richard D. , Sandoval Raudel , Vogel Stephen M. , Malik Asrar B.
Published: December 2017

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca2+ chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca2+ to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca2+ chelation and reassembly of the VE-cadherin complex within 5 min after Ca2+ repletion. We determined the125I-labeled albumin permeability-surface area product and capillary filtration coefficient ( K fc) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased 125I-albumin permeability-surface area product and K fc in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca2+. An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K fc induced by Ca2+chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.
Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages
Authors: Shen Han-Ming , Zhang Zhuo , Zhang Qi-Feng , Ong Choon-Nam
Published: December 2017

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.
IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils
Authors: Shim Jae Jeong , Dabbagh Karim , Ueki Iris F. , Dao-Pick Trang , Burgel Pierre-Regis , Takeyama Kiyoshi , Tam Dominic Cheng-Wei , Nadel Jay A.
Published: December 2017

Mucus hypersecretion contributes to the morbidity and mortality in acute asthma. Both T helper 2 (Th2) cytokines and epidermal growth factor receptor (EGFR) signaling have been implicated in allergen-induced goblet cell (GC) metaplasia. Present results show that a cascade of EGFR involving neutrophils is implicated in interleukin (IL)-13-induced mucin expression in GC. Treatment with a selective EGFR tyrosine kinase inhibitor prevented IL-13-induced GC metaplasia dose dependently and completely. Instillation of IL-13 also induced tumor necrosis factor-α protein expression, mainly in infiltrating neutrophils. Control airway epithelium contained few leukocytes, but intratracheal instillation of IL-13 resulted in time-dependent leukocyte recruitment by IL-13-induced IL-8-like chemoattractant expression in airway epithelium. Pretreatment with an inhibitor of leukocytes in the bone marrow (cyclophosphamide) or with a blocking antibody to IL-8 prevented both IL-13-induced leukocyte recruitment and GC metaplasia. These findings indicate that EGFR signaling is involved in IL-13-induced mucin production. They suggest a potential therapeutic role for inhibitors of the EGFR cascade in the hypersecretion that occurs in acute asthma.
Labeling of vagal motoneurons and central afferents after injection of cholera toxin B into the airway lumen
Authors: Pérez Fontán J. Julio , Velloff Christine R.
Published: December 2017

We tested the hypothesis that application of the subunit B of cholera toxin (CTB) to the airway mucosa would produce labeling of neuronal somata and sensory fibers in the medulla oblongata. Using125I-CTB as a tracer, we demonstrated first that CTB is transported across the tracheal epithelium, but once in the airway wall, it remains confined to the subepithelial space and lamina propria. Despite the rarity of intrinsic neurons in these areas, intraluminal CTB labeled ≈10–60 neurons/rat in the nucleus ambiguus and a smaller number of neurons in the dorsal motor nucleus of the vagus. Well-defined sensory fiber terminals were also labeled in the commissural, medial, and ventrolateral subnuclei of the nucleus of the tractus solitarius. Approximately 50 and 90% of the neurons labeled by intraluminal CTB were also labeled by injections of FluoroGold into the tracheal adventitia and lung parenchyma, respectively. These findings demonstrate that a substantial number of medullary vagal motoneurons innervate targets in the vicinity of the airway epithelium. These neurons do not appear to be segregated anatomically from vagal motoneurons that project to deeper layers of the airway wall or lung parenchyma.
Muc1 mucins on the cell surface are adhesion sites forPseudomonas aeruginosa
Authors: Lillehoj E. P. , Hyun S. W. , Kim B. T. , Zhang X. G. , Lee D. I. , Rowland S. , Kim K. C.
Published: December 2017

Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237–244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.
Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14
Authors: Maus Ulrich , Herold Susanne , Muth Heidrun , Maus Regina , Ermert Leander , Ermert Monika , Weissmann Norbert , Rosseau Simone , Seeger Werner , Grimminger Friedrich , Lohmeyer Jürgen
Published: December 2017

The evaluation of monocytes recruited into the alveolar space under both physiological and inflammatory conditions is hampered by difficulties in discriminating these cells from resident alveolar macrophages (rAMs). Using the intravenous injected fluorescent dye PKH26, which accumulated in rAMs without labeling blood leukocytes, we developed a technique that permits the identification, isolation, and functional analysis of monocytes recruited into lung alveoli of mice. Alveolar deposition of murine JE, the homologue of human monocyte chemoattractant protein (MCP)-1 (JE/MCP-1), in mice provoked an alveolar influx of monocytes that were recovered by bronchoalveolar lavage and separated from PKH26-stained rAMs by flow cytometry. Alveolar recruited monocytes showed a blood monocytic phenotype as assessed by cell surface expression of F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. In contrast, CD14 was markedly upregulated on alveolar recruited monocytes together with increased tumor necrosis factor-α message, discriminating this monocyte population from peripheral blood monocytes and rAMs. Thus monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 keep phenotypic features of blood monocytes but upregulate CD14 and are “primed” for enhanced responsiveness to endotoxin with increased cytokine expression.
Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression
Authors: Resta Thomas C. , Kanagy Nancy L. , Walker Benjimen R.
Published: December 2017

Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso- N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17β-estradiol (E2β), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E2β attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E2β. Finally, responses to S-nitroso- N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E2β on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity.
Mechanism of cooperative effects of rhinovirus and atopic sensitization on airway responsiveness
Authors: Grunstein Michael M. , Hakonarson Hakon , Hodinka Richard L. , Maskeri Neil , Kim Cecilia , Chuang Sing
Published: December 2017

To elucidate the mechanistic interplay between rhinovirus (RV) exposure and atopic sensitization in regulating airway smooth muscle (ASM) responsiveness, isolated rabbit ASM tissue and cultured human ASM cells were passively sensitized with sera from atopic asthmatic or nonatopic nonasthmatic (control) subjects in the absence and presence of inoculation with RV serotype 16. Relative to control subjects, atopic asthmatic serum-sensitized and RV-inoculated ASM exhibited significantly increased contractility to acetylcholine, impaired relaxation to isoproterenol, and enhanced release of the proinflammatory cytokine interleukin-1β. These effects were potentiated in atopic asthmatic serum-sensitized ASM concomitantly inoculated with RV and inhibited by pretreating the tissues with monoclonal blocking antibodies against intercellular adhesion molecule (ICAM)-1 (CD54), the host receptor for RV serotype 16, or lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), the endogenous counterreceptor for ICAM-1. Moreover, RV inoculation was found to potentiate the induction of mRNA and surface protein expression of FcεRII (CD23), the low-affinity receptor for IgE, in atopic asthmatic serum-sensitized ASM. Collectively, these observations provide new evidence demonstrating that 1) RV exposure and atopic sensitization act cooperatively to potentiate induction of proasthmatic changes in ASM responsiveness in association with upregulated proinflammatory cytokine release and FcεRII expression and 2) the effects of RV exposure and atopic sensitization are mediated by cooperative ICAM-1-coupled LFA-1 signaling in the ASM itself.
IFN-β mediates coordinate expression of antigen-processing genes in RSV-infected pulmonary epithelial cells
Authors: Jamaluddin Mohammad , Wang Shaofei , Garofalo Roberto P. , Elliott Todd , Casola Antonella , Baron Samuel , Brasier Allan R.
Published: December 2017

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) clear respiratory tract infections caused by the pneumovirus respiratory syncytial virus (RSV) and also mediate vaccine-induced pulmonary injury. Herein we examined the mechanism for RSV-induced MHC class I presentation. Like infectious viruses, conditioned medium from RSV-infected cells (RSV-CM) induces naive cells to coordinately express a gene cluster encoding the transporter associated with antigen presentation 1 (TAP1) and low molecular mass protein (LMP) 2 and LMP7. Neutralization of RSV-CM with antibodies to interferon (IFN)-β largely blocked TAP1/LMP2/LMP7expression, whereas anti-interleukin-1 antibodies were without effect, and recombinant IFN-β increased TAP1/LMP2/LMP7 expression to levels produced by RSV-CM. LMP2, LMP7, and TAP1 expression were required for MHC class I upregulation because the irreversible proteasome inhibitor lactacystin or transfection with a competitive TAP1 inhibitor blocked inducible class I expression. We conclude that RSV infection coordinately increases MHC class I expression and proteasome activity through the paracrine action of IFN-β to induce expression of the TAP1/LMP2/LMP7 locus, an event that may be important in the initiation of CTL-mediated lung injury.
Ion exchange activity in pulmonary artery smooth muscle cells: the response to hypoxia
Authors: Madden Jane A. , Ray Daniel E. , Keller Peter A. , Kleinman Jack G.
Published: December 2017

