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Sample records for airway cells role

  1. Role of ROCK2 in CD4(+) cells in allergic airways responses in mice.

    PubMed

    Kasahara, D I; Mathews, J A; Ninin, F M C; Wurmbrand, A P; Liao, J K; Shore, S A

    2017-02-01

    Rho kinases (ROCKs) contribute to allergic airways disease. ROCKs also play a role in lymphocyte proliferation and migration. To determine the role of ROCK2 acting within CD4(+) cells in allergic airways responses. ROCK2-haploinsufficient (ROCK2(+/-) ) and wild-type mice were sensitized with ovalbumin (OVA). ROCK2(+/-) mice then received either CD4(+) cells from ROCK2-sufficient OVA TCR transgenic (OT-II) mice or saline i.v. 48 h before challenge with aerosolized OVA. Wild-type mice received saline before challenge. Allergic airways responses were measured 48 h after the last challenge. Allergic airways responses were also assessed in mice lacking ROCK2 only in CD4(+) cells (ROCK2(CD)(4Cre) mice) vs. control (CD4-Cre and ROCK2(flox/flox) ) mice. OVA-induced increases in bronchoalveolar lavage lymphocytes, eosinophils, IL-13, IL-5, and eotaxin were reduced in ROCK2(+/-) vs. wild-type mice, as were airway hyperresponsiveness and mucous hypersecretion. In ROCK2(+/-) mice, adoptive transfer with CD4(+) cells from OT-II mice restored effects of OVA on lymphocytes, eosinophils, IL-13, IL-5, and mucous hypersecretion to wild-type levels, whereas eotaxin and airway hyperresponsiveness were not affected. ROCK2 inhibitors reduced IL-13-induced release of eotaxin from airway smooth muscle (ASM), similar to effects of these inhibitors on ASM contractility. Despite the ability of adoptive transfer to restore allergic airways inflammation in ROCK2-insufficient mice, allergic inflammation was not different in ROCK2(CD)(4Cre) vs. control mice. ROCK2 contributes to allergic airways responses likely via effects within ASM cells and within non-lymphocyte cells involved in lymphocyte activation and migration into the airways. © 2016 John Wiley & Sons Ltd.

  2. New Role of Adult Lung c-kit+ Cells in a Mouse Model of Airway Hyperresponsiveness

    PubMed Central

    Cappetta, Donato; Urbanek, Konrad; Esposito, Grazia; Matteis, Maria; Sgambato, Manuela; Tartaglione, Gioia; Rossi, Francesco

    2016-01-01

    Structural changes contribute to airway hyperresponsiveness and airflow obstruction in asthma. Emerging evidence points to the involvement of c-kit+ cells in lung homeostasis, although their potential role in asthma is unknown. Our aim was to isolate c-kit+ cells from normal mouse lungs and to test whether these cells can interfere with hallmarks of asthma in an animal model. Adult mouse GFP-tagged c-kit+ cells, intratracheally delivered in the ovalbumin-induced airway hyperresponsiveness, positively affected airway remodeling and improved airway function. In bronchoalveolar lavage fluid of cell-treated animals, a reduction in the number of inflammatory cells and in IL-4, IL-5, and IL-13 release, along with an increase of IL-10, was observed. In MSC-treated mice, the macrophage polarization to M2-like subset may explain, at least in part, the increment in the level of anti-inflammatory cytokine IL-10. After in vitro stimulation of c-kit+ cells with proinflammatory cytokines, the indoleamine 2,3-dioxygenase and TGFβ were upregulated. These data, together with the increased apoptosis of inflammatory cells in vivo, indicate that c-kit+ cells downregulate immune response in asthma by influencing local environment, possibly by cell-to-cell contact combined to paracrine action. In conclusion, intratracheally administered c-kit+ cells reduce inflammation, positively modulate airway remodeling, and improve function. These data document previously unrecognized properties of c-kit+ cells, able to impede pathophysiological features of experimental airway hyperresponsiveness. PMID:28090152

  3. Pro-inflammatory role of natural killer cells in the development of allergic airway disease.

    PubMed

    Mathias, C B; Guernsey, L A; Zammit, D; Brammer, C; Wu, C A; Thrall, R S; Aguila, H L

    2014-04-01

    Natural Killer (NK) cells have been implicated in the development of allergic airway inflammation. However, the in vivo role of NK cells has not been firmly established due to the lack of animal models with selective deficiencies in NK cells. To determine the specific contribution of NK cells in a murine model of allergic airway disease (AAD). The role of NK cells in AAD was studied using NK-deficient (NKD) mice, perforin(-/-) mice, and mice depleted of Ly49A/D/G(+) NK cell subsets in an ovalbumin-induced model of allergic airway disease (OVA-AAD). Induction of OVA-AAD in C57BL/6 wild-type (WT) mice resulted in the expansion of airway NK cells and the development of pronounced airway eosinophilia. In the absence of NK cells or specific subsets of NK cells, either in NKD mice, or after the depletion of Ly49A/D/G(+) NK cells, the development of OVA-AAD was significantly impaired as seen by decreased airway inflammation and eosinophilia, decreased secretion of the Th2 cytokines IL-4, IL-5 and IL-13 and diminished OVA-specific antibody production. Furthermore, while OVA-exposure induced a dramatic expansion of dendritic cells (DCs) in WT mice, their induction was significantly attenuated in NKD mice. Development of OVA-AAD in perforin(-/-) mice suggested that the proinflammatory role of NK cells is not dependent on perforin-mediated cytotoxicity. Lastly, induction of allergic disease by OVA-specific CD4 T cells from WT but not NK-depleted or NKD mice in RAG(-/-) recipients, demonstrates that NK cells are essential for T cell priming. Our data demonstrate that conventional NK cells play an important and distinct role in the development of AAD. The presence of activated NK cells has been noted in patients with asthma. Understanding the mechanisms by which NK cells regulate allergic disease is therefore an important component of treatment approaches. © 2014 John Wiley & Sons Ltd.

  4. Contrasting roles for the receptor for advanced glycation end-products on structural cells in allergic airway inflammation vs. airway hyperresponsiveness.

    PubMed

    Taniguchi, Akihiko; Miyahara, Nobuaki; Waseda, Koichi; Kurimoto, Etsuko; Fujii, Utako; Tanimoto, Yasushi; Kataoka, Mikio; Yamamoto, Yasuhiko; Gelfand, Erwin W; Yamamoto, Hiroshi; Tanimoto, Mitsune; Kanehiro, Arihiko

    2015-10-15

    The receptor for advanced glycation end-products (RAGE) is a multiligand receptor that belongs to the immunoglobulin superfamily. RAGE is reported to be involved in various inflammatory disorders; however, studies that address the role of RAGE in allergic airway disease are inconclusive. RAGE-sufficient (RAGE+/+) and RAGE-deficient (RAGE-/-) mice were sensitized to ovalbumin, and airway responses were monitored after ovalbumin challenge. RAGE-/- mice showed reduced eosinophilic inflammation and goblet cell metaplasia, lower T helper type 2 (Th2) cytokine production from spleen and peribronchial lymph node mononuclear cells, and lower numbers of group 2 innate lymphoid cells in the lung compared with RAGE+/+ mice following sensitization and challenge. Experiments using irradiated, chimeric mice showed that the mice expressing RAGE on radio-resistant structural cells but not hematopoietic cells developed allergic airway inflammation; however, the mice expressing RAGE on hematopoietic cells but not structural cells showed reduced airway inflammation. In contrast, absence of RAGE expression on structural cells enhanced innate airway hyperresponsiveness (AHR). In the absence of RAGE, increased interleukin (IL)-33 levels in the lung were detected, and blockade of IL-33 receptor ST2 suppressed innate AHR in RAGE-/- mice. These data identify the importance of RAGE expressed on lung structural cells in the development of allergic airway inflammation, T helper type 2 cell activation, and group 2 innate lymphoid cell accumulation in the airways. RAGE on lung structural cells also regulated innate AHR, likely through the IL-33-ST2 pathway. Thus manipulating RAGE represents a novel therapeutic target in controlling allergic airway responses. Copyright © 2015 the American Physiological Society.

  5. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia and airway mucus formation in vitro and in vivo

    PubMed Central

    Gundavarapu, Sravanthi; Wilder, Julie A.; Mishra, Neerad C.; Rir-sima-ah, Jules; Langley, Raymond J.; Singh, Shashi P.; Saeed, Ali Imran; Jaramillo, Richard J.; Gott, Katherine M.; Peña-Philippides, Juan Carlos; Harrod, Kevin S.; McIntosh, J. Michael; Buch, Shilpa; Sopori, Mohan L.

    2012-01-01

    Background Airway mucus hypersecretion is a key pathophysiological feature in number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. Objectives Characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. Methods IL-13 and gamma-aminobutyric acid receptors (GABAARs) are implicated in airway mucus. We examined the role of IL-13 and GABAARs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells, and secondhand cigarette smoke and/or ovalbumin-induced mucus formation in vivo. Results Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABAAR antagonist picrotoxin (PIC). Airway epithelial cells express α7/α9/α10 nicotinic acetylcholine receptors (nAChRs) and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells and/or in mouse airways. Conclusions Nicotine-induced airway mucus formation is independent of IL-13 and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various pro-mucoid agents, including IL-13. In the absence of nicotine, acetylcholine may be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABAARα2 in the MUC5AC pathway. Acetylcholine and α-7-nAChRs may serve as therapeutic targets to control airway mucus. PMID:22578901

  6. Type 2 innate lymphoid cells: friends or foes-role in airway allergic inflammation and asthma.

    PubMed

    Pishdadian, Abbas; Varasteh, Abdol-Reza; Sankian, Mojtaba

    2012-01-01

    Innate-like lymphocytes (ILLs) and innate lymphoid cells (ILCs) are two newly characterized families of lymphocytes with limited and no rearranged antigen receptors, respectively. These soldiers provide a first line of defense against foreign insults by triggering a prompt innate immune response and bridging the gap of innate and adaptive immunity. Type 2 innate lymphoid cells (ILCs2) are newly identified members of the ILC family that play a key role in type 2 immune responses by prompt production of type 2 cytokines (especially IL-5 and IL-13) in response to antigen-induced IL-25/33 and by recruiting type 2 "immune franchise." Regarding the two different roles of type 2 cytokines, helminth expulsion and type 2-related diseases, here we review the latest advances in ILC2 biology and examine the pivotal role of resident ILCs2 in allergen-specific airway inflammation and asthma.

  7. Role of Allergen Source-Derived Proteases in Sensitization via Airway Epithelial Cells

    PubMed Central

    Matsumura, Yasuhiro

    2012-01-01

    Protease activity is a characteristic common to many allergens. Allergen source-derived proteases interact with lung epithelial cells, which are now thought to play vital roles in both innate and adaptive immune responses. Allergen source-derived proteases act on airway epithelial cells to induce disruption of the tight junctions between epithelial cells, activation of protease-activated receptor-2, and the production of thymic stromal lymphopoietin. These facilitate allergen delivery across epithelial layers and enhance allergenicity or directly activate the immune system through a nonallergic mechanism. Furthermore, they cleave regulatory cell surface molecules involved in allergic reactions. Thus, allergen source-derived proteases are a potentially critical factor in the development of allergic sensitization and appear to be strongly associated with heightened allergenicity. PMID:22523502

  8. Pivotal role of c-Fos in nitric oxide synthase 2 expression in airway epithelial cells

    PubMed Central

    Chambellan, Arnaud; Leahy, Rachel; Xu, Weiling; Cruickshank, Paul J.; Janocha, Allison; Szabo, Katalin; Cannady, Steven B.; Comhair, Suzy A.A.; Erzurum, Serpil C.

    2010-01-01

    The regulation of nitric oxide synthase 2 (NOS2) in airway epithelial cells plays a key role in the innate host response to a wide variety of microbial agents and also participates in the generation of pathologic airway inflammation. Among the important signalling cascades that direct NOS2 gene expression are nuclear factor κB (NFκB) and interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (STAT-1). Previous studies suggest activator protein-1 (AP-1), in particular c-Fos component of AP-1, influences NOS2 expression. We investigated the effect of c-Fos modulation using RNA interference siRNA on NOS2 gene expression. A549 cells stably transfected with a plasmid overexpressing a c-Fos siRNA construct (FOSi) resulted in a decrease of NOS2 protein inducibility by IFN γ. In contrast, classical IFN γ inducible signal transduction pathways interferon regulated factor-1 (IRF-1) and pSTAT-1 were activated at a similar magnitude in FOSi and control cells. DNA–protein binding assays showed that c-Fos binding was present in wild type cells, but reduced in FOSi clones. FOSi clones had activation of NFκB detectable by DNA–protein binding assays, which may have contributed to a decrease of NOS2 expression. Overall, these studies indicate that c-Fos is a requisite and specific component for inducible NOS2 expression. PMID:19135542

  9. B cells play key roles in th2-type airway immune responses in mice exposed to natural airborne allergens.

    PubMed

    Drake, Li Yin; Iijima, Koji; Hara, Kenichiro; Kobayashi, Takao; Kephart, Gail M; Kita, Hirohito

    2015-01-01

    Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH-/- mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH-/- mice. The allergen-induced expansion of CD4+ T cells was impaired in the lungs and draining lymph nodes of JH-/- mice. Furthermore, lymphocytes from JH-/- mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.

  10. Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

    PubMed

    Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

    2005-09-01

    Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

  11. Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

    PubMed

    Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

    2005-01-01

    Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

  12. Role of non-coding RNAs in maintaining primary airway smooth muscle cells

    PubMed Central

    2014-01-01

    Background The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases. Objective We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS). Methods mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR. Results A small number of miRNAs (including miR-150, −371-5p, −718, −940, −1181, −1207-5p, −1915, and −3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA ‘sponges’ for 4 miRNAs (miR-150, −371-5p, −940, −1207-5p). Conclusion This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). PMID:24886442

  13. A critical role for dendritic cells in the evolution of IL-1β-mediated murine airway disease

    PubMed Central

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Goodsell, Amanda; Ma, Royce; Moermans, Catherine; McKnelly, Kate J.; Baron, Jody L.; Krummel, Matthew F.; Nishimura, Stephen L.

    2015-01-01

    Chronic airway inflammation and fibrosis, known as airway remodeling, are defining features of chronic obstructive pulmonary disease (COPD) and are refractory to current treatments. How and if chronic inflammation contributes to airway fibrosis remains controversial. Here, we use a model of COPD airway disease utilizing adenoviral (Ad) delivery of IL-1β to determine that adaptive T-cell immunity is required for airway remodeling since mice deficient in α/β T-cells (tcra −/−) are protected. Dendritic cells (DCs) accumulate around COPD airways and are critical to prime adaptive immunity, but have not been shown to directly influence airway remodeling. We show that DC depletion or deficiency in the crucial DC chemokine receptor, ccr6, both protect from Ad-IL-1β-induced airway adaptive T-cell immune responses, and fibrosis in mice. These results provide evidence that chronic airway inflammation, mediated by accumulation of α/β T-cells and driven by DCs, is critical to airway fibrosis. PMID:25786688

  14. A critical role for dendritic cells in the evolution of IL-1β-mediated murine airway disease.

    PubMed

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Goodsell, Amanda; Ma, Royce; Moermans, Catherine; McKnelly, Kate J; Baron, Jody L; Krummel, Matthew F; Nishimura, Stephen L

    2015-04-15

    Chronic airway inflammation and fibrosis, known as airway remodeling, are defining features of chronic obstructive pulmonary disease and are refractory to current treatments. How and whether chronic inflammation contributes to airway fibrosis remain controversial. In this study, we use a model of chronic obstructive pulmonary disease airway disease utilizing adenoviral delivery of IL-1β to determine that adaptive T cell immunity is required for airway remodeling because mice deficient in α/β T cells (tcra(-/-)) are protected. Dendritic cells (DCs) accumulate around chronic obstructive pulmonary disease airways and are critical to prime adaptive immunity, but they have not been shown to directly influence airway remodeling. We show that DC depletion or deficiency in the crucial DC chemokine receptor ccr6 both protect from adenoviral IL-1β-induced airway adaptive T cell immune responses and fibrosis in mice. These results provide evidence that chronic airway inflammation, mediated by accumulation of α/β T cells and driven by DCs, is critical to airway fibrosis. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. [Function and modulation of type Ⅱ innate lymphoid cells and their role in chronic upper airway inflammatory diseases].

    PubMed

    Liu, Y; Liu, Z

    2017-02-07

    Type Ⅱ innate lymphoid cells (ILC2) is a family of innate immune lymphocytes, which provide effective immune responses to cytokines. ILC2 are regulated by the nuclear transcription factor ROR alpha and GATA3, secreting cytokines IL-5 and IL-13, etc. Animal models have shown that ILC2 are involved in allergic diseases, such as asthma and atopic dermatitis, and also play a very important role in the metabolic balance. In addition, recent reports suggest that ILC2 not only play a role in the initial stages of the disease, but also can lead to chronic pathological changes in the disease, such as fibrosis, and may have an effect on acquired immunity. This paper mainly focus in the role and regulation of ILC2 cells, and review the research status of ILC2 in the field of chronic upper airway inflammatory diseases including allergic rhinitis and chronic rhinosinusitis.