The purposes of this study were to determine 1) the presence of the major ion transport activities that regulate cytoplasmic pH (pHc) in cat pulmonary artery smooth muscle cells, i.e., Na+/H+ and the Na+-dependent and -independent Cl−/HCO3 − exchange, 2) whether pHc changes in cells from small (SPAs) and large (LPAs) pulmonary arteries during hypoxia, and 3) whether changes in pHc are due to changes in the balance of exchange activities. Exchange activities as defined by physiological maneuvers rather than molecular identity were ascertained with fluorescence microscopy to document changes in the ratio of the pHc indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Steady-state pHc was higher in LPA than in SPA normoxic smooth muscle cells. SPAs and LPAs possessed all three transport activities; in HCO3 −-containing normoxic solutions, Cl−/HCO3 − exchange rather than Na+/H+ exchange set the level of pHc; in HCO3 −-containing hypoxic solutions, pHc increased in SPA and decreased in LPA cells; altering the baseline pHc of a cell type to that of the other did not change the direction of the pHc response during hypoxia. The absence of Na+ prevented hypoxia-induced alkalinization in SPA cells; in both cell types, inhibiting the Cl−/HCO3 − exchange activities reversed the normal direction of pHc changes during hypoxia.
Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersensitivity to substance P
Authors: Kwan Marilyn L. , Gómez Antonio D. , Baluk Peter , Hashizume Hiroya , McDonald Donald M.
Published: December 2017

Angiogenesis and microvascular remodeling are features of chronic airway inflammation caused by Mycoplasma pulmonis infection in rats. As airway blood vessels undergo remodeling, they become unusually sensitive to substance P-induced plasma leakage. Here we determined whether the remodeled vessels are leaky under baseline conditions, whether their heightened sensitivity is specific to substance P, and whether the leakage is reversible. Four weeks after infection, the amount of baseline leakage of Evans blue in the tracheal mucosa was two to five times the normal level. Gaps < 1 μm in diameter were located between endothelial cells in some remodeled vessels. Substance P, but not platelet-activating factor or 5-hydroxytryptamine, produced an exaggerated leakage response. Inhalation of the β2-adrenergic receptor agonist salmeterol reduced the leakage by <60%. We conclude that the blood vessel remodeling after M. pulmonis infection is associated with microvascular leakiness due, in part, to the formation of endothelial gaps. This leakage is accompanied by an abnormal sensitivity to substance P but not to platelet-activating factor or 5-hydroxytryptamine and can be reduced by β2-agonists.
Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis
Authors: Harrington Elizabeth O. , Smeglin Anthony , Newton Julie , Ballard Gajarah , Rounds Sharon
Published: December 2017

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130CAS. No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130CASproteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130CASproteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.
GM-CSF regulates protein and lipid catabolism by alveolar macrophages
Authors: Yoshida Mitsuhiro , Ikegami Machiko , Reed Jacquelyn A. , Chroneos Zissis C. , Whitsett Jeffrey A.
Published: December 2017

Metabolism of surfactant protein (SP) A and dipalmitoylphosphatidylcholine (DPPC) was assessed in alveolar macrophages isolated from granulocyte-macrophage colony-stimulated factor (GM-CSF) gene-targeted [GM(−/−)] mice, wild-type mice, and GM(−/−) mice expressing GM-CSF under control of the SP-C promoter element (SP-C-GM). Although binding and uptake of125I-SP-A were significantly increased in alveolar macrophages from GM(−/−) compared with wild type or SP-C-GM mice, catabolism of125I-SP-A was markedly decreased in GM(−/−) mice. Association of [3H]DPPC with alveolar macrophages from GM(−/−), wild-type, and SP-C-GM mice was similar; however, catabolism of DPPC was markedly reduced in cells from GM(−/−) mice. Fluorescence-activated cell sorter analysis demonstrated decreased catabolism of rhodamine-labeled dipalmitoylphosphatidylethanolamine by alveolar macrophages from GM(−/−) mice. GM-CSF deficiency was associated with increased SP-A uptake by alveolar macrophages but with impaired surfactant lipid and SP-A degradation. These findings demonstrate the important role of GM-CSF in the regulation of alveolar macrophage lipid and SP-A catabolism.
Type 2 rhinovirus infection of cultured human tracheal epithelial cells: role of LDL receptor
Authors: Suzuki Tomoko , Yamaya Mutsuo , Kamanaka Masahito , Jia Yu X. , Nakayama Katsutoshi , Hosoda Masayoshi , Yamada Norihiro , Nishimura Hidekazu , Sekizawa Kiyohisa , Sasaki Hidetada
Published: December 2017

To examine the role of the low-density lipoprotein (LDL) receptor on minor group human rhinovirus (RV) infection, primary cultures of human tracheal epithelial cells were infected with a minor group (RV2) or a major group (RV14) RV. Viral infection was confirmed by showing with PCR that viral titers in supernatants and lysates from infected cells increased with time. RV2 and RV14 increased expression of mRNA and protein of the LDL receptor on the cells and the cytokine production. RV2 induced activation of transcription factors SP1 and nuclear factor-κB (NF-κB). An antibody to the LDL receptor inhibited RV2 infection and RV2-induced cytokine production without an effect on RV14 infection and RV14-induced cytokine production. These findings imply that RV2 upregulates LDL receptor expression on airway epithelial cells, thereby increasing susceptibility to minor group RV infection. LDL receptor expression and cytokine production may be mediated, in part, via activation of transcription factors by RV2. These events may be important in airway inflammation after minor group RV infection in asthma.
Terminal sialylation is altered in airway cells with impaired CFTR-mediated chloride transport
Authors: Kube Dianne , Adams Lynn , Perez Aura , Davis Pamela B.
Published: December 2017

Reduced terminal sialylation at the surface of airway epithelial cells from patients with cystic fibrosis may predispose them to bacterial infection. To determine whether a lack of chloride transport or misprocessing of mutant cystic fibrosis transmembrane conductance regulator (CFTR) is critical for the alterations in glycosylation, we studied a normal human tracheal epithelial cell line (9/HTEo−) transfected with the regulatory (R) domain of CFTR, which blocks CFTR-mediated chloride transport; ΔF508 CFTR, which is misprocessed, wild-type CFTR; or empty vector. Reduced cAMP-stimulated chloride transport is seen in the R domain and ΔF508 transfectants. These two cell lines had consistent, significantly reduced binding of elderberry bark lectin, which recognizes terminal sialic acid in the α-2,6 configuration. Binding of other lectins, including Maakia amurensis lectin, which recognizes sialic acid in the α-2,3 configuration, was comparable in all cell lines. Because the cell surface change occurred in R domain-transfected cells, which continue to express wild-type CFTR, it cannot be related entirely to misprocessed or overexpressed CFTR. It is associated most closely with reduced CFTR activity.
Complement-dependent immune complex-induced bronchial inflammation and hyperreactivity
Authors: Lukacs Nicholas W. , Glovsky M. Michael , Ward Peter A.
Published: December 2017

Bronchoconstriction responses in the airway are caused by multiple insults and are the hallmark symptom in asthma. In an acute lung injury model in mice, IgG immune complex deposition elicited severe airway hyperreactivity that peaked by 1 h, was maintained at 4 h, and was resolved by 24 h. The depletion of complement with cobra venom factor (CVF) markedly reduced the hyperreactive airway responses, suggesting that complement played an important role in the response. Blockade of C5a with specific antisera also significantly reduced airway hyperreactivity in this acute lung model. Complement depletion by CVF treatment significantly reduced tumor necrosis factor and histamine levels in bronchoalveolar lavage fluids, correlating with reductions in airway hyperreactivity. To further examine the role of specific complement requirement, we initiated the immune complex response in C5-sufficient and C5-deficient congenic animals. The airway hyperreactivity response was partially reduced in the C5-deficient mice. Complement depletion with CVF attenuated airway hyperreactivity in the C5-sufficient mice but had a lesser effect on the airway hyperreactive response and histamine release in bronchoalveolar lavage fluids in C5-deficient mice. These data indicate that acute lung injury in mice after deposition of IgG immune complexes induced airway hyperreactivity that is C5 and C5a dependent.
Nitric oxide production in the hypoxic lung
Authors: Le Cras Timothy D. , McMurtry Ivan F.
Published: December 2017

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2to be in the physiological range, it has been suggested that O2substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.
Lysosomal-type PLA2and turnover of alveolar DPPC
Authors: Fisher Aron B. , Dodia Chandra
Published: December 2017

This study evaluated the role of a lysosomal-type phospholipase A2(aiPLA2) in the degradation of internalized dipalmitoylphosphatidylcholine (DPPC) and in phospholipid synthesis by the rat lung. Uptake and degradation of DPPC were measured in isolated perfused rat lungs over 3 h following endotracheal instillation of [3H]DPPC in mixed unilamellar liposomes plus or minus MJ33, a specific inhibitor of lung aiPLA2. Uptake of DPPC was calculated from total tissue-associated radiolabel, and degradation was calculated from the sum of radiolabel in degradation products. Both uptake and degradation were markedly stimulated by addition of 8-bromo-cAMP to the perfusate. MJ33 had no effect on DPPC uptake but decreased DPPC degradation at 3 h by ∼40–50%. The effect of MJ33 on lung synthesis of DPPC was evaluated with intact rats over a 12- to 24-h period following intravenous injection of radiolabeled palmitate and choline. MJ33 treatment decreased palmitate incorporation into disaturated phosphatidylcholine of lamellar bodies and surfactant by ∼65% at 24 h but had no effect on choline incorporation. This result is compatible with inhibition of the deacylation/reacylation pathway for DPPC synthesis. These results obtained with intact rat lungs indicate that aiPLA2is a major enzyme for degradation of internalized DPPC and also has an important role in DPPC synthesis.
Reduction of BCNU toxicity to lung cells by high-level expression ofO6-methylguanine-DNA methyltransferase
Authors: Wu Min , Kelley Mark R. , Hansen W. Kent , Martin William J.
Published: December 2017