  16. A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

    PubMed Central

    Liang, Lihua; Woodward, Owen M.; Chen, Zhaohui; Cotter, Robert; Guggino, William B.

    2011-01-01

    Ca2+ activated Cl− channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl− secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca2+ and Cl− secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells. PMID:21765932

  17. Role of γδ T cells in exacerbated airway inflammation during reinfection of neonatally primed mice in adulthood.

    PubMed

    Wu, Jianqi; Xu, Lei; Han, Xu; Hu, Haiyan; Qi, Feifei; Bai, Song; Chai, Ruonan; Teng, Yuee; Liu, Beixing

    2017-08-16

    Age at primary infection with respiratory syncytial virus (RSV) is a crucial factor in determining the outcome of reinfection. However, how neonatal RSV infection affects the immune system and renders the host more susceptible to reinfection in later life is poorly understood. In the present study, by using BALB/c mice that were first infected with RSV as neonates, the role of γδ T cells in the development of airway inflammation during reinfection in adulthood was investigated. We found that neonatal RSV infection resulted in an aggravated infiltration of mononuclear cells in bronchoalveolar lavage (BAL) fluids, in parallel with a significant increase in the levels of type 2 cytokines in lungs on day 4 after reinfection. Since the numbers of total γδ T cells as well as activated γδ T cells, particularly IL-4-, IL-5-, and IL-13-producing γδ T cells, were enhanced markedly in the lungs of neonatally primed mice, we speculate that γδ T cells might participate in the augmented airway inflammation seen during reinfection. Indeed, depletion of γδ T cells attenuated the severity of lung histopathology during reinfection. Meanwhile, treatment of neonatal mice with anti-TCRδ mAb diminished not only the numbers of neutrophils, eosinophils, and lymphocytes, but also the levels of IL-4, IL-5, and IL-13 in the lungs after reinfection in adulthood, suggesting that γδ T cells, particularly Th2-type γδ T cells might play a critical role in exacerbating the pulmonary tissue pathology during reinfection of adult mice that were first infected as neonates. © 2017 Wiley Periodicals, Inc.

  18. Eosinophils induce airway smooth muscle cell proliferation.

    PubMed

    Halwani, Rabih; Vazquez-Tello, Alejandro; Sumi, Yuki; Pureza, Mary Angeline; Bahammam, Ahmed; Al-Jahdali, Hamdan; Soussi-Gounni, Abdelillah; Mahboub, Bassam; Al-Muhsen, Saleh; Hamid, Qutayba

    2013-04-01

    Asthma is characterized by eosinophilic airway inflammation and remodeling of the airway wall. Features of airway remodeling include increased airway smooth muscle (ASM) mass. However, little is known about the interaction between inflammatory eosinophils and ASM cells. In this study, we investigated the effect of eosinophils on ASM cell proliferation. Eosinophils were isolated from peripheral blood of mild asthmatics and non-asthmatic subjects and co-cultured with human primary ASM cells. ASM proliferation was estimated using Ki-67 expression assay. The expression of extracellular matrix (ECM) mRNA in ASM cells was measured using quantitative real-time PCR. The role of eosinophil derived Cysteinyl Leukotrienes (CysLTs) in enhancing ASM proliferation was estimated by measuring the release of leukotrienes from eosinophils upon their direct contact with ASM cells using ELISA. This role was confirmed either by blocking eosinophil-ASM contact or co-culturing them in the presence of leukotrienes antagonist. ASM cells co-cultured with eosinophils, isolated from asthmatics, but not non-asthmatics, had a significantly higher rate of proliferation compared to controls. This increase in ASM proliferation was independent of their release of ECM proteins but dependent upon eosinophils release of CysLTs. Eosinophil-ASM cell to cell contact was required for CysLTs release. Preventing eosinophil contact with ASM cells using anti-adhesion molecules antibodies, or blocking the activity of eosinophil derived CysLTs using montelukast inhibited ASM proliferation. Our results indicated that eosinophils contribute to airway remodeling during asthma by enhancing ASM cell proliferation and hence increasing ASM mass. Direct contact of eosinophils with ASM cells triggers their release of CysLTs which enhance ASM proliferation. Eosinophils, and their binding to ASM cells, constitute a potential therapeutic target to interfere with the series of biological events leading to airway remodeling

  19. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

    PubMed

    Gundavarapu, Sravanthi; Wilder, Julie A; Mishra, Neerad C; Rir-Sima-Ah, Jules; Langley, Raymond J; Singh, Shashi P; Saeed, Ali Imran; Jaramillo, Richard J; Gott, Katherine M; Peña-Philippides, Juan Carlos; Harrod, Kevin S; McIntosh, J Michael; Buch, Shilpa; Sopori, Mohan L

    2012-09-01

    Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo. Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both. Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  20. Airway epithelial cells: current concepts and challenges.

    PubMed

    Crystal, Ronald G; Randell, Scott H; Engelhardt, John F; Voynow, Judith; Sunday, Mary E

    2008-09-15

    The adult human bronchial tree is covered with a continuous layer of epithelial cells that play a critical role in maintaining the conduit for air, and which are central to the defenses of the lung against inhaled environmental concomitants. The epithelial sheet functions as an interdependent unit with the other lung components. Importantly, the structure and/or function of airway epithelium is deranged in major lung disorders, including chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Investigations regarding the airway epithelium have led to many advances over the past few decades, but new developments in genetics and stem cell/progenitor cell biology have opened the door to understanding how the airway epithelium is developed and maintained, and how it responds to environmental stress. This article provides an overview of the current state of knowledge regarding airway epithelial stem/progenitor cells, gene expression, cell-cell interactions, and less frequent cell types, and discusses the challenges for future areas of investigation regarding the airway epithelium in health and disease.

  1. The human airway epithelial basal cell transcriptome.

    PubMed

    Hackett, Neil R; Shaykhiev, Renat; Walters, Matthew S; Wang, Rui; Zwick, Rachel K; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G

    2011-05-04

    The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.

  2. The Human Airway Epithelial Basal Cell Transcriptome

    PubMed Central

    Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

    2011-01-01

    Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

  3. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  4. Mast Cell-Airway Smooth Muscle Crosstalk

    PubMed Central

    Kaur, Davinder; Doe, Camille; Woodman, Lucy; Heidi Wan, Wing-Yan; Sutcliffe, Amanda; Hollins, Fay

    2012-01-01

    Background: The mast cell localization to airway smooth muscle (ASM) bundle in asthma is important in the development of disordered airway physiology. Thymic stromal lymphopoietin (TSLP) is expressed by airway structural cells. Whether it has a role in the crosstalk between these cells is uncertain. We sought to define TSLP expression in bronchial tissue across the spectrum of asthma severity and to investigate the TSLP and TSLP receptor (TSLPR) expression and function by primary ASM and mast cells alone and in coculture. Methods: TSLP expression was assessed in bronchial tissue from 18 subjects with mild to moderate asthma, 12 with severe disease, and nine healthy control subjects. TSLP and TSLPR expression in primary mast cells and ASM was assessed by immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assay, and its function was assessed by calcium imaging. The role of TSLP in mast cell and ASM proliferation, survival, differentiation, synthetic function, and contraction was examined. Results: TSLP expression was increased in the ASM bundle in mild-moderate disease. TSLP and TSLPR were expressed by mast cells and ASM and were functional. Mast cell activation by TSLP increased the production of a broad range of chemokines and cytokines, but did not affect mast cell or ASM proliferation, survival, or contraction. Conclusions: TSLP expression by the bronchial epithelium and ASM was upregulated in asthma. TSLP promoted mast cell synthetic function, but did not contribute to other functional consequences of mast cell-ASM crosstalk. PMID:22052771

  5. Leukocytic cell sources of airway tissue kallikrein

    PubMed Central

    Lauredo, Isabel T.; Forteza, Rosanna M.; Botvinnikova, Yelena; Abraham, William M.

    2008-01-01

    Lung tissue kallikrein (TK) is a serine proteinase that putatively plays a role in the pathophysiology of asthma by generating kallidin and bradykinin, mediators that contribute to airway hyperresponsiveness. In previous studies we observed biphasic increases in TK activity in bronchoalveolar lavage fluid following airway allergen challenge in allergic sheep. Although glandular TK is likely a major source of the initial increase in TK, the sources of the late increases in TK that are associated with the development of airway hyperresponsiveness may be dependent on activated resident and recruited inflammatory cells including alveolar macrophages (AMs) and neutrophils (PMNs). These cells increase concomitantly with the late increases in TK activity. To test this hypothesis, we obtained AMs from bronchoalveolar lavage fluid and PMNs and monocytes (precursors of AMs) from sheep blood and determined whether these cells contained TK and whether these same cells could release TK upon activation. Using confocal microscopy, immunocytochemical techniques, and enzyme activity assays, we found that all three cell types contained and secreted TK. All three cell types demonstrated basal release of TK, which could be increased after stimulation with zymosan. In addition, PMNs also released TK in the presence of phorbol ester, suggesting multiple secretory pathways in these cells. Further-more, we showed that human monocytes also contain and secrete TK. We conclude that in the airways, monocytes, PMNs, and AMs may contribute to increased TK activity. Knowing the sources of TK in the airways could be important in understanding the mechanisms of inflammation that contribute to the pathophysiology of asthma and may help in the development of new therapies to control the disease. PMID:14660481

  6. A role for CCL28-CCR3 in T-cell homing to the human upper airway mucosa.

    PubMed

    Danilova, E; Skrindo, I; Gran, E; Hales, B J; Smith, W A; Jahnsen, J; Johansen, F E; Jahnsen, F L; Baekkevold, E S

    2015-01-01

    Lymphocyte recruitment to peripheral tissues is fundamental for immune surveillance and homeostasis, but the chemokines and chemokine receptors responsible for tissue-specific homing of T cells to the upper airway mucosa have not been determined. To address this, we analyzed the chemokines expressed in the normal human nasal mucosa and found that CCL28 is preferentially expressed at a high level on the lumenal face of vascular endothelial cells in the mucosa. Analysis of the cognate chemokine receptors revealed that close to 50% of the CD4(+) T cells in the human nasal mucosa expressed the CCL28 receptor CCR3, whereas CCR3 was hardly detectable on T cells in the small intestine and skin. In the circulation, CCR3(+) T cells comprised a small subset that did not express homing receptors to the intestine or skin. Moreover, depletion of CCR3(+)CD4(+) T cells abrogated the proliferative response of human blood CD4(+) T cells against the opportunistic nasopharyngeal pathogen Haemophilus influenzae, indicating that the CCR3(+)CD4(+) T-cell subset in the circulation contains antigen specificities relevant for the upper airways. Together, these findings indicate that CCL28-CCR3 interactions are involved in the homeostatic trafficking of CD4(+) T cells to the upper airways.

  7. Adalimumab ameliorates OVA-induced airway inflammation in mice: Role of CD4(+) CD25(+) FOXP3(+) regulatory T-cells.

    PubMed

    Elsakkar, Mohamed G; Sharaki, Olla A; Abdallah, Dina M; Mostafa, Dalia K; Shekondali, Fadia T

    2016-09-05

    Asthma is a chronic inflammatory heterogeneous disorder initiated by a dysregulated immune response which drives disease development in susceptible individuals. Though T helper 2 (TH2) biased responses are usually linked to eosinophilic asthma, other Th cell subsets induce neutrophilic airway inflammation which provokes the most severe asthmatic phenotypes. A growing evidence highlights the role of T regulatory (Treg) cells in damping abnormal Th responses and thus inhibiting allergy and asthma. Therefore, strategies to induce or augment Treg cells hold promise for treatment and prevention of allergic airway inflammation. Recently, the link between Tumor necrosis factor-α (TNF-α) and Treg has been uncovered, and TNF-α antagonists are increasingly used in many autoimmune diseases. Yet, their benefits in allergic airway inflammation is not clarified. We investigated the effect of Adalimumab, a TNF-α antagonist, on Ovalbumin (OVA)-induced allergic airway inflammation in CD1 mice and explored its impact on Treg cells. Our results showed that Adalimumab treatment attenuated the OVA-induced increase in serum IgE, TH2 and TH1 derived inflammatory cytokines (IL-4 and IFN-γ, respectively) in bronchoalveolar lavage (BAL) fluid, suppressed recruitment of inflammatory cells in BAL fluid and lung, and inhibited BAL fluid neutrophilia. It also ameliorated goblet cell metaplasia and bronchial fibrosis. Splenocytes flow cytometry revealed increased percentage of CD4(+) CD25(+) FOXP3(+) Treg cells by Adalimumab that was associated with increase in their suppressive activity as shown by elevated BAL fluid IL-10. We conclude that the beneficial effects of Adalimumab in this CD1 neutrophilic model of allergic airway inflammation are attributed to augmentation of Treg cell number and activity.

  8. A possible role for CD8+ and non-CD8+ cell granzyme B in early small airway wall remodelling in centrilobular emphysema.

    PubMed

    Kim, Won-Dong; Chi, Hyun-Sook; Choe, Kang-Hyeon; Oh, Yeon-Mok; Lee, Sang-Do; Kim, Kyu-Rae; Yoo, Kwang-Ha; Ngan, David A; Elliott, W Mark; Granville, David J; Sin, Don D; Hogg, James C

    2013-05-01

    CD8(+) cell infiltration and apoptosis of airway epithelial cells are increased in chronic obstructive pulmonary disease (COPD). CD8(+) T cells induce apoptosis by releasing granzymes, which can also cause extracellular matrix degradation and remodelling. Granzyme B levels and T cells expressing granzyme B are increased in bronchoalveolar lavage fluid of COPD patients, which suggests that granzyme B may contribute to the pathogenesis of COPD. This study provides quantitation of granzyme B-positive cells in relation to CD8(+) cells in the small airway walls of emphysema. Antibodies against CD8 and granzyme B were used to identify CD8(+) and granzyme B(+) cells. Volume fraction (Vv) of CD8(+) and granzyme B(+) cells were quantitated by the point counting method in the small airways of 13 non-smoker control subjects and 46 emphysema patients (14 panlobular emphysema (PLE) and 32 centrilobular emphysema (CLE) lungs). Immunohistochemical detection of macrophage scavenger receptor was also performed in randomly selected cases. The Vv of CD8(+) and granzyme B(+) cells in CLE was greater than those in control and PLE (both P < 0.001) subjects. The Vv of granzyme B(+) cells was greater than that of CD8(+) cells (P = 0.006), and not all CD8(+) cells were positive for granzyme B in CLE subjects. Monocytes expressing both granzyme B and macrophage scavenger receptor and granulocytes expressing granzyme B were identified. Upregulation of granzyme B in CD8(+) and non-CD8(+) cells is an early phenomenon of small airway wall remodelling in centrilobular emphysema and suggests its possible role in the pathogenesis of COPD. © 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.

  9. Mast cells play an important role in Chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway

    PubMed Central

    Chiba, Norika; Shimada, Kenichi; Chen, Shuang; Jones, Heather D.; Alsabeh, Randa; Slepenkin, Anatoly V.; Peterson, Ellena; Crother, Timothy R.; Arditi, Moshe

    2015-01-01

    Mast cells are known as central players in allergy and anaphylaxis, and play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae (Cpn) is an important human pathogen, but it is unclear what role mast cells play during Cpn infection. We infected C57BL/6 (WT) and mast cell-deficient mice, Kitw-sh/w-sh (Wsh), with Cpn. Wsh mice showed improved survival than WT, with fewer cells in Wsh BALF despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT. Cromolyn, a mast cell stabilizer, reduced BAL cells and bacterial burden similar to Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BAL cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation while Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of MMP-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during Cpn infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for Cpn. PMID:25754739

  10. Responses of well-differentiated nasal epithelial cells exposed to particles: Role of the epithelium in airway inflammation

    SciTech Connect

    Auger, Floriane; Gendron, Marie-Claude; Chamot, Christophe; Marano, Francelyne; Dazy, Anne-Catherine . E-mail: dazy@paris7.jussieu.fr

    2006-09-15

    Numerous epidemiological studies support the contention that ambient air pollution particles can adversely affect human health. To explain the acute inflammatory process in airways exposed to particles, a number of in vitro studies have been performed on cells grown submerged on plastic and poorly differentiated, and on cell lines, the physiology of which is somewhat different from that of well-differentiated cells. In order to obtain results using a model system in which epithelial cells are similar to those of the human airway in vivo, apical membranes of well-differentiated human nasal epithelial (HNE) cells cultured in an air-liquid interface (ALI) were exposed for 24 h to diesel exhaust particles (DEP) and Paris urban air particles (PM{sub 2.5}). DEP and PM{sub 2.5} (10-80 {mu}g/cm{sup 2}) stimulated both IL-8 and amphiregulin (ligand of EGFR) secretion exclusively towards the basal compartment. In contrast, there was no IL-1{beta} secretion and only weak non-reproducible secretion of TNF-{alpha}. IL-6 and GM-CSF were consistently stimulated towards the apical compartment and only when cells were exposed to PM{sub 2.5}. ICAM-1 protein expression on cell surfaces remained low after particle exposure, although it increased after TNF-{alpha} treatment. Internalization of particles, which is believed to initiate oxidative stress and proinflammatory cytokine expression, was restricted to small nanoparticles ({<=} 40 nm). Production of reactive oxygen species (ROS) was detected, and DEP were more efficient than PM{sub 2.5}. Collectively, our results suggest that airway epithelial cells exposed to particles augment the local inflammatory response in the lung but cannot alone initiate a systemic inflammatory response.

  11. Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells.

    PubMed

    Wages, Phillip A; Silbajoris, Robert; Speen, Adam; Brighton, Luisa; Henriquez, Andres; Tong, Haiyan; Bromberg, Philip A; Simmons, Steven O; Samet, James M

    2014-01-01

    Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn(2+)) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn(2+) toxicity is not fully understood. H2O2 and Zn(2+) have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn(2+) to cause cellular H2O2 production. To determine the role of Zn(2+)-induced H2O2 production in the human airway epithelial cell response to Zn(2+) exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50µM Zn(2+) for 5min in the presence of 1µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn(2+) exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn(2+)-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn(2+)-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn(2+) leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms.

  12. Mast cells play an important role in chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway.

    PubMed

    Chiba, Norika; Shimada, Kenichi; Chen, Shuang; Jones, Heather D; Alsabeh, Randa; Slepenkin, Anatoly V; Peterson, Ellena; Crother, Timothy R; Arditi, Moshe

    2015-04-15

    Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.

  13. Mature cystic fibrosis airway neutrophils suppress T cell function: evidence for a role of arginase 1 but not programmed death-ligand 1.

    PubMed

    Ingersoll, Sarah A; Laval, Julie; Forrest, Osric A; Preininger, Marcela; Brown, Milton R; Arafat, Dalia; Gibson, Greg; Tangpricha, Vin; Tirouvanziam, Rabindra

    2015-06-01

    Bacteria colonize cystic fibrosis (CF) airways, and although T cells with appropriate Ag specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T cell function therein. Programmed death-ligand 1 (PD-L1), which exerts T cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1(hi) and PD-L1(lo) subsets, whereas healthy control (HC) airway PMNs were uniformly PD-L1(hi). Blood PMNs incubated in CF airway fluid lost PD-L1 over time; in coculture, Ab blockade of PD-L1 failed to inhibit the suppression of T cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T cell suppression in CF, likely hampering resolution of infection and inflammation.

  14. MATURE CYSTIC FIBROSIS AIRWAY NEUTROPHILS SUPPRESS T-CELL FUNCTION: EVIDENCE FOR A ROLE OF ARGINASE 1, BUT NOT PROGRAMMED DEATH-LIGAND 1

    PubMed Central

    Ingersoll, Sarah A.; Laval, Julie; Forrest, Osric A.; Preininger, Marcela; Brown, Milton R.; Arafat, Dalia; Gibson, Greg; Tangpricha, Vin; Tirouvanziam, Rabindra

    2015-01-01

    Bacteria colonize cystic fibrosis (CF) airways, and while T cells with appropriate antigen specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T-cell function therein. Programmed Death-Ligand 1 (PD-L1), which exerts T-cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1hi and PD-L1lo subsets, while healthy control (HC) airway PMNs were uniformly PD-L1hi. Blood PMNs incubated in CF airway fluid lost PD-L1 over time, and in coculture, antibody blockade of PD-L1 failed to inhibit the suppression of T-cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T-cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T-cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T-cell suppression in CF, likely hampering resolution of infection and inflammation. PMID:25926674

  15. Resolution of cell-mediated airways diseases

    PubMed Central

    2010-01-01

    "Inflammation resolution" has of late become a topical research area. Activation of resolution phase mechanisms, involving select post-transcriptional regulons, transcription factors, 'autacoids', and cell phenotypes, is now considered to resolve inflammatory diseases. Critical to this discourse on resolution is the elimination of inflammatory cells through apoptosis and phagocytosis. For major inflammatory diseases such as asthma and COPD we propose an alternative path to apoptosis for cell elimination. We argue that transepithelial migration of airway wall leukocytes, followed by mucociliary clearance, efficiently and non-injuriously eliminates pro-inflammatory cells from diseased airway tissues. First, it seems clear that numerous infiltrated granulocytes and lymphocytes can be speedily transmitted into the airway lumen without harming the epithelial barrier. Then there are a wide range of 'unexpected' findings demonstrating that clinical improvement of asthma and COPD is not only associated with decreasing numbers of airway wall inflammatory cells but also with increasing numbers of these cells in the airway lumen. Finally, effects of inhibition of transepithelial migration support the present hypothesis. Airway inflammatory processes have thus been much aggravated when transepithelial exit of leukocytes has been inhibited. In conclusion, the present hypothesis highlights risks involved in drug-induced inhibition of transepithelial migration of airway wall leukocytes. It helps interpretation of common airway lumen data, and suggests approaches to treat cell-mediated airway inflammation. PMID:20540713

  16. Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3.

    PubMed

    Kim, Dusik; Kim, Juyeon; Burghardt, Beáta; Best, Len; Steward, Martin C

    2014-07-15

    Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.

  17. Myb permits multilineage airway epithelial cell differentiation

    PubMed Central

    Pan, Jie-hong; Adair-Kirk, Tracy L.; Patel, Anand C.; Huang, Tao; Yozamp, Nicholas S.; Xu, Jian; Reddy, E. Premkumar; Byers, Derek E.; Pierce, Richard A.; Holtzman, Michael J.; Brody, Steven L.

    2014-01-01

    The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63+ basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps is poorly defined. Here we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb+ cells were identified as p63− and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63− population with failed maturation of Foxj1+ ciliated cells, as well as Scbg1a1+ and Muc5ac+ secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb+ cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63− Myb+ population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease. PMID:25103188

  18. Lysophosphatidylcholine plays critical role in allergic airway disease manifestation

    PubMed Central

    Bansal, Preeti; Gaur, Shailendera Nath; Arora, Naveen

    2016-01-01

    Phospholipase A2 (sPLA2), pivotal for allergic and inflammatory response, hydrolyses phosphatidylcholine (PC) to lysophosphatidylcholine (LPC). In present study, the role of LPC in allergic airway disease manifestation was studied using mouse model. Balb/c mice were immunized using cockroach extract (CE) and LPC release was blocked by sPLA2 inhibitor. Airway hyperresponse (AHR), lung-histology, total and differential leukocyte count (TLC&DLC), Th2 type cytokines, sPLA2 activity and LPC levels in bronchoalveolar lavage fluid (BALF) were measured. Exogenous LPC was given to the mice with or without CE sensitization, to demonstrate its role in allergic airway disease manifestation. Anti-CD1d antibody was given to study the involvement of natural killer T (NKT) cells in LPC induced response. AHR, lung-inflammation, TLC, DLC, Th2 type cytokines, sPLA2 activity and LPC levels were increased on CE challenge. sPLA2 activity and LPC release was blocked by sPLA2-inhibitor, which decreased AHR, and inflammatory parameters. Exogenous LPC with or without CE sensitization increased above parameters. CE challenge or LPC exposure increased LY49C+TCRβ+ NKT cells in BALF and spleen, which was reduced by anti-CD1d antibody, accompanied with reduction in AHR and allergic airway inflammation parameters. Conclusively, LPC induces allergic airway disease manifestation and it does so probably via CD1d-restricted LY49C+TCRβ+ NKT cells. PMID:27282246

  19. Role of Aspergillus fumigatus in Triggering Protease-Activated Receptor-2 in Airway Epithelial Cells and Skewing the Cells toward a T-helper 2 Bias

    PubMed Central

    Homma, Tetsuya; Kato, Atsushi; Bhushan, Bharat; Norton, James E.; Suh, Lydia A.; Carter, Roderick G.; Gupta, Dave S.

    2016-01-01

    Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1–biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C– and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho–IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C–induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF

  20. Central Role of Cellular Senescence in TSLP-Induced Airway Remodeling in Asthma

    PubMed Central

    Wu, Jinxiang; Dong, Fangzheng; Wang, Rui-An; Wang, Junfei; Zhao, Jiping; Yang, Mengmeng; Gong, Wenbin; Cui, Rutao; Dong, Liang

    2013-01-01

    Background Airway remodeling is a repair process that occurs after injury resulting in increased airway hyper-responsiveness in asthma. Thymic stromal lymphopoietin (TSLP), a vital cytokine, plays a critical role in orchestrating, perpetuating and amplifying the inflammatory response in asthma. TSLP is also a critical factor in airway remodeling in asthma. Objectives To examine the role of TSLP-induced cellular senescence in airway remodeling of asthma in vitro and in vivo. Methods Cellular senescence and airway remodeling were examined in lung specimens from patients with asthma using immunohischemical analysis. Both small molecule and shRNA approaches that target the senescent signaling pathways were used to explore the role of cellular senescence in TSLP-induced airway remodeling in vitro. Senescence-Associated β-galactosidase (SA-β-Gal) staining, and BrdU assays were used to detect cellular senescence. In addition, the Stat3-targeted inhibitor, WP1066, was evaluated in an asthma mouse model to determine if inhibiting cellular senescence influences airway remodeling in asthma. Results Activation of cellular senescence as evidenced by checkpoint activation and cell cycle arrest was detected in airway epithelia samples from patients with asthma. Furthermore, TSLP-induced cellular senescence was required for airway remodeling in vitro. In addition, a mouse asthma model indicates that inhibiting cellular senescence blocks airway remodeling and relieves airway resistance. Conclusion TSLP stimulation can induce cellular senescence during airway remodeling in asthma. Inhibiting the signaling pathways of cellular senescence overcomes TSLP-induced airway remodeling. PMID:24167583

  1. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

    PubMed Central

    Juncadella, Ignacio J.; Kadl, Alexandra; Sharma, Ashish K.; Shim, Yun M.; Hochreiter-Hufford, Amelia; Borish, Larry; Ravichandran, Kodi S.

    2013-01-01

    Lung epithelial cells can influence immune responses to airway allergens1,2. Airway epithelial cells also undergo apoptosis after encountering environmental allergens3; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells1,4,5. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens. PMID:23235830

  2. Role of Rho kinase isoforms in murine allergic airway responses.

    PubMed

    Zhu, M; Liu, P-Y; Kasahara, D I; Williams, A S; Verbout, N G; Halayko, A J; Fedulov, A; Shoji, T; Williams, E S; Noma, K; Shore, S A; Liao, J K

    2011-10-01

    Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses.

  3. Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Huang, Junwei; Shan, Jiajie; Kim, Dusik; Liao, Jie; Evagelidis, Alexandra; Alper, Seth L; Hanrahan, John W

    2012-01-01

    Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl−/HCO3− exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (Isc). We have studied the role of AE2 in Cl− and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced ≥90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na+–K+–2Cl− cotransporter) or NBCe1 (Na+–nHCO3− cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO3−] as compared with the control lines. Unstimulated equivalent short-circuit current (Ieq) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both Ieq and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl−/HCO3− exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl− removal was reduced by ∼80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl− loading during cAMP-stimulated secretion of Cl− and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway

  4. Role of sarcoplasmic reticulum Ca2+ content in Ca2+ entry of bovine airway smooth muscle cells.

    PubMed

    Bazán-Perkins, Blanca; Flores-Soto, Edgar; Barajas-López, Carlos; Montaño, Luis M

    2003-10-01

    Depletion of intracellular Ca(2+) stores induces the opening of an unknown Ca(2+ )entry pathway to the cell. We measured the intracellular free-Ca(2+) concentration ([Ca(2+)]i) at different sarcoplasmic reticulum (SR) Ca(2+) content in fura-2-loaded smooth muscle cells isolated from bovine tracheas. The absence of Ca(2+) in the extracellular medium generated a time-dependent decrement in [Ca(2+)]i which was proportional to the reduction in the SR-Ca(2+) content. This SR-Ca(2+) level was indirectly determined by measuring the amount of Ca(2+) released by caffeine. Ca(2+) restoration at different times after Ca(2+)-free incubation (2, 4, 6 and 10 min) induced an increment of [Ca(2+)]i. This increase in [Ca(2+)]i was considered as Ca(2+) entry to the cell. The rate of this entry was slow (~0.3 nM/s) when SR-Ca(2+) content was higher than 50% (2 and 4 min in Ca(2+)-free medium), and significantly ( p<0.01) accelerated (>1.0 nM/s) when SR-Ca(2+) content was lower than 50% (6 and 10 min in Ca(2+)-free medium). Thapsigargin significantly induced a higher rate of this Ca(2+) entry ( p<0.01). Variations in Ca(2+) influx after SR-Ca(2+) depletion were estimated more directly by a Mn(2+) quench approach. Ca(2+) restoration to the medium 4 min after Ca(2+) removal did not modify the Mn(2+) influx. However, when Ca(2+) was added after 10 min in Ca(2+)-free medium, an increment of Mn(2+) influx was observed, corroborating an increase in Ca(2+) entry. The fast Ca(2+) influx was Ni(2+) sensitive but was not affected by other known capacitative Ca(2+) entry blockers such as La(3+), Mg(2+), SKF 96365 and 2-APB. It was also not affected by the blockage of L-type Ca2(+) channels with methoxyverapamil or by the sustained K(+)-induced depolarisation. The slow Ca(2+) influx was only sensitive to SKF 96365. In conclusion, our results indicate that in bovine airway smooth muscle cells Ca(2+) influx after SR-Ca(2+) depletion has two rates: A) The slow Ca(2+) influx, which occurred in cells

  5. Comments to Role of upper airway ultrasound in airway management.

    PubMed

    Lien, Wan-Ching

    2017-01-01

    Tracheal ultrasound can be an alternative diagnostic tool in airway management, besides traditional confirmatory methods such as capnography and auscultation. The standard image is a hyperechoic air-mucosa (A-M) interface with a reverberation artifact posteriorly (comet-tail artifact). If the second A-M interface appears, which we call a "double-tract sign," esophageal intubation is considered.

  6. Mast cells in airway diseases and interstitial lung disease.

    PubMed

    Cruse, Glenn; Bradding, Peter

    2016-05-05

    Mast cells are major effector cells of inflammation and there is strong evidence that mast cells play a significant role in asthma pathophysiology. There is also a growing body of evidence that mast cells contribute to other inflammatory and fibrotic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. This review discusses the role that mast cells play in airway diseases and highlights how mast cell microlocalisation within specific lung compartments and their cellular interactions are likely to be critical for their effector function in disease.

  7. A pathogenic role for the integrin CD103 in experimental allergic airways disease.

    PubMed

    Fear, Vanessa S; Lai, Siew Ping; Zosky, Graeme R; Perks, Kara L; Gorman, Shelley; Blank, Fabian; von Garnier, Christophe; Stumbles, Philip A; Strickland, Deborah H

    2016-11-01

    The integrin CD103 is the αE chain of integrin αEβ7 that is important in the maintenance of intraepithelial lymphocytes and recruitment of T cells and dendritic cells (DC) to mucosal surfaces. The role of CD103 in intestinal immune homeostasis has been well described, however, its role in allergic airway inflammation is less well understood. In this study, we used an ovalbumin (OVA)-induced, CD103-knockout (KO) BALB/c mouse model of experimental allergic airways disease (EAAD) to investigate the role of CD103 in disease expression, CD4(+) T-cell activation and DC activation and function in airways and lymph nodes. We found reduced airways hyper-responsiveness and eosinophil recruitment to airways after aerosol challenge of CD103 KO compared to wild-type (WT) mice, although CD103 KO mice showed enhanced serum OVA-specific IgE levels. Following aerosol challenge, total numbers of effector and regulatory CD4(+) T-cell subsets were significantly increased in the airways of WT but not CD103 KO mice, as well as a lack of DC recruitment into the airways in the absence of CD103. While total airway DC numbers, and their in vivo allergen capture activity, were essentially normal in steady-state CD103 KO mice, migration of allergen-laden airway DC to draining lymph nodes was disrupted in the absence of CD103 at 24 h after aerosol challenge. These data support a role for CD103 in the pathogenesis of EAAD in BALB/c mice through local control of CD4(+) T cell and DC subset recruitment to, and migration from, the airway mucosa during induction of allergic inflammation.

  8. [Research progress on role of chemokine receptor CCR3 signaling in allergic airway diseases].

    PubMed

    Xu, Yi; Liu, Yuehui

    2012-12-01

    Allergic airway diseases have been identified as chronic inflammatory diseases of respiratory membranes, characterized by infiltration of many inflammatory cells, especially eosinophils. The expression of CCR3 is abundant on the cell surface of eosinophils. Increased accumulation of CCR3-driven inflammatory cells is thought to favor the development of allergy. In this review, we survey the properties of CCR3 and its ligands and highlight the roles of CCR3 signaling in allergic airway diseases.

  9. Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

    PubMed Central

    Knox, Alan J.

    2015-01-01

    Human airway smooth muscle cells (HASMC) contribute to asthma pathophysiology through an increased smooth muscle mass and elevated cytokine/chemokine output. Little is known about how HASMC and the airway epithelium interact to regulate chronic airway inflammation and remodeling. Amphiregulin is a member of the family of epidermal growth factor receptor (EGFR) agonists with cell growth and proinflammatory roles and increased expression in the lungs of asthma patients. Here we show that bradykinin (BK) stimulation of HASMC increases amphiregulin secretion in a mechanism dependent on BK-induced COX-2 expression, increased PGE2 output, and the stimulation of HASMC EP2 and EP4 receptors. Conditioned medium from BK treated HASMC induced CXCL8, VEGF, and COX-2 mRNA and protein accumulation in airway epithelial cells, which were blocked by anti-amphiregulin antibodies and amphiregulin siRNA, suggesting a paracrine effect of HASMC-derived amphiregulin on airway epithelial cells. Consistent with this, recombinant amphiregulin induced CXCL8, VEGF, and COX-2 in airway epithelial cells. Finally, we found that conditioned media from amphiregulin-stimulated airway epithelial cells induced amphiregulin expression in HASMC and that this was dependent on airway epithelial cell COX-2 activity. Our study provides evidence of a dynamic axis of interaction between HASMC and epithelial cells that amplifies CXCL8, VEGF, COX-2, and amphiregulin production. PMID:26047642

  10. The role of γδ T cells in airway epithelial injury and bronchial responsiveness after chlorine gas exposure in mice

    PubMed Central

    Koohsari, Hossein; Tamaoka, Meiyo; Campbell, Holly R; Martin, James G

    2007-01-01

    Background Acute exposure to chlorine (Cl2) gas causes epithelial injury and airway dysfunction. γδ T cells are present in the mucosal surface of the airways and may contribute to the injury/repair response of the epithelium. Methods C57Bl/6J (wild type) and TCR-δ-/- mice exposed to Cl2 (400 ppm) for 5 minutes underwent measurements of airway responses to i.v. methacholine (MCh) at 1, 3, and 5 days after exposure. Bronchoalveolar lavage was performed to determine epithelial and leukocyte counts, and protein content. Tissue repair was assessed by proliferating cell nuclear antigen (PCNA) immunoreactivity and by expression of keratinocyte growth factor (KGF) mRNA by real-time PCR. Results Wild type mice developed a greater degree of airway hyperresponsiveness to MCh at 1 day post exposure to Cl2 compared with TCR-δ-/- mice. Epithelial cell counts in BAL after Cl2 exposure were greater in TCR-δ-/- mice, but macrophages showed a later peak and granulocyte numbers were lower in TCR-δ-/- than in wild type mice. Both groups had increased levels of total protein content in BAL after Cl2 exposure that resolved after 3 and 5 days, respectively. Epithelial proliferating cell nuclear antigen staining was increased at 1 and 3 days post exposure and was similar in the two groups. KGF mRNA was constitutively expressed in both groups and did not increase significantly after Cl2 but expression was lower in TCR-δ-/- mice. Conclusion The severity of airway epithelial injury after Cl2 is greater in TCR-δ-/- mice but the inflammatory response and the change in airway responsiveness to methacholine are reduced. The rates of epithelial regeneration are comparable in both groups. PMID:17343743

  11. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

  12. Differential Roles of Hydrogen Peroxide in Adaptive and Inflammatory Gene Expression Induced by Exposure of Human Airway Epithelial Cells to Zn2+

    EPA Science Inventory

    Oxidant stress is believed to play an important role in particulate matter (PM)–mediated toxicity in the respiratory tract. Zinc (Zn2+) is a ubiquitous component of PM that has been shown to induce adverse responses such as inflammatory and adaptive gene expression in airway epit...

  13. Differential Roles of Hydrogen Peroxide in Adaptive and Inflammatory Gene Expression Induced by Exposure of Human Airway Epithelial Cells to Zn2+

    EPA Science Inventory

    Oxidant stress is believed to play an important role in particulate matter (PM)–mediated toxicity in the respiratory tract. Zinc (Zn2+) is a ubiquitous component of PM that has been shown to induce adverse responses such as inflammatory and adaptive gene expression in airway epit...

  14. Epithelium-derived chemokines induce airway smooth muscle cell migration.

    PubMed

    Takeda, N; Sumi, Y; Préfontaine, D; Al Abri, J; Al Heialy, N; Al-Ramli, W; Michoud, M-C; Martin, J G; Hamid, Q

    2009-07-01

    The remodelling of airway smooth muscle (ASM) associated with asthma severity may involve the migration of ASM cells towards the epithelium. However, little is known about the mechanisms of cell migration and the effect of epithelial-derived mediators on this process. The main objective of the current study is to assess the effects of epithelial-derived chemokines on ASM cell migration. Normal human ASM cells were incubated with supernatants from cells of the bronchial epithelial cell line BEAS-2B and normal human bronchial epithelial (NHBE) cells. To induce chemokine production, epithelial cells were treated with TNF-alpha. Chemokine expression by epithelial cells was evaluated by quantitative real-time PCR, ELISA and membrane antibody array. To identify the role of individual chemokines in ASM cell migration, we performed migration assays with a modified Boyden chamber using specific neutralizing antibodies to block chemokine effects. Supernatants from BEAS-2B cells treated with TNF-alpha increased ASM cell migration; migration was increased 1.6 and 2.5-fold by supernatant from BEAS-2B cells treated with 10 and 100 ng/mL TNF-alpha, respectively. Protein levels in supernatants and mRNA expression by BEAS-2B cells of regulated on activation, normal T cell expressed and secreted (RANTES) and IL-8 were significantly increased by 100 ng/mL TNF-alpha treatment. The incubation of supernatant with antibodies to RANTES or IL-8 significantly reduced ASM cell migration, and the combined antibodies further inhibited the cell migration. The migratory effects of supernatants and inhibiting effects of RANTES and/or IL-8 were confirmed also using NHBE cells. The results show that chemokines from airway epithelial cells cause ASM cell migration and might potentially play a role in the process of airway remodelling in asthma.

  15. Characterization of Side Population Cells from Human Airway Epithelium

    PubMed Central

    Hackett, Tillie-Louise; Shaheen, Furquan; Johnson, Andrew; Wadsworth, Samuel; Pechkovsky, Dmitri V.; Jacoby, David B.; Kicic, Anthony; Stick, Stephen M.; Knight, Darryl A.

    2010-01-01

    The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45− SP, CD45+ SP, and non-SP cells were isolated and sorted. CD45− SP cells made up 0.12% ± 0.01% of the total epithelial cell population in normal airway but 4.1% ± 0.06% of the epithelium in asthmatic airways. All CD45− SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45− SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45− SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and longterm proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. PMID:18653771

  16. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  17. [The role of SDF-1/CXCR4 on airway inflammation and airway remodeling in a rat asthma model].

    PubMed

    Mo, Biwen; Li, Jie; Wei, Jianghong; Wang, Changming; Zeng, Jinrong; Wang, Jiying; Huang, Jianwei

    2015-01-01

    To explore the roles of stromal cell-derived factor 1 (SDF-1) and C-X-C chemokine receptor 4 (CXCR4) on airway inflammation and airway remodeling in rat asthma models. Eighteen female SD rats were randomly divided into 3 groups (n = 6): control group, asthmatic 4 weeks group and asthmatic 8 weeks group. The rats were sensitized and inhaled ovalbumin (OVA). After the asthma model was successfully established, the airway pressure was measured. The methods of HE staining and Image-Pro Plus image analysis software were used to detect the changes of eosinophils (EOS), the perimeter of inner bronchial lumen, the wall area, the area of bronchial smooth muscle and the number of smooth muscle cells of airway walls. RT-PCR and Western-blot were used to detect the expression of SDF-1 and CXCR4 in lung tissues among the 3 groups.Immunohistochemistry was used to detect the expression of SDF-1 in airway walls. Compared with the control group, the airway responsiveness, the count of EOS, the area of bronchial wall, the area of bronchial smooth muscle, the number of smooth muscle cells of airway walls in the asthmatic 4 weeks and asthmatic 8 weeks were significantly increased, and significant difference between the 2 asthmatic groups was also observed in the above indexes (P < 0.01) .RT-PCR showed that compared with the control group (SDF-1 was 0.146 ± 0.003 and CXCR4 was 0.281 ± 0.002) , the expression of SDF-1 (0.583 ± 0.004 and 0.724 ± 0.008) and CXCR4 (0.467 ± 0.003 and 0.655 ± 0.002) in lung tissues in the asthmatic 4 weeks and asthmatic 8 weeks were significantly increased (P < 0.01) . In addition, compared with the asthmatic 4 weeks group, the expression of SDF-1 and CXCR4 in lung tissues in the 8 weeks asthmatic group were significantly increased (P < 0.01) . Compared with the control group (0.180 ± 0.009) , the expression of SDF-1 in airway walls in the asthmatic 4 weeks and asthmatic 8 weeks groups (0.270 ± 0.006 and 0.350 ± 0.009) were significantly increased

  18. B Cell Activating Transcription Factor (BATF) plays a critical role in the pathogenesis of anti-MHC induced Obliterative Airway Disease

    PubMed Central

    Xu, Z.; Ramachandran, S.; Gunasekaran, M.; Nayak, D.; Benshoff, N.; Hachem, R.; Gelman, A.; Mohanakumar, T.

    2016-01-01

    Antibodies (Abs) against MHC results in T helper-17 (Th17) mediated immunity against lung self-antigens (SAgs), K-α1 tubulin and collagen V and Obliterative Airway Disease (OAD). Since B cell activating transcription factor (BATF) controls Th17 and autoimmunity, we proposed that BATF may play a critical role in OAD. Anti-H2Kb was administered intrabronchially into Batf−/− and C57BL/6 mice. Histopathology of the lungs on days 30 and 45 following Abs administration to Batf−/− mice resulted in decreased cellular infiltration, epithelial metaplasia, fibrosis and obstruction. There was lack of Abs to SAgs, reduction of SAgs specific IL17 T cells, IL-6, IL-23, IL-17, IL-1β, FGF-6 and CXCL12 and decreased Janus kinase 2, STAT3, and Retinoid-related orphan receptor gamma-T. Further, miR-301a, a regulator of Th17, was reduced in Batf−/− mice in contrast to up regulation of miR-301a and down regulation of PIAS3 in anti-MHC induced OAD animals. We also demonstrate increase in miR-301a in the bronchoalveolar lavage cells from lung transplant (LTx) recipients with Abs to HLA. This was accompanied by reduction in PIAS3 mRNA. Therefore, we conclude that BATF plays a critical role in the immune responses to SAgs and pathogenesis of anti-MHC induced rejection. Targeting BATF should be considered for preventing chronic rejection following human LTx. PMID:26844425

  19. B Cell-Activating Transcription Factor Plays a Critical Role in the Pathogenesis of Anti-Major Histocompatibility Complex-Induced Obliterative Airway Disease.

    PubMed

    Xu, Z; Ramachandran, S; Gunasekaran, M; Nayak, D; Benshoff, N; Hachem, R; Gelman, A; Mohanakumar, T

    2016-04-01

    Antibodies (Abs) against major histocompatibility complex (MHC) results in T helper-17 (Th17)-mediated immunity against lung self-antigens (SAgs), K-α1 tubulin and collagen V and obliterative airway disease (OAD). Because B cell-activating transcription factor (BATF) controls Th17 and autoimmunity, we proposed that BATF may play a critical role in OAD. Anti-H2K(b) was administered intrabronchially into Batf (-/-) and C57BL/6 mice. Histopathology of the lungs on days 30 and 45 after Ab administration to Batf (-/-) mice resulted in decreased cellular infiltration, epithelial metaplasia, fibrosis, and obstruction. There was lack of Abs to SAgs, reduction of Sag-specific interleukin (IL)-17 T cells, IL-6, IL-23, IL-17, IL-1β, fibroblast growth factor-6, and CXCL12 and decreased Janus kinase 2, signal transducer and activator of transcription 3 (STAT3), and retinoid-related orphan receptor γT. Further, micro-RNA (miR)-301a, a regulator of Th17, was reduced in Batf (-/-) mice in contrast to upregulation of miR-301a and downregulation of protein inhibitor of activated STAT3 (PIAS3) in anti-MHC-induced OAD animals. We also demonstrate an increase in miR-301a in the bronchoalveolar lavage cells from lung transplant recipients with Abs to human leukocyte antigen. This was accompanied by reduction in PIAS3 mRNA. Therefore, we conclude that BATF plays a critical role in the immune responses to SAgs and pathogenesis of anti-MHC-induced rejection. Targeting BATF should be considered for preventing chronic rejection after human lung transplantation.

  20. Tryptase does not alter transepithelial conductance or paracellular permeability in human airway epithelial cells.

    PubMed

    Chang, Eugene H; Lee, John H; Zabner, Joseph

    2010-01-01

    Cell tight junction proteins create a barrier between airway epithelial cells to limit paracellular transport from the apical to basolateral surface. This barrier can impede the entry of respiratory pathogens and toxins from the airway lumen into the systemic circulation. Mast cell-mediated inflammation in the human airway can cause a disruption of this barrier. Tryptase is one of the major mediators released by mast cells and has been studied extensively in diseases such as asthma, reflux, and sinusitis. We hypothesize that tryptase may play a role in airway paracellular permeability by disrupting the cell tight junction proteins. We tested this hypothesis by applying tryptase on the apical and basolateral surface to primary human airway epithelia grown in an air-liquid interface and measured changes in the transepithelial conductance and paracellular permeability of the membrane during short (every minute) and longer (over hours) time courses. We then immunostained the cell membranes for occludins and claudins to observe for changes in the structure of the tight junctions after tryptase application. Our data show that tryptase does not alter paracellular permeability in human airway cells over minutes or hours, and that tryptase does not alter the structure of the cell junction. Tryptase alone does not alter paracellular permeability in human airway cells. Tryptase may be altering the epithelial membrane independent of the cell tight junction pathway or other mast cell mediators may play a role in permeability.

  1. [Expression and role of sugar chains on airway mucus during the exacerbation of airway inflammation].

    PubMed

    Ishibashi, Yuji; Inouye, Yoshio; Taniguchi, Akiyoshi

    2012-01-01

    Human bronchial mucins, such as MUC5AC, have traditionally been defined as a family of high-molecular weight glycoproteins. Changes in the contents of sugar chains on MUC5AC are among the fundamental features in inflammatory respiratory disease. The changes have been shown to lead to unfavorable alterations in the viscosity of mucus, resulting in impairment of mucociliary transport, vulnerability to viral/bacterial infection as sugar chains play an important role in adhesion of some viruses and bacteria to the epithelium, and finally inflammatory cell infiltration in the airway. Recently, we found that expression of some glycosyltransferases associated with the contents and structure of sugar chains is regulated by phosphatidylinositol-phospholipase (PI-PL) C signaling in cells. L-Carbocisteine, a mucoregulatory drug, normalized or balanced fucosylated and sialylated sugar chains, such as sialyl Lewis x through inhibition of PI-PL C signaling. We prepared MUC5AC fusion protein with tandem repeats associated with MUC5AC, and confirmed that L-carbocisteine inhibited the increases in viscosity associated with sialyl Lewis x expression levels. In addition, the clinical study (2008) noted that L-carbocisteine reduced the frequency of common colds and exacerbation of symptoms in patients with COPD. These favorable effects in patients may be due to normalization of sugar chain contents on mucins. We suggest that the inhibitory effect on infection of airway epithelial cells by rhinoviruses, respiratory syncytial virus, and influenza viruses by treatment with L-carbocisteine may also be based on the regulation of sugar chain contents or structures on mucins.

  2. Airway management considerations in children with I-cell disease.

    PubMed

    Mallen, Jonathan; Highstein, Mallory; Smith, Lee; Cheng, Jeffrey

    2015-05-01

    Inclusion-cell disease (mucolipidosis II/I-cell disease) is a lysosomal storage disease characterized by a constellation of physical findings which complicate airway management. There is currently a deficit of published literature describing appropriate strategies for acute management of these children's airways. This paper details emergency and anesthetic airway management concerns and potential solutions in a small series of children with I-cell disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Role of ADAM and ADAMTS metalloproteinases in airway diseases

    PubMed Central

    2009-01-01

    Lungs are exposed to the outside environment and therefore to toxic and infectious agents or allergens. This may lead to permanent activation of innate immune response elements. A Disintegrin And Metalloproteinases (ADAMs) and ADAMs with Thrombospondin motifs (ADAMTS) are proteinases closely related to Matrix Metalloproteinases (MMPs). These multifaceted molecules bear metalloproteinase and disintegrin domains endowing them with features of both proteinases and adhesion molecules. Proteinases of the ADAM family are associated to various physiological and pathological processes and display a wide spectrum of biological effects encompassing cell fusion, cell adhesion, "shedding process", cleavage of various substrates from the extracellular matrix, growth factors or cytokines... This review will focus on the putative roles of ADAM/ADAMTS proteinases in airway diseases such as asthma and COPD. PMID:20034386

  4. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    PubMed

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  5. [Role of phosphorylation of MARCKS-PSD in the secretion of MUC5AC induced by cold temperatures in human airway epithelial cells].

    PubMed

    Li, Minchao; Perelman, Juliy M; Zhou, Xiangdong

    2012-05-01

    To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC. Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively. Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05). TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.

  6. NHERF1 and CFTR restore tight junction organisation and function in cystic fibrosis airway epithelial cells: role of ezrin and the RhoA/ROCK pathway.

    PubMed

    Castellani, Stefano; Guerra, Lorenzo; Favia, Maria; Di Gioia, Sante; Casavola, Valeria; Conese, Massimo

    2012-11-01

    Tight junctions (TJs) restrict the transit of ions and molecules through the paracellular route and act as a barrier to regulate access of inflammatory cells into the airway lumen. The pathophysiology of cystic fibrosis (CF) lung disease is characterised by abnormal ion and fluid transport across the epithelium and polymorphonuclear (PMN) leukocyte-dominated inflammatory response. Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) is a protein involved in PKA-dependent activation of CFTR by interacting with CFTR via its PDZ domains and with ezrin via its C-terminal domain. We have previously found that the NHERF1-overexpression dependent rescue CFTR-dependent chloride secretion is due to the re-organisation of the actin cytoskeleton network induced by the formation of the multiprotein complex NHERF1-RhoA-ezrin-actin. In this context, we here studied whether NHERF1 and CFTR are involved in the organisation and function of TJs. F508del CFBE41o⁻ monolayers presented nuclear localisation of zonula occludens (ZO-1) and occludin as well as disorganisation of claudin 1 and junction-associated adhesion molecule 1 as compared with wild-type 16HBE14o⁻ monolayers, paralleled by increased permeability to dextrans and PMN transmigration. Overexpression of either NHERF1 or CFTR in CFBE41o⁻ cells rescued TJ proteins to their proper intercellular location and decreased permeability and PMN transmigration, while this effect was not achieved by overexpressing either NHERF1 deprived of ezrin-binding domain. Further, expression of a phospho-dead ezrin mutant, T567A, increased permeability in both 16HBE14o⁻ cells and in a CFBE clone stably overexpressing NHERF1 (CFBE/sNHERF1), whereas a constitutively active form of ezrin, T567D, achieved the opposite effect in CFBE41o⁻ cells. A dominant-negative form of RhoA (RhoA-N19) also disrupted ZO-1 localisation at the intercellular contacts dislodging it to the nucleus and increased permeability in CFBE/sNHERF1. The inhibitor Y27632 of

  7. The role of bronchoscopy in the diagnosis of airway disease

    PubMed Central

    Dixon, Jennifer; Tieu, Brandon H.

    2016-01-01

    Endoscopy of the airway is a valuable tool for the evaluation and management of airway disease. It can be used to evaluate many different bronchopulmonary diseases including airway foreign bodies, tumors, infectious and inflammatory conditions, airway stenosis, and bronchopulmonary hemorrhage. Traditionally, options for evaluation were limited to flexible and rigid bronchoscopy. Recently, more sophisticated technology has led to the development of endobronchial ultrasound (EBUS) and electromagnetic navigational bronchoscopy (ENB). These technological advances, combined with increasing provider experience have resulted in a higher diagnostic yield with endoscopic biopsies. This review will focus on the role of bronchoscopy, including EBUS, ENB, and rigid bronchoscopy in the diagnosis of bronchopulmonary diseases. In addition, it will cover the anesthetic considerations, equipment, diagnostic yield, and potential complications. PMID:28149583

  8. Cell proliferation contributes to PNEC hyperplasia after acute airway injury.

    PubMed

    Stevens, T P; McBride, J T; Peake, J L; Pinkerton, K E; Stripp, B R

    1997-03-01

    Pulmonary neuroendocrine cells (PNECs) are airway epithelial cells that are capable of secreting a variety of neuropeptides. PNECs are scattered throughout the bronchial tree either as individual cells or clusters of cells termed neuroepithelial bodies (NEBs). PNECs and their secretory peptides have been considered to play a role in fetal lung development. Although the normal physiological function of PNECs and neuropeptides in normal adult lungs and in repair from lung injury is not known, PNEC hyperplasia has been associated with chronic lung diseases, such as bronchopulmonary dysplasia, and with chronic exposures, such as hypoxia, tobacco smoke, nitrosamines, and ozone. To evaluate changes in PNEC number and distribution after acute airway injury, FVB/n mice were treated with either naphthalene or vehicle. Naphthalene is an aromatic hydrocarbon that, at the dose used in this study, selectively destroys nonciliated bronchial epithelial cells (Clara cells) through cytochrome P-450-mediated metabolic activation into cytotoxic epoxides. PNECs were identified by immunohistochemical analysis of calcitonin gene-related peptide-like immunoreactivity (CGRP-IR). Proliferating cells were marked with [(3)H]thymidine incorporation. Acute naphthalene toxicity results in PNEC hyperplasia that is detectable after 5 days of recovery. PNEC hyperplasia is characterized by increased numbers of NEBs without significant changes in the number of isolated PNECs and by increased [(3)H]thymidine labeling of CGRP-IR cells. These data show that cell proliferation contributes to PNEC hyperplasia after acute airway injury and suggest that PNECs may be capable of more rapidly increasing their number in response to injury than previously recognized.

  9. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  10. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  11. Adoptive transfer of induced-Treg cells effectively attenuates murine airway allergic inflammation.

    PubMed

    Xu, Wei; Lan, Qin; Chen, Maogen; Chen, Hui; Zhu, Ning; Zhou, Xiaohui; Wang, Julie; Fan, Huimin; Yan, Chun-Song; Kuang, Jiu-Long; Warburton, David; Togbe, Dieudonnée; Ryffel, Bernhard; Zheng, Song-Guo; Shi, Wei

    2012-01-01

    Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4(+)FoxP3(+)) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.

  12. Expression of ligands for Siglec-8 and Siglec-9 in human airways and airway cells

    PubMed Central

    Jia, Yi; Yu, Huifeng; Fernandes, Steve M.; Wei, Yadong; Gonzalez-Gil, Anabel; Motari, Mary G.; Vajn, Katarina; Stevens, Whitney W.; Peters, Anju T.; Bochner, Bruce S.; Kern, Robert C.; Schleimer, Robert P.; Schnaar, Ronald L.

    2015-01-01

    Background Balanced activation and inhibition of the immune system ensures pathogen clearance while avoiding hyperinflammation. Siglecs, sialic acid binding proteins found on subsets of immune cells, often inhibit inflammation: Siglec-8 on eosinophils and Siglec-9 on neutrophils engage sialoglycan ligands on airways to diminish ongoing inflammation. The identities of human siglec ligands and their expression during inflammation are largely unknown. Objective The histological distribution, expression and molecular characteristics of siglec ligands were explored in healthy and inflamed human upper airways and in a cellular model of airway inflammation. Methods Normal and chronically inflamed upper airway tissues were stained for siglec ligands. The ligands were extracted from normal and inflamed tissues and from human Calu-3 cells for quantitative analysis by siglec blotting and isolation by siglec capture. Results Siglec-8 ligands were expressed on a subpopulation of submucosal gland cells of human inferior turbinate, whereas Siglec-9 ligands were expressed more broadly (submucosal glands, epithelium, connective tissue); both were significantly upregulated in chronic rhinosinusitis patients. Human airway (Calu-3) cells expressed Siglec-9 ligands on mucin 5B under inflammatory control via the NF-κB pathway, and mucin 5B carried sialoglycan ligands of Siglec-9 on human upper airway tissue. Conclusion Inflammation results in upregulation of immune inhibitory Siglec-8 and Siglec-9 sialoglycan ligands on human airways. Siglec-9 ligands were upregulated via the NF-κB pathway resulting in their enhanced expression on mucin 5B. Siglec sialoglycan ligand expression in inflamed cells and tissues may contribute to the control of airway inflammation. PMID:25747723

  13. NOTCH1 is required for regeneration of Clara cells during repair of airway injury.

    PubMed

    Xing, Yiming; Li, Aimin; Borok, Zea; Li, Changgong; Minoo, Parviz

    2012-05-01

    The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the targets of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells, and repair and re-establishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell-fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/enhancer of split-5 (Hes5) and paired-box-containing gene 6 (Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild-type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression.

  14. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells

    PubMed Central

    Konishi, Satoshi; Gotoh, Shimpei; Tateishi, Kazuhiro; Yamamoto, Yuki; Korogi, Yohei; Nagasaki, Tadao; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Ito, Isao; Tsukita, Sachiko; Mishima, Michiaki

    2015-01-01

    Summary Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1+-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM+ VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a “9 + 2” microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine. PMID:26724905

  15. Pulmonary neuroendocrine cells, airway innervation, and smooth muscle are altered in Cftr null mice.

    PubMed

    Pan, Jie; Luk, Catherine; Kent, Geraldine; Cutz, Ernest; Yeger, Herman

    2006-09-01

    The amine- and peptide-producing pulmonary neuroendocrine cells (PNEC) are widely distributed within the airway mucosa of mammalian lung as solitary cells and innervated clusters, neuroepithelial bodies (NEB), which function as airway O2 sensors. These cells express Cftr and hence could play a role in the pathophysiology of cystic fibrosis (CF) lung disease. We performed confocal microscopy and morphometric analysis on lung sections from Cftr-/- (null), Cftr+/+, and Cftr+/- (control) mice at developmental stages E20, P5, P9, and P30 to determine the distribution, frequency, and innervation of PNEC/NEB, innervation and cell mass of airway smooth muscle, and neuromuscular junctions using synaptic vesicle protein 2, smooth muscle actin, and synaptophysin markers, respectively. The mean number of PNEC/NEB in Cftr-/- mice was significantly reduced compared with control mice at E20, whereas comparable or increased numbers were observed postnatally. NEB cells in Cftr null mice showed a significant reduction in intracorpuscular nerve endings compared with control mice, which is consistent with an intrinsic abnormality of the PNEC system. The airways of Cftr-/- mice showed reduced density (approximately 20-30%) of smooth muscle innervation, decreased mean airway smooth muscle mass (approximately 35%), and reduced density (approximately 20%) of nerve endings compared with control mice. We conclude that the airways of Cftr-/- mice exhibit heretofore unappreciated structural alterations affecting cellular and neural components of the PNEC system and airway smooth muscle and its innervation resulting in blunted O2 sensing and reduced airway tonus. Cftr could play a role in the development of the PNEC system, lung innervation, and airway smooth muscle.

  16. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans.

  17. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  18. Bystander suppression of allergic airway inflammation by lung resident memory CD8+ T cells

    NASA Astrophysics Data System (ADS)

    Marsland, Benjamin J.; Harris, Nicola L.; Camberis, Mali; Kopf, Manfred; Hook, Sarah M.; Le Gros, Graham

    2004-04-01

    CD8+ memory T cells have recently been recognized as playing a key role in natural immunity against unrelated viral infections, a phenomenon referred to as "heterologous antiviral immunity." We now provide data that the cellular immunological interactions that underlie such heterologous immunity can play an equally important role in regulating T helper 2 immune responses and protecting mucosal surfaces from allergen-induced inflammation. Our data show that CD8+ T cells, either retained in the lung after infection with influenza virus, or adoptively transferred via the intranasal route can suppress allergic airway inflammation. The suppression is mediated by IFN-, which acts to reduce the activation level, T helper 2 cytokine production, airways hyperresponsiveness, and migration of allergen-specific CD4+ T cells into the lung, whereas the systemic and draining lymph node responses remain unchanged. Of note, adoptive transfer of previously activated transgenic CD8+ T cells conferred protection against allergic airway inflammation, even in the absence of specific-antigen. Airway resident CD8+ T cells produced IFN- when directly exposed to conditioned media from activated dendritic cells or the proinflammatory cytokines IL-12 and IL-18. Taken together these data indicate that effector/memory CD8+ T cells present in the airways produce IFN- after inflammatory stimuli, independent of specific-antigen, and as a consequence play a key role in modifying the degree and frequency of allergic responses in the lung.

  19. Intelectin is required for IL-13-induced monocyte chemotactic protein-1 and -3 expression in lung epithelial cells and promotes allergic airway inflammation

    PubMed Central

    Gu, Naibing; Kang, Guannan; Jin, Chang'E; Xu, Yongjian; Zhang, Zhenxiang; Erle, David J.

    2010-01-01

    Asthma is characterized by airway inflammation, mucus overproduction, airway hyperreactivity, and peribronchial fibrosis. Intelectin has been shown to be increased in airway epithelium of asthmatics. However, the role of intelectin in the pathogenesis of asthma is unknown. Airway epithelial cells can secrete chemokines such as monocyte chemotactic protein (MCP)-1 and -3 that play crucial roles in asthmatic airway inflammation. We hypothesized that intelectin plays a role in allergic airway inflammation by regulating chemokine expression. In a mouse allergic asthma model, we found that mRNA expression of intelectin-2 as well as MCP-1 and -3 in mouse lung was increased very early (within 2 h) after allergen challenge. Expression of intelectin protein was localized to mucous cells in airway epithelium. Treatment of MLE12 mouse lung epithelial cells with interleukin IL-13, a critical mediator of allergic airway disease, induced expression of intelectin-1 and -2 as well as MCP-1 and -3. When IL-13-induced intelectin-1 and -2 expression was inhibited by RNA interference, IL-13-induced extracellular signal-regulated kinase 1/2 phosphorylation and MCP-1 and -3 production by MLE12 cells was inhibited. Furthermore, inhibition of intelectin expression by airway transfection with shRNA targeting intelectin-1 and -2 attenuated allergen-induced airway inflammation. We conclude that intelectin, a molecule expressed by airway epithelial cells and upregulated in asthma, is required for IL-13-induced MCP-1 and -3 production in mouse lung epithelial cells and contributes to allergic airway inflammation. PMID:19965981

  20. Intelectin is required for IL-13-induced monocyte chemotactic protein-1 and -3 expression in lung epithelial cells and promotes allergic airway inflammation.

    PubMed

    Gu, Naibing; Kang, Guannan; Jin, Chang'E; Xu, Yongjian; Zhang, Zhenxiang; Erle, David J; Zhen, Guohua

    2010-03-01

    Asthma is characterized by airway inflammation, mucus overproduction, airway hyperreactivity, and peribronchial fibrosis. Intelectin has been shown to be increased in airway epithelium of asthmatics. However, the role of intelectin in the pathogenesis of asthma is unknown. Airway epithelial cells can secrete chemokines such as monocyte chemotactic protein (MCP)-1 and -3 that play crucial roles in asthmatic airway inflammation. We hypothesized that intelectin plays a role in allergic airway inflammation by regulating chemokine expression. In a mouse allergic asthma model, we found that mRNA expression of intelectin-2 as well as MCP-1 and -3 in mouse lung was increased very early (within 2 h) after allergen challenge. Expression of intelectin protein was localized to mucous cells in airway epithelium. Treatment of MLE12 mouse lung epithelial cells with interleukin IL-13, a critical mediator of allergic airway disease, induced expression of intelectin-1 and -2 as well as MCP-1 and -3. When IL-13-induced intelectin-1 and -2 expression was inhibited by RNA interference, IL-13-induced extracellular signal-regulated kinase 1/2 phosphorylation and MCP-1 and -3 production by MLE12 cells was inhibited. Furthermore, inhibition of intelectin expression by airway transfection with shRNA targeting intelectin-1 and -2 attenuated allergen-induced airway inflammation. We conclude that intelectin, a molecule expressed by airway epithelial cells and upregulated in asthma, is required for IL-13-induced MCP-1 and -3 production in mouse lung epithelial cells and contributes to allergic airway inflammation.

  1. Allergic airway inflammation induces the migration of dendritic cells into airway sensory ganglia

    PubMed Central

    2014-01-01

    Background A neuroimmune crosstalk between dendritic cells (DCs) and airway nerves in the lung has recently been reported. However, the presence of DCs in airway sensory ganglia under normal and allergic conditions has not been explored so far. Therefore, this study aims to investigate the localisation, distribution and proliferation of DCs in airway sensory ganglia under allergic airway inflammation. Methods Using the house dust mite (HDM) model for allergic airway inflammation BALB/c mice were exposed to HDM extract intranasally (25 μg/50 μl) for 5 consecutive days a week over 7 weeks. With the help of the immunohistochemistry, vagal jugular-nodose ganglia complex (JNC) sections were analysed regarding their expression of DC-markers (MHC II, CD11c, CD103), the neuronal marker PGP 9.5 and the neuropeptide calcitonin gene-related peptide (CGRP) and glutamine synthetase (GS) as a marker for satellite glia cells (SGCs). To address the original source of DCs in sensory ganglia, a proliferation experiment was also carried in this study. Results Immune cells with characteristic DC-phenotype were found to be closely located to SGCs and vagal sensory neurons under physiological conditions. The percentage of DCs in relation to neurons was significantly increased by allergic airway inflammation in comparison to the controls (HDM 51.38 ± 2.38% vs. control 28.16 ± 2.86%, p < 0.001). The present study also demonstrated that DCs were shown to proliferate in jugular-nodose ganglia, however, the proliferation rate of DCs is not significantly changed in the two treated animal groups (proliferating DCs/ total DCs: HDM 0.89 ± 0.38%, vs. control 1.19 ± 0.54%, p = 0.68). Also, increased number of CGRP-positive neurons was found in JNC after allergic sensitisation and challenge (HDM 31.16 ± 5.41% vs. control 7.16 ± 1.53%, p < 0.001). Conclusion The present findings suggest that DCs may migrate from outside into the ganglia to interact with

  2. α7 Nicotinic Acetylcholine Receptor Regulates Airway Epithelium Differentiation by Controlling Basal Cell Proliferation

    PubMed Central

    Maouche, Kamel; Polette, Myriam; Jolly, Thomas; Medjber, Kahina; Cloëz-Tayarani, Isabelle; Changeux, Jean-Pierre; Burlet, Henriette; Terryn, Christine; Coraux, Christelle; Zahm, Jean-Marie; Birembaut, Philippe; Tournier, Jean-Marie

    2009-01-01

    Airway epithelial basal cells are known to be critical for regenerating injured epithelium and maintaining tissue homeostasis. Recent evidence suggests that the α7 nicotinic acetylcholine receptor (nAChR), which is highly permeable to Ca2+, is involved in lung morphogenesis. Here, we have investigated the potential role of the α7 nAChR in the regulation of airway epithelial basal cell proliferation and the differentiation of the human airway epithelium. In vivo during fetal development and in vitro during the regeneration of the human airway epithelium, α7 nAChR expression coincides with epithelium differentiation. Inactivating α7 nAChR function in vitro increases cell proliferation during the initial steps of the epithelium regeneration, leading to epithelial alterations such as basal cell hyperplasia and squamous metaplasia, remodeling observed in many bronchopulmonary diseases. The regeneration of the airway epithelium after injury in α7−/− mice is delayed and characterized by a transient hyperplasia of basal cells. Moreover, 1-year-old α7−/− mice more frequently present basal cells hyperplasia. Modulating nAChR function or expression shows that only α7 nAChR, as opposed to heteropentameric αxβy nAChRs, controls the proliferation of human airway epithelial basal cells. These findings suggest that α7 nAChR is a key regulator of the plasticity of the human airway epithelium by controlling basal cell proliferation and differentiation pathway and is involved in airway remodeling during bronchopulmonary diseases. PMID:19808646

  3. Pluripotent allospecific CD8+ effector T cells traffic to lung in murine obliterative airway disease.

    PubMed

    West, Erin E; Lavoie, Tera L; Orens, Jonathan B; Chen, Edward S; Ye, Shui Q; Finkelman, Fred D; Garcia, Joe G N; McDyer, John F

    2006-01-01

    Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+ IFN-gamma+ T cells were detected in airway allografts, with significant coexpression of TNF-alpha and granzyme B. Therefore, using IFN-gamma as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-gamma was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.

  4. Cyclooxygenase 2: its regulation, role and impact in airway inflammation.

    PubMed

    Rumzhum, N N; Ammit, A J

    2016-03-01

    Cyclooxygenase 2 (COX-2: official gene symbol - PTGS2) has long been regarded as playing a pivotal role in the pathogenesis of airway inflammation in respiratory diseases including asthma. COX-2 can be rapidly and robustly expressed in response to a diverse range of pro-inflammatory cytokines and mediators. Thus, increased levels of COX-2 protein and prostanoid metabolites serve as key contributors to pathobiology in respiratory diseases typified by dysregulated inflammation. But COX-2 products may not be all bad: prostanoids can exert anti-inflammatory/bronchoprotective functions in airways in addition to their pro-inflammatory actions. Herein, we outline COX-2 regulation and review the diverse stimuli known to induce COX-2 in the context of airway inflammation. We discuss some of the positive and negative effects that COX-2/prostanoids can exert in in vitro and in vivo models of airway inflammation, and suggest that inhibiting COX-2 expression to repress airway inflammation may be too blunt an approach; because although it might reduce the unwanted effects of COX-2 activation, it may also negate the positive effects. Evidence suggests that prostanoids produced via COX-2 upregulation show diverse actions (and herein we focus on prostaglandin E2 as a key example); these can be either beneficial or deleterious and their impact on respiratory disease can be dictated by local concentration and specific interaction with individual receptors. We propose that understanding the regulation of COX-2 expression and associated receptor-mediated functional outcomes may reveal number of critical steps amenable to pharmacological intervention. These may prove invaluable in our quest towards future development of novel anti-inflammatory pharmacotherapeutic strategies for the treatment of airway diseases. © 2015 John Wiley & Sons Ltd.

  5. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  6. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  7. The Role of Bitter and Sweet Taste Receptors in Upper Airway Immunity.

    PubMed

    Workman, Alan D; Palmer, James N; Adappa, Nithin D; Cohen, Noam A

    2015-12-01

    Over the past several years, taste receptors have emerged as key players in the regulation of innate immune defenses in the mammalian respiratory tract. Several cell types in the airway, including ciliated epithelial cells, solitary chemosensory cells, and bronchial smooth muscle cells, all display chemoresponsive properties that utilize taste receptors. A variety of bitter products secreted by microbes are detected with resultant downstream inflammation, increased mucous clearance, antimicrobial peptide secretion, and direct bacterial killing. Genetic variation of bitter taste receptors also appears to play a role in the susceptibility to infection in respiratory disease states, including that of chronic rhinosinusitis. Ongoing taste receptor research may yield new therapeutics that harness innate immune defenses in the respiratory tract and may offer alternatives to antibiotic treatment. The present review discusses taste receptor-protective responses and analyzes the role these receptors play in mediating airway immune function.

  8. The Role of Bitter and Sweet Taste Receptors in Upper Airway Immunity

    PubMed Central

    Workman, Alan D.; Palmer, James N.; Adappa, Nithin D.

    2016-01-01

    Over the past several years, taste receptors have emerged as key players in the regulation of innate immune defenses in the mammalian respiratory tract. Several cell types in the airway, including ciliated epithelial cells, solitary chemosensory cells, and bronchial smooth muscle cells, all display chemoresponsive properties that utilize taste receptors. A variety of bitter products secreted by microbes are detected with resultant downstream inflammation, increased mucous clearance, antimicrobial peptide secretion, and direct bacterial killing. Genetic variation of bitter taste receptors also appears to play a role in the susceptibility to infection in respiratory disease states, including that of chronic rhinosinusitis. Ongoing taste receptor research may yield new therapeutics that harness innate immune defenses in the respiratory tract and may offer alternatives to antibiotic treatment. The present review discusses taste receptor-protective responses and analyzes the role these receptors play in mediating airway immune function. PMID:26492878

  9. Oxidant-mediated ciliary dysfunction. Possible role in airway disease

    SciTech Connect

    Burman, W.J.; Martin, W.J. 2d.

    1986-03-01

    The effects of reactive species of oxygen on the airway are not well known. This study examined the effects of hydrogen peroxide (H2O2) on the structure and function of the airway epithelium. Tracheal rings were prepared from 200 g male rats. Damage to the airway epithelium was assayed by monitoring the ciliary beat frequency, the release of 51Cr, and histology. H2O2 at concentrations of 1.0 mM and above caused a very rapid decrease in ciliary beat frequency. After ten minutes' exposure to 1.0 mM, the ciliary beat frequency was 72 +/- 20 percent of control. Release of 51Cr was a less sensitive measure with significant release occurring after four hours of exposure to ciliotoxic concentrations of H2O2. Histologic changes were not evident within the experimental time period. All toxic effects of H2O2 were completely blocked by catalase. This study shows that H2O2 causes a rapid decline in ciliary activity and suggests that oxidant-mediated ciliary dysfunction could play a role in the pathogenesis of airway disease. The ciliary beat frequency provides a sensitive, physiologically relevant parameter for the in vitro study of these diseases.

  10. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  11. Theophylline Represses IL-8 Secretion from Airway Smooth Muscle Cells Independently of Phosphodiesterase Inhibition. Novel Role as a Protein Phosphatase 2A Activator.

    PubMed

    Patel, Brijeshkumar S; Rahman, Md Mostafizur; Rumzhum, Nowshin N; Oliver, Brian G; Verrills, Nicole M; Ammit, Alaina J

    2016-06-01

    Theophylline is an old drug experiencing a renaissance owing to its beneficial antiinflammatory effects in chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Multiple modes of antiinflammatory action have been reported, including inhibition of the enzymes that degrade cAMP-phosphodiesterase (PDE). Using primary cultures of airway smooth muscle (ASM) cells, we recently revealed that PDE4 inhibitors can potentiate the antiinflammatory action of β2-agonists by augmenting cAMP-dependent expression of the phosphatase that deactivates mitogen-activated protein kinase (MAPK)-MAPK phosphatase (MKP)-1. Therefore, the aim of this study was to address whether theophylline repressed cytokine production in a similar, PDE-dependent, MKP-1-mediated manner. Notably, theophylline did not potentiate cAMP release from ASM cells treated with the long-acting β2-agonist formoterol. Moreover, theophylline (0.1-10 μM) did not increase formoterol-induced MKP-1 messenger RNA expression nor protein up-regulation, consistent with the lack of cAMP generation. However, theophylline (at 10 μM) was antiinflammatory and repressed secretion of the neutrophil chemoattractant cytokine IL-8, which is produced in response to TNF-α. Because theophylline's effects were independent of PDE4 inhibition or antiinflammatory MKP-1, we then wished to elucidate the novel mechanisms responsible. We investigated the impact of theophylline on protein phosphatase (PP) 2A, a master controller of multiple inflammatory signaling pathways, and show that theophylline increases TNF-α-induced PP2A activity in ASM cells. Confirmatory results were obtained in A549 lung epithelial cells. PP2A activators have beneficial effects in ex vivo and in vivo models of respiratory disease. Thus, our study is the first to link theophylline with PP2A activation as a novel mechanism to control respiratory inflammation.

  12. Biofilm-dependent airway infections: a role for ambroxol?

    PubMed

    Cataldi, M; Sblendorio, V; Leo, A; Piazza, O

    2014-08-01

    Biofilms are a key factor in the development of both acute and chronic airway infections. Their relevance is well established in ventilator associated pneumonia, one of the most severe complications in critically ill patients, and in cystic fibrosis, the most common lethal genetic disease in Caucasians. Accumulating evidence suggests that biofilms could have also a role in chronic obstructive pulmonary disease and their involvement in bronchiectasis has been proposed as well. When they grow in biofilms, microorganisms become multidrug-resistant. Therefore the treatment of biofilm-dependent airway infections is problematic. Indeed, it still largely based on measures aiming to prevent the formation of biofilms or remove them once that they are formed. Here we review recent evidence suggesting that the mucokinetic drug ambroxol has specific anti-biofilm properties. We also discuss how additional pharmacological properties of this drug could be beneficial in biofilm-dependent airway infections. Specifically, we review the evidence showing that: 1-ambroxol exerts anti-inflammatory effects by inhibiting at multiple levels the activity of neutrophils, and 2-it improves mucociliary clearance by interfering with the activity of airway epithelium ion channels and transporters including sodium/bicarbonate and sodium/potassium/chloride cotransporters, cystic fibrosis transmembrane conductance regulator and aquaporins. As a whole, the data that we review here suggest that ambroxol could be helpful in biofilm-dependent airway infections. However, considering the limited clinical evidence available up to date, further clinical studies are required to support the use of ambroxol in these diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells.

    PubMed Central

    Kelley, T J; Drumm, M L

    1998-01-01

    It has been reported that exhaled nitric oxide levels are reduced in cystic fibrosis (CF) patients. We have examined the inducible isoform of nitric oxide synthase (iNOS) in the airways by immunostaining and found that iNOS is constitutively expressed in the airway epithelia of non-CF mouse and human tissues but essentially absent in the epithelium of CF airways. We explored potential consequences of lost iNOS expression and found that iNOS inhibition significantly increases mouse nasal trans-epithelial potential difference, and hindered the ability of excised mouse lungs to prevent growth of Pseudomonas aeruginosa. The absence of continuous nitric oxide production in epithelial cells of CF airways may play a role in two CF-associated characteristics: hyperabsorption of sodium and susceptibility to bacterial infections. PMID:9739054

  14. Post-transcriptional regulation of interleukin-10 in peripheral B cells of airway allergy patients

    PubMed Central

    Luo, Xiang-Qian; Yang, Shao-Bo; Qiu, Shu-Qi; Xie, Rui-Di; Yang, Li-Tao; Ke, Yu-Xing; Zhao, Hong-Xia; Geng, Xiao-Rui; Yang, Gui; Liu, Zhi-Qiang; Liu, Jiang-Qi; Wang, Shuai; Liu, Da-Bo; Liu, Jun

    2016-01-01

    The dysfunction of peripheral immune tolerance plays an important role in the pathogenesis of allergic diseases. Recent reports indicate that micro RNA (miR)-98 is associated with the process of aberrant immune responses. This study aims to test a hypothesis that miR-98 is associated with the pathogenesis of airway allergy via interfering with the development of regulatory B cells (Breg). In this study, patients with airway allergy were recruited into this study. The frequency of Bregs was assessed by flow cytometry. The levels of miR-98 in peripheral B cells were determined by RT-qPCR. A cell-culture model of B cells was developed to test the role of miR-98 in the repressing of interleukin (IL)-10 in B cells. The results showed that the levels of IL-10 in peripheral B cells were significantly lower in patients with airway allergy as compared with healthy subjects. High levels of miR-98 (one of the miR-98 members) were detected in peripheral B cells of patients with airway allergy, which was mimicked by stimulating B cells with IL-4. Histone acetyltransferase p300 was involved in the IL-4-induced miR-98 expression. miR-98 mediated the IL-4-inhibited IL-10 expression in B cells. In conclusion, miR-98 affects the expression of IL-10 in B cells and may be a novel therapeutic target for the treatment of allergic diseases. PMID:28078048

  15. Airway epithelial cell expression of interleukin-6 in transgenic mice. Uncoupling of airway inflammation and bronchial hyperreactivity.

    PubMed Central

    DiCosmo, B F; Geba, G P; Picarella, D; Elias, J A; Rankin, J A; Stripp, B R; Whitsett, J A; Flavell, R A

    1994-01-01

    We produced transgenic mice which overexpress human IL-6 in the airway epithelial cells. Transgenic mice develop a mononuclear cell infiltrate adjacent to large and mid-sized airways. Immunohistochemistry reveals these cells to be predominantly CD4+ cells, MHC class II+ cells, and B220+ cells. Transgenic mice and nontransgenic mice had similar baseline respiratory system resistance (0.47 +/- 0.06 vs 0.43 +/- 0.04 cmH2O/ml per s at 9 wk of age, P = NS and 0.45 +/- 0.07 vs 0.43 +/- 0.09 cmH2O/ml per s at 17 wk of age, P = NS). Transgenic mice, however, required a significantly higher log dose of methacholine to produce a 100% increase in respiratory system resistance as compared with non-transgenic littermates (1.34 +/- 0.24 vs 0.34 +/- 0.05 mg/ml, P < or = 0.01). We conclude that the expression of human IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine. These findings demonstrate an uncoupling of IL-6-induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology. Images PMID:7962549

  16. Role of IRE1α/XBP-1 in Cystic Fibrosis Airway Inflammation

    PubMed Central

    Ribeiro, Carla M. P.; Lubamba, Bob A.

    2017-01-01

    Cystic fibrosis (CF) pulmonary disease is characterized by chronic airway infection and inflammation. The infectious and inflamed CF airway environment impacts on the innate defense of airway epithelia and airway macrophages. The CF airway milieu induces an adaptation in these cells characterized by increased basal inflammation and a robust inflammatory response to inflammatory mediators. Recent studies have indicated that these responses depend on activation of the unfolded protein response (UPR). This review discusses the contribution of airway epithelia and airway macrophages to CF airway inflammatory responses and specifically highlights the functional importance of the UPR pathway mediated by IRE1/XBP-1 in these processes. These findings suggest that targeting the IRE1/XBP-1 UPR pathway may be a therapeutic strategy for CF airway disease. PMID:28075361

  17. Mast cell mediators in citric acid-induced airway constriction of guinea pigs

    SciTech Connect

    Lin, C.-H.; Lai, Y.-L. . E-mail: tiger@ha.mc.ntu.edu.tw

    2005-08-15

    We demonstrated previously that mast cells play an important role in citric acid (CA)-induced airway constriction. In this study, we further investigated the underlying mediator(s) for this type of airway constriction. At first, to examine effects caused by blocking agents, 67 young Hartley guinea pigs were divided into 7 groups: saline + CA; methysergide (serotonin receptor antagonist) + CA; MK-886 (leukotriene synthesis inhibitor) + CA; mepyramine (histamine H{sub 1} receptor antagonist) + CA; indomethacin (cyclooxygenase inhibitor) + CA; cromolyn sodium (mast cell stabilizer) + CA; and compound 48/80 (mast cell degranulating agent) + CA. Then, we tested whether leukotriene C{sub 4} (LTC{sub 4}) or histamine enhances CA-induced airway constriction in compound 48/80-pretreated guinea pigs. We measured dynamic respiratory compliance (Crs) and forced expiratory volume in 0.1 s (FEV{sub 0.1}) during either baseline or recovery period. In addition, we detected histamine level, an index of pulmonary mast cell degranulation, in bronchoalveolar lavage (BAL) samples. Citric acid aerosol inhalation caused decreases in Crs and FEV{sub 0.1}, indicating airway constriction in the control group. This airway constriction was significantly attenuated by MK-886, mepyramine, cromolyn sodium, and compound 48/80, but not by either methysergide or indomethacin. Both LTC{sub 4} and histamine infusion significantly increased the magnitude of CA-induced airway constriction in compound 48/80-pretreated guinea pigs. Citric acid inhalation caused significant increase in histamine level in the BAL sample, which was significantly suppressed by compound 48/80. These results suggest that leukotrienes and histamine originating from mast cells play an important role in CA inhalation-induced noncholinergic airway constriction.

  18. Highly Differentiated Human Airway Epithelial Cells: a Model to Study Host cell-parasite Interactions in Pertussis

    PubMed Central

    Guevara, Claudia; Zhang, Chengxian; Gaddy, Jennifer A.; Iqbal, Junaid; Guerra, Julio; Greenberg, David P.; Decker, Michael D.; Carbonetti, Nicholas; Starner, Timothy D.; McCray, Paul B.; Mooi, Frits R.

    2017-01-01

    Background Bordetella pertussis colonizes the human respiratory mucosa. Most studies on B. pertussis adherence have relied on cultured mammalian cells that lack key features present in differentiated human airway cells or on animal models that are not natural hosts of B. pertussis. The objectives of this work are to evaluate B. pertussis infection on highly differentiated human airway cells in vitro and to show the role of B. pertussis fimbriae in cell adherence. Methods Primary human airway epithelial (PHAE) cells from human bronchi and a human bronchial epithelial (HBE) cell line were grown in vitro under air-liquid interface conditions. Results PHAE and HBE cells infected with B. pertussis wild type strain revealed bacterial adherence to cell’s apical surface and bacterial induced cytoskeleton changes and cell detachment. Mutations in the major fimbrial subunits Fim2/3 or in the minor fimbrial adhesin subunit FimD affected B. pertussis adherence to predominantly HBE cells. This cell model recapitulates the morphologic features of the human airway infected by B. pertussis and confirms the role of fimbriae in B. pertussis adherence. Furthemore, HBE cells show that fimbrial subunits, and specifically FimD adhesin, are critical in B. pertussis adherence to airway cells. Conclusions The relevance of this model to study host-parasite interaction in pertussis lies in the striking physiologic and morphologic similarity between the PHAE and HBE cells and the human airway ciliated and goblet cells in vivo. These cells can proliferate in vitro, differentiate, and express the same genetic profile as human respiratory cells in vivo. PMID:26492208

  19. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

    PubMed

    Burke, Michael C; Li, Feng-Qian; Cyge, Benjamin; Arashiro, Takeshi; Brechbuhl, Heather M; Chen, Xingwang; Siller, Saul S; Weiss, Matthew A; O'Connell, Christopher B; Love, Damon; Westlake, Christopher J; Reynolds, Susan D; Kuriyama, Ryoko; Takemaru, Ken-Ichi

    2014-10-13

    Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

  20. Insights into Group 2 Innate Lymphoid Cells in Human Airway Disease.

    PubMed

    Karta, Maya R; Broide, David H; Doherty, Taylor A

    2016-01-01

    Recent discoveries have led to the identification of a novel group of immune cells, the innate lymphoid cells (ILCs). The members of this group are divided into three subpopulations: ILC1s, ILC2s, and ILC3s. ILC2s produce Th2 cytokines, IL-4, IL-5, and IL-13, upon activation by epithelial cell-derived cytokines, lipid mediators (cysteinyl leukotrienes and prostaglandin D2), and TNF family member TL1A and promote structural and immune cell responses in the airways after antigen exposure. In addition, ILC2 function is also influenced by inducible T cell costimulator (ICOS)/ICOS-ligand (ICOS-L) interactions via direct contact between immune cells. The most common airway antigens are allergens and viruses which are highly linked to the induction of airway diseases with underlying type 2 inflammation including asthma and allergic rhinitis. Based on recent findings linking ILC2s and airway Th2 responses, there is intensive investigation into the role of ILC2s in human disease with the hope of a better understanding of the pathophysiology and the discovery of novel potential therapeutic targets. This review summarizes the recent advances made in elucidating ILC2 involvement in human Th2 airway disease.

  1. [Regulation of airway stem cell proliferation in idiopathic pulmonary fibrosis].

    PubMed

    Yang, S X; Wu, Q; Sun, X; Li, X; Li, K; Xu, L; Li, Y; Zhang, Q Y; Zhang, Y C; Chen, H Y

    2016-09-01

    To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis. Lung cell suspension was prepared from β-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-β inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed. The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-β receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%. During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

  2. Role of mitochondrial hydrogen peroxide induced by intermittent hypoxia in airway epithelial wound repair in vitro.

    PubMed

    Hamada, Satoshi; Sato, Atsuyasu; Hara-Chikuma, Mariko; Satooka, Hiroki; Hasegawa, Koichi; Tanimura, Kazuya; Tanizawa, Kiminobu; Inouchi, Morito; Handa, Tomohiro; Oga, Toru; Muro, Shigeo; Mishima, Michiaki; Chin, Kazuo

    2016-05-15

    The airway epithelium acts as a frontline barrier against various environmental insults and its repair process after airway injury is critical for the lung homeostasis restoration. Recently, the role of intracellular reactive oxygen species (ROS) as transcription-independent damage signaling has been highlighted in the wound repair process. Both conditions of continuous hypoxia and intermittent hypoxia (IH) induce ROS. Although IH is important in clinical settings, the roles of IH-induced ROS in the airway repair process have not been investigated. In this study, we firstly showed that IH induced mitochondrial hydrogen peroxide (H2O2) production and significantly decreased bronchial epithelial cell migration, prevented by catalase treatment in a wound scratch assay. RhoA activity was higher during repair process in the IH condition compared to in the normoxic condition, resulting in the cellular morphological changes shown by immunofluorescence staining: round cells, reduced central stress fiber numbers, pronounced cortical actin filament distributions, and punctate focal adhesions. These phenotypes were replicated by exogenous H2O2 treatment under the normoxic condition. Our findings confirmed the transcription-independent role of IH-induced intracellular ROS in the bronchial epithelial cell repair process and might have significant implications for impaired bronchial epithelial cell regeneration.

  3. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

    PubMed Central

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M.; Collins, Jane E.; Davies, Donna E.; Morgan, Hywel; Swindle, Emily J.

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  4. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells.

    PubMed Central

    Scott, P H; Belham, C M; al-Hafidh, J; Chilvers, E R; Peacock, A J; Gould, G W; Plevin, R

    1996-01-01

    In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation. PMID:8836145

  5. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  6. Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

    PubMed Central

    Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

    2016-01-01

    Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

  7. The role bronchoscopy in the diagnosis of airway disease in children

    PubMed Central

    Soyer, Tutku

    2016-01-01

    Bronchoscopy is endoscopic examination of airways that allows both diagnostic and interventional procedures in the evaluation of airway disease in children. It can be performed with either rigid or flexible instruments, depending on the particular needs of patients and skills of bronchoscopist. In addition to visualization of airways, bronchoscopy enables to obtain specimens from lungs and distal airways. Bronchoalveolar lavage (BAL) yields samples from surfaces of the alveoli and aids differential diagnosis of various pulmonary disease. Foreign body removal and examination of anatomy and dynamics of airways are also common indications of bronchoscopy in children. Improvement in the technology, endoscopic instrumentation allows detailed evaluation and interventional manipulation of airway lesions in small children. Although bronchoscopy is considered as a safe procedure, obstruction of airway may challenge and require special endoscopic skills with appropriate instrumentation. This review is aimed to outline the role of bronchoscopy in diagnosis airway disease in children. PMID:28066622

  8. Putting the Squeeze on Airway Epithelia

    PubMed Central

    Park, Jin-Ah; Fredberg, Jeffrey J.

    2015-01-01

    Asthma is characterized by chronic inflammation, airway hyperresponsiveness, and progressive airway remodeling. The airway epithelium is known to play a critical role in the initiation and perpetuation of these processes. Here, we review how excessive epithelial stress generated by bronchoconstriction is sufficient to induce airway remodeling, even in the absence of inflammatory cells. PMID:26136543

  9. TLR4 signalling in pulmonary stromal cells is critical for inflammation and immunity in the airways.

    PubMed

    Perros, Frederic; Lambrecht, Bart N; Hammad, Hamida

    2011-09-24

    Inflammation of the airways, which is often associated with life-threatening infection by Gram-negative bacteria or presence of endotoxin in the bioaerosol, is still a major cause of severe airway diseases. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to protein rich fluid leak in the alveolar space. Mammalian Toll-like receptors (TLRs) are important signalling receptors in innate host defense. Among these receptors, TLR4 plays a critical role in the response to endotoxin. Lungs are a complex compartmentalized organ with separate barriers, namely the alveolar-capillary barrier, the microvascular endothelium, and the alveolar epithelium. An emerging theme in the field of lung immunology is that structural cells (SCs) of the airways such as epithelial cells (ECs), endothelial cells, fibroblasts and other stromal cells produce activating cytokines that determine the quantity and quality of the lung immune response. This review focuses on the role of TLR4 in the innate and adaptive immune functions of the pulmonary SCs.

  10. TLR4 signalling in pulmonary stromal cells is critical for inflammation and immunity in the airways

    PubMed Central

    2011-01-01

    Inflammation of the airways, which is often associated with life-threatening infection by Gram-negative bacteria or presence of endotoxin in the bioaerosol, is still a major cause of severe airway diseases. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to protein rich fluid leak in the alveolar space. Mammalian Toll-like receptors (TLRs) are important signalling receptors in innate host defense. Among these receptors, TLR4 plays a critical role in the response to endotoxin. Lungs are a complex compartmentalized organ with separate barriers, namely the alveolar-capillary barrier, the microvascular endothelium, and the alveolar epithelium. An emerging theme in the field of lung immunology is that structural cells (SCs) of the airways such as epithelial cells (ECs), endothelial cells, fibroblasts and other stromal cells produce activating cytokines that determine the quantity and quality of the lung immune response. This review focuses on the role of TLR4 in the innate and adaptive immune functions of the pulmonary SCs. PMID:21943186

  11. Epithelial Cell Proliferation Contributes to Airway Remodeling in Severe Asthma

    PubMed Central

    Cohen, Lance; E, Xueping; Tarsi, Jaime; Ramkumar, Thiruvamoor; Horiuchi, Todd K.; Cochran, Rebecca; DeMartino, Steve; Schechtman, Kenneth B.; Hussain, Iftikhar; Holtzman, Michael J.; Castro, Mario

    2007-01-01

    Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. Measurements and Main Results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p = 0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p = 0.002) and Ki67 was increased (p < 0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p < 0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p = 0.002), suggesting increased cell death. Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe

  12. Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium

    SciTech Connect

    Rennolds, Jessica; Malireddy, Smitha; Hassan, Fatemat; Tridandapani, Susheela; Parinandi, Narasimham; Boyaka, Prosper N.; Cormet-Boyaka, Estelle

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cadmium induces secretion of IL-6 and IL-8 by two distinct pathways. Black-Right-Pointing-Pointer Cadmium increases NAPDH oxidase activity leading to Erk activation and IL-8 secretion. Black-Right-Pointing-Pointer Curcumin prevents cadmium-induced secretion of both IL-6 and IL-8 by airway cells. Black-Right-Pointing-Pointer Curcumin could be use to suppress lung inflammation due to cadmium inhalation. -- Abstract: Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmium promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-{kappa}B dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation.

  13. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  14. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  15. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    PubMed Central

    2009-01-01

    Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide. PMID:19650888

  16. Lung airway-surveilling CXCR3(hi) memory CD8(+) T cells are critical for protection against influenza A virus.

    PubMed

    Slütter, Bram; Pewe, Lecia L; Kaech, Susan M; Harty, John T

    2013-11-14

    Inducing memory CD8(+) T cells specific for conserved antigens from influenza A virus (IAV) is a potential strategy for broadly protective vaccines. Here we show that memory CD8(+) T cells in the airways played an important role in early control of IAV. Expression of the chemokine receptor CXCR3 was critical for memory CD8(+) T cells to populate the airways during the steady state and vaccination approaches were designed to favor the establishment of memory CD8(+) T cells in the airways. Specifically, we found that interleukin-12 (IL-12) signaling shortly after immunization limited CXCR3 expression on memory CD8(+) T cells. Neutralization of IL-12 or adjuvants that did not induce high amounts of IL-12 enhanced CXCR3 expression, sustained airway localization of memory CD8(+) T cells, and resulted in superior protection against IAV.

  17. Regulation of human airway smooth muscle cell migration and relevance to asthma.

    PubMed

    Salter, Brittany; Pray, Cara; Radford, Katherine; Martin, James G; Nair, Parameswaran

    2017-08-16

    Airway remodelling is an important feature of asthma pathogenesis. A key structural change inherent in airway remodelling is increased airway smooth muscle mass. There is emerging evidence to suggest that the migration of airway smooth muscle cells may contribute to cellular hyperplasia, and thus increased airway smooth muscle mass. The precise source of these cells remains unknown. Increased airway smooth muscle mass may be collectively due to airway infiltration of myofibroblasts, neighbouring airway smooth muscle cells in the bundle, or circulating hemopoietic progenitor cells. However, the relative contribution of each cell type is not well understood. In addition, although many studies have identified pro and anti-migratory agents of airway smooth muscle cells, whether these agents can impact airway remodelling in the context of human asthma, remains to be elucidated. As such, further research is required to determine the exact mechanism behind airway smooth muscle cell migration within the airways, how much this contributes to airway smooth muscle mass in asthma, and whether attenuating this migration may provide a therapeutic avenue for asthma. In this review article, we will discuss the current evidence with respect to the regulation of airway smooth muscle cell migration in asthma.

  18. Immunologically Induced Alterations of Airway Smooth Muscle Cell Membrane

    NASA Astrophysics Data System (ADS)

    Souhrada, M.; Souhrada, J. F.

    1984-08-01

    Active and passive sensitization, both in vivo and in vitro, caused significant hyperpolarization of airway smooth muscle cell preparations isolated from guinea pigs. An increase in the contribution of the electrogenic Na+ pump to the resting membrane potential was responsible for this change. Hyperpolarization, as induced by passive sensitization, was not prevented by agents that inhibit specific mediators of anaphylaxis but was abolished when serum from sensitized animals was heated. The heat-sensitive serum factor, presumably reaginic antibodies, appears to be responsible for the membrane hyperpolarization of airway smooth muscle cells after sensitization.

  19. Store-operated Ca2+ channels in airway epithelial cell function and implications for asthma

    PubMed Central

    Samanta, Krishna; Parekh, Anant B.

    2016-01-01

    The epithelial cells of the lung are at the interface of a host and its environment and are therefore directly exposed to the inhaled air-borne particles. Rather than serving as a simple physical barrier, airway epithelia detect allergens and other irritants and then help organize the subsequent immune response through release of a plethora of secreted signals. Many of these signals are generated in response to opening of store-operated Ca2+ channels in the plasma membrane. In this review, we describe the properties of airway store-operated channels and their role in regulating airway epithelial cell function. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377718

  20. Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer.

    PubMed

    Watkins, D Neil; Berman, David M; Burkholder, Scott G; Wang, Baolin; Beachy, Philip A; Baylin, Stephen B

    2003-03-20

    Embryonic signalling pathways regulate progenitor cell fates in mammalian epithelial development and cancer. Prompted by the requirement for sonic hedgehog (Shh) signalling in lung development, we investigated a role for this pathway in regeneration and carcinogenesis of airway epithelium. Here we demonstrate extensive activation of the hedgehog (Hh) pathway within the airway epithelium during repair of acute airway injury. This mode of Hh signalling is characterized by the elaboration and reception of the Shh signal within the epithelial compartment, and immediately precedes neuroendocrine differentiation. We reveal a similar pattern of Hh signalling in airway development during normal differentiation of pulmonary neuroendocrine precursor cells, and in a subset of small-cell lung cancer (SCLC), a highly aggressive and frequently lethal human tumour with primitive neuroendocrine features. These tumours maintain their malignant phenotype in vitro and in vivo through ligand-dependent Hh pathway activation. We propose that some types of SCLC might recapitulate a critical, Hh-regulated event in airway epithelial differentiation. This requirement for Hh pathway activation identifies a common lethal malignancy that may respond to pharmacological blockade of the Hh signalling pathway.

  1. TLR2 Regulates Gap Junction Intercellular Communication in Airway Cells

    PubMed Central

    Martin, Francis J.; Prince, Alice S.

    2009-01-01

    The innate immune response to inhaled bacteria, such as the opportunist Pseudomonas aeruginosa, is initiated by TLR2 displayed on the apical surface of airway epithelial cells. Activation of TLR2 is accompanied by an immediate Ca2+ flux that is both necessary and sufficient to stimulate NF-κB and MAPK proinflammatory signaling to recruit and activate polymorphonuclear leukocytes in the airway. In human airway cells gap junction channels were found to provide a regulated conduit for the movement of Ca2+ from cell to cell. In response to TLR2 stimulation, by either lipid agonists or P. aeruginosa, gap junctions functioned to transiently amplify proinflammatory signaling by communicating Ca2+ fluxes from stimulated to adjacent, non-stimulated cells thus increasing epithelial CXCL8 production. P. aeruginosa stimulation also induced tyrosine phosphorylation of Connexin 43 and association with c-Src, events linked to the closure of these channels. By 4 hours post bacterial stimulation, gap junction communication was decreased indicating an autoregulatory control of the connexins. Thus, gap junction channels comprised of Connexin 43 and other connexins in airway cells provide a mechanism to coordinate and regulate the epithelial immune response even in the absence of signals from the immune system. PMID:18354224

  2. The Role of Capital Productivity in British Airways' Financial Recovery

    NASA Technical Reports Server (NTRS)

    Morrell, Peter

    1999-01-01

    British Airways (BA) was privatised in 1987, but its financial recovery occurred a number of years earlier. This recovery was sustained throughout the early 1990s economic recession, a period when few major airlines were operating profitably. This paper examines the role of productivity developments at British Airways from the early 1980s through 1996. The emphasis is on capital productivity and investment, but changes in capital intensity and labour productivity are also evaluated. Various measures are considered for both capital and labour productivity: outputs are measured in available tonne-kms (ATKS) and revenue tonne-kms (RTKs), with the former preferred over the latter two measures, after adjustment for work performed by BA for others. Capital inputs are measured in equivalent lease costs adjusted to constant prices with a different treatment of flight and ground equipment or assets. Labour inputs are derived from total payroll costs deflated by a UK wage price index. The airline made considerable capital investments over the period and at the same time went through two major processes of labour restructuring. This resulted in a gradual increase in capital intensity, relative high labour productivity growth, but poor capital productivity performance. However, capital investment played an important role in the airline's sustained labour and total factor productivity over the whole period.

  3. The Role of Capital Productivity in British Airways' Financial Recovery

    NASA Technical Reports Server (NTRS)

    Morrell, Peter

    1999-01-01

    British Airways (BA) was privatized in 1987, but its financial recovery occurred a number of years earlier, This recovery was sustained throughout the early 1990s economic recession, a period when few major airlines were operating profitably. This paper examines the role of productivity developments at British Airways from the early 1980s through 1996. The emphasis is on capital productivity and investment, but changes in capital intensity and labour productivity are also evaluated. Various measures are considered for both capital and labour productivity: outputs are measured in available tonne-kms (ATKs) and revenue tonne-kms (RTKs), with the former preferred over the latter two measures, after adjustment for work performed by BA for others. Capital inputs are measured in equivalent lease costs adjusted to constant prices with a different treatment of flight and ground equipment or assets. Labour inputs are derived from total payroll costs deflated by a UK wage price index. The airline made considerable capital investments over the period and at the same time went through two major processes of labour restructuring. This resulted in a gradual increase in capital intensity, relative high labour productivity growth, but poor capital productivity performance, However, capital investment played an important role in the airline's sustained labour and total factor productivity over the whole period.

  4. The Role of Capital Productivity in British Airways' Financial Recovery

    NASA Technical Reports Server (NTRS)

    Morrell, Peter

    1999-01-01

    British Airways (BA) was privatized in 1987, but its financial recovery occurred a number of years earlier, This recovery was sustained throughout the early 1990s economic recession, a period when few major airlines were operating profitably. This paper examines the role of productivity developments at British Airways from the early 1980s through 1996. The emphasis is on capital productivity and investment, but changes in capital intensity and labour productivity are also evaluated. Various measures are considered for both capital and labour productivity: outputs are measured in available tonne-kms (ATKs) and revenue tonne-kms (RTKs), with the former preferred over the latter two measures, after adjustment for work performed by BA for others. Capital inputs are measured in equivalent lease costs adjusted to constant prices with a different treatment of flight and ground equipment or assets. Labour inputs are derived from total payroll costs deflated by a UK wage price index. The airline made considerable capital investments over the period and at the same time went through two major processes of labour restructuring. This resulted in a gradual increase in capital intensity, relative high labour productivity growth, but poor capital productivity performance, However, capital investment played an important role in the airline's sustained labour and total factor productivity over the whole period.

  5. Airway Epithelium Interactions with Aeroallergens: Role of Secreted Cytokines and Chemokines in Innate Immunity

    PubMed Central

    Gandhi, Vivek D.; Vliagoftis, Harissios

    2015-01-01

    Airway epithelial cells are the first line of defense against the constituents of the inhaled air, which include allergens, pathogens, pollutants, and toxic compounds. The epithelium not only prevents the penetration of these foreign substances into the interstitium, but also senses their presence and informs the organism’s immune system of the impending assault. The epithelium accomplishes the latter through the release of inflammatory cytokines and chemokines that recruit and activate innate immune cells at the site of assault. These epithelial responses aim to eliminate the inhaled foreign substances and minimize their detrimental effects to the organism. Quite frequently, however, the innate immune responses of the epithelium to inhaled substances lead to chronic and high level release of pro-inflammatory mediators that may mediate the lung pathology seen in asthma. The interactions of airway epithelial cells with allergens will be discussed with particular focus on interactions-mediated epithelial release of cytokines and chemokines and their role in the immune response. As pollutants are other major constituents of inhaled air, we will also discuss how pollutants may alter the responses of airway epithelial cells to allergens. PMID:25883597

  6. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    PubMed

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  7. Pulmonary Stress Induced by Hyperthermia: Role of Airway Sensory Nerves

    DTIC Science & Technology

    2014-12-01

    Hyperthermia, asthma, airway constriction, cough , dyspnea 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...obtained from our completed study of thermal stress generated various airway dysfunctions (airway constriction, cough , etc.) in patients with allergic...airway irritation and cough in patients with allergic rhinitis Results obtained from this study series have been published in the journal Respiratory

  8. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  9. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  10. Vanishing Bronchus After Lung Transplantation: The Role of Sequential Airway Dilatations

    PubMed Central

    Alraiyes, Abdul Hamid; Inaty, Hanine; Machuzak, Michael S.

    2017-01-01

    Background: Airway complications after lung transplant play an important role in patient survival. Early recognition and treatment of these complications are necessary to help ensure that patients who receive lung transplants have good outcomes. Case Report: A 61-year-old female with a history of pulmonary venous occlusive disease presented to our hospital for a double-lung transplant. Her postoperative course was complicated by severe primary graft dysfunction. Airway examination showed significant mucosal ischemia distal to the anastomosis bilaterally with diffuse narrowing of all distal bronchial segments. Repeat bronchoscopies with debridement of necrotic material and balloon dilatation of stenotic airways were performed to maintain airway patency. Conclusion: Post–lung transplant airway necrosis and stenosis mandate early identification and treatment. Repetitive bronchoscopies with sequential balloon dilatations are mandatory to prevent future airway stenosis and airway vanishing. PMID:28331451

  11. The role of reactive oxygen and nitrogen species in airway epithelial gene expression.

    PubMed Central

    Martin, L D; Krunkosky, T M; Voynow, J A; Adler, K B

    1998-01-01

    The body first encounters deleterious inhaled substances, such as allergens, industrial particles, pollutants, and infectious agents, at the airway epithelium. When this occurs, the epithelium and its resident inflammatory cells respond defensively by increasing production of cytokines, mucus, and reactive oxygen and nitrogen species (ROS/RNS). As inflammation in the airway increases, additional infiltrating cells increase the level of these products. Recent interest has focused on ROS/RNS as potential modulators of the expression of inflammation-associated genes important to the pathogenesis of various respiratory diseases. ROS/RNS appear to play a variety of roles that lead to changes in expression of genes such as interleukin-6 and intercellular adhesion molecule 1. By controlling this regulation, the reactive species can serve as exogenous stimuli, as intercellular signaling molecules, and as modulators of the redox state in epithelial cells. Unraveling the molecular mechanisms affected by ROS/RNS acting in these capacities should aid in the understanding of how stimulated defense mechanisms within the airway can lead to disease. Images Figure 1 PMID:9788898

  12. [Regeneration of airway epithelium].

    PubMed

    Adam, D; Perotin, J-M; Lebargy, F; Birembaut, P; Deslée, G; Coraux, C

    2014-04-01

    Epithelial regeneration is a complex process. It can lead to the remodeling of the airway epithelium as in asthma, COPD or cystic fibrosis. The development of in vivo and in vitro models has allowed the analysis of remodeling mechanisms and showed the role of components of extracellular matrix, proteases, cytokines and growth factors. Airway epithelial progenitors and stems cells have been studied in these models. However, their identification remains difficult. Identification and characterization of airway epithelial progenitor/stem-cells, and a better knowledge of the regeneration process may allow the development of new therapeutic strategies for airway epithelial reconstitution. Copyright © 2013 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  13. Local blockade of epithelial PDL-1 in the airways enhances T cell function and viral clearance during influenza virus infection.

    PubMed

    McNally, Beth; Ye, Fang; Willette, Meredith; Flaño, Emilio

    2013-12-01

    In order to maintain the gas exchange function of the lung following influenza virus infection, a delicate orchestration of positive and negative regulatory pathways must be maintained to attain viral eradication while minimizing local inflammation. The programmed death receptor 1 ligand/programmed death receptor 1 (PDL-1/PD-1) pathway plays an important immunoregulatory role, particularly in the context of T cell function. Here, we have shown that influenza virus infection of primary airway epithelial cells strongly enhances PDL-1 expression and does so in an alpha interferon receptor (IFNAR) signaling-dependent manner. PD-1 is expressed primarily on effector T cells in the lung, compared to effector memory and central memory cells, and shortly after influenza virus infection, an increased number of PD-1(+) T cells are recruited to the airways. Using in vitro cocultures of airway epithelial cells and T cells and in vivo models of influenza virus infection, we have demonstrated that blockade of airway epithelial PDL-1 improves CD8 T cell function, defined by increased production of gamma interferon (IFN-γ) and granzyme B and expression of CD107ab. Furthermore, PDL-1 blockade in the airways served to accelerate influenza virus clearance and enhance infection recovery. Our findings suggest that local manipulation of the PDL-1/PD-1 axis in the airways may represent a therapeutic alternative during acute influenza virus infection.

  14. Ca2+ signaling in airway epithelial cells facilitates leukocyte recruitment and transepithelial migration

    PubMed Central

    Chun, Jarin; Prince, Alice

    2009-01-01

    In airway cells, TLR2 stimulation by bacterial products activates Ca2+ fluxes that signal leukocyte recruitment to the lung and facilitates transepithelial migration into the airway lumen. TLR2 is apically displayed on airway cells, where it senses bacterial stimuli. Biochemical and genetic approaches demonstrate that TLR2 ligands stimulate release of Ca2+ from intracellular stores by activating TLR2 phosphorylation by c-Src and recruiting PI3K and PLCγ to affect Ca2+ release through IP3Rs. This Ca2+ release plays a pivotal role in signaling TLR2-dependent NF-κB activation and chemokine expression to recruit PMNs to the lung. In addition, TLR2-initiated Ca2+ release activates Ca2+-dependent proteases, calpains, which cleave the transmembrane proteins occludin and E-cadherin to promote PMN transmigration. This review highlights recent findings that demonstrate a central role for Ca2+ signaling in airway epithelial cells to induce proinflammatory gene transcription and to initiate junctional changes that accommodate transmigration of recruited PMNs. PMID:19605699

  15. CD38 and airway hyper-responsiveness: studies on human airway smooth muscle cells and mouse models.

    PubMed

    Guedes, Alonso G P; Deshpande, Deepak A; Dileepan, Mythili; Walseth, Timothy F; Panettieri, Reynold A; Subramanian, Subbaya; Kannan, Mathur S

    2015-02-01

    Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.

  16. Aeroallergen challenge promotes dendritic cell proliferation in the airways.

    PubMed

    Veres, Tibor Z; Voedisch, Sabrina; Spies, Emma; Valtonen, Joona; Prenzler, Frauke; Braun, Armin

    2013-02-01

    Aeroallergen provocation induces the rapid accumulation of CD11c(+)MHC class II (MHC II)(+) dendritic cells (DCs) in the lungs, which is driven by an increased recruitment of blood-derived DC precursors. Recent data show, however, that well-differentiated DCs proliferate in situ in various tissues. This may also contribute to their allergen-induced expansion; therefore, we studied DC proliferation in the airways of mice in the steady state and after local aeroallergen provocation. Confocal whole-mount microscopy was used to visualize proliferating DCs in different microanatomical compartments of the lung. We demonstrate that in the steady state, CD11c(+)MHC II(+) DCs proliferate in both the epithelial and subepithelial layers of the airway mucosa as well as in the lung parenchyma. A 1-h pulse of the nucleotide 5-ethynyl-2'-deoxyuridine was sufficient to label 5% of DCs in both layers of the airway mucosa. On the level of whole-lung tissue, 3-5% of both CD11b(+) and CD11b(-) DC populations and 0.3% of CD11c(+)MHC II(low) lung macrophages incorporated 5-ethynyl-2'-deoxyuridine. Aeroallergen provocation caused a 3-fold increase in the frequency of locally proliferating DCs in the airway mucosa. This increase in mucosal DC proliferation was later followed by an elevation in the number of DCs. The recruitment of monocyte-derived inflammatory DCs contributed to the increasing number of DCs in the lung parenchyma, but not in the airway mucosa. We conclude that local proliferation significantly contributes to airway DC homeostasis in the steady state and that it is the major mechanism underlying the expansion of the mucosal epithelial/subepithelial DC network in allergic inflammation.

  17. Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice.

    PubMed

    Williams, C M; Galli, S J

    2000-08-07

    The importance of mast cells in the development of the allergen-induced airway hyperreactivity and inflammation associated with asthma remains controversial. We found that genetically mast cell-deficient WBB6F(1)-W/W(v) mice that were sensitized to ovalbumin (OVA) without adjuvant, then challenged repetitively with antigen intranasally, exhibited much weaker responses in terms of bronchial hyperreactivity to aerosolized methacholine, lung tissue eosinophil infiltration, and numbers of proliferating cells within the airway epithelium than did identically treated WBB6F(1)-+/+ normal mice. However, W/W(v) mice that had undergone selective reconstitution of tissue mast cells with in vitro-derived mast cells of congenic +/+ mouse origin exhibited airway responses that were very similar to those of the +/+ mice. By contrast, W/W(v) mice that were sensitized with OVA emulsified in alum and challenged with aerosolized OVA exhibited levels of airway hyperreactivity and lung tissue eosinophil infiltration that were similar to those of the corresponding +/+ mice. Nevertheless, these W/W(v) mice exhibited significantly fewer proliferating cells within the airway epithelium than did identically treated +/+ mice. These results show that, depending on the "asthma model" investigated, mast cells can either have a critical role in, or not be essential for, multiple features of allergic airway responses in mice.

  18. Differentiated transplant derived airway epithelial cell cytokine secretion is not regulated by cyclosporine

    PubMed Central

    2011-01-01

    Background While lung transplantation is an increasingly utilized therapy for advanced lung diseases, chronic rejection in the form of Bronchiolitis Obliterans Syndrome (BOS) continues to result in significant allograft dysfunction and patient mortality. Despite correlation of clinical events with eventual development of BOS, the causative pathophysiology remains unknown. Airway epithelial cells within the region of inflammation and fibrosis associated with BOS may have a participatory role. Methods Transplant derived airway epithelial cells differentiated in air liquid interface culture were treated with IL-1β and/or cyclosporine, after which secretion of cytokines and growth factor and gene expression for markers of epithelial to mesenchymal transition were analyzed. Results Secretion of IL-6, IL-8, and TNF-α, but not TGF-β1, was increased by IL-1β stimulation. In contrast to previous studies using epithelial cells grown in submersion culture, treatment of differentiated cells in ALI culture with cyclosporine did not elicit cytokine or growth factor secretion, and did not alter IL-6, IL-8, or TNF-α production in response to IL-1β treatment. Neither IL-1β nor cyclosporine elicited expression of markers of the epithelial to mesenchymal transition E-cadherin, EDN-fibronectin, and α-smooth muscle actin. Conclusion Transplant derived differentiated airway epithelial cell IL-6, IL-8, and TNF-α secretion is not regulated by cyclosporine in vitro; these cells thus may participate in local inflammatory responses in the setting of immunosuppression. Further, treatment with IL-1β did not elicit gene expression of markers of epithelial to mesenchymal transition. These data present a model of differentiated airway epithelial cells that may be useful in understanding epithelial participation in airway inflammation and allograft rejection in lung transplantation. PMID:21477368

  19. Adhesion of Streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter.

    PubMed

    Mushtaq, Naseem; Ezzati, Majid; Hall, Lucinda; Dickson, Iain; Kirwan, Michael; Png, Ken M Y; Mudway, Ian S; Grigg, Jonathan

    2011-05-01

    Epidemiologic studies report an association between pneumonia and urban particulate matter (PM) less than 10 microns (μm) in aerodynamic diameter (PM(10)). Streptococcus pneumoniae is a common cause of bacterial pneumonia worldwide. To date, the mechanism whereby urban PM enhances vulnerability to S pneumoniae infection is unclear. Adhesion of S pneumoniae to host cells is a prerequisite for infection. Host-expressed proteins, including the receptor for platelet-activating factor (PAFR), are co-opted by S pneumoniae to adhere to lower airway epithelial cells. To define whether inhalable urban PM enhances the adhesion of S pneumoniae to airway epithelial cells. A549 cells were cultured with PM(10) from Leicester (United Kingdom [UK]) and PM(10) and PM less than 2.5 μm in aerodynamic diameter (PM(2.5)) from Accra (Ghana), then infected with S pneumoniae strain D39. Pneumococcal adhesion to human primary bronchial epithelial cells was also assessed. Bacterial adhesion was determined by quantitative culture and confocal microscopy. The role of oxidative stress was assessed by N-acetyl cysteine, and the role of PAFR was assessed by mRNA transcript level, receptor expression, and receptor blocking. PM(10) (UK) increased S pneumoniae adhesion to both A549 airway epithelial cells and human primary bronchial epithelial cells. PM(10) (Ghana) and PM(2.5) (Ghana) also increased adhesion. Culture of A549 cells by PM(10) (UK) increased PAFR mRNA transcript level and PAFR expression. PM(10) (