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O6position of guanine. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O6position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 μM BCNU had a cell survival rate of 12.5 ± 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 ± 2.7% ( P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.
Glutamine protects mitochondrial structure and function in oxygen toxicity
Authors: Ahmad Shama , White Carl W. , Chang Ling-Yi , Schneider Barbara K. , Allen Corrie B.
Published: December 2017

Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.
Conebulization of surfactant and urokinase restores gas exchange in perfused lungs with alveolar fibrin formation
Authors: Schermuly Ralph Theo , Günther Andreas , Ermert Monika , Ermert Leander , Ghofrani Hossein Ardeschir , Weissmann Norbert , Grimminger Friedrich , Seeger Werner , Walmrath Dieter
Published: December 2017

Alveolar fibrin generation has been suggested to possess strong surfactant-inhibitory potency. In perfused rabbit lungs, fibrin formation in the alveolar space was induced by sequential ultrasonic aerosolization of fibrinogen and thrombin, and the efficacy of rescue administration of surfactant and urokinase was investigated. Ventilation-perfusion (V˙a/Q˙) distribution was assessed by the multiple inert gas elimination technique. Aerosolization of fibrinogen (∼20 mg/kg body wt) increased shunt flow to ∼7%. Sequential nebulization of fibrinogen and thrombin (1.3 U/kg body wt) caused alveolar fibrin deposition, documented immunohistologically, and provoked marked shunt flow, progressing to ∼22% at the end of the experiments. The hemodynamics were virtually unchanged. Rescue aerosolization of natural bovine surfactant (15 mg/kg body wt) or urokinase-type plasminogen activator (4,500 U/kg body wt), undertaken after fibrin formation, improved gas exchange but progressive shunt flow still occurred (efficacy, surfactant > urokinase). In contrast, conebulization of surfactant and urokinase reversed shunt flow to ∼7%, with an increased appearance of normalV˙a/Q˙ matching. We conclude that alveolar fibrin formation is a potent surfactant-inhibitory mechanism in intact lungs, provoking severe V˙a/Q˙ mismatch with a predominance of shunt flow, and that rescue aerosolization of surfactant plus urokinase may offer restoration of gas exchange under these conditions.
Activation of class IA PI3K stimulates DNA synthesis in human airway smooth muscle cells
Authors: Krymskaya Vera P. , Ammit Alaina J. , Hoffman Rebecca K. , Eszterhas Andrew J. , Panettieri Reynold A.
Published: December 2017

The precise mechanisms that regulate increases in airway smooth muscle (ASM) mass in asthma are unknown. This study determined whether class IA phosphatidylinositol 3-kinase (PI3K) is sufficient to stimulate DNA synthesis and characterized the PI3K isoforms expressed in human ASM cells. ASM cells express class IA, II, and III PI3K but not class IB. Because thrombin induces ASM cell proliferation, we investigated whether thrombin can stimulate class IA PI3K. Transient transfection of ASM cells with hemagglutinin-tagged p85 PI3K followed by immunostaining revealed that in quiescent cells, p85 was expressed diffusely in the cytoplasm and after stimulation with thrombin p85 translocated to the cell membrane. Microinjection of ASM cells with a dominant negative class IA PI3K inhibited thrombin-induced DNA synthesis by 30% and epidermal growth factor (EGF)- or serum-induced DNA synthesis by 13 and 28%, respectively ( P < 0.05 by χ2 analysis). In parallel experiments, transfection or microinjection of cells with constitutively active PI3K markedly increased DNA synthesis in transfected cells 10.5-fold and in microinjected cells 12.7-fold ( P < 0.05 by χ2 analysis) compared with cells transfected or microinjected with control plasmid. Interestingly, constitutively active PI3K augmented EGF-induced DNA synthesis but had little effect on that induced by serum or thrombin in ASM cells. Collectively, these data suggest that class IA PI3K is activated by thrombin and is sufficient to induce ASM cell growth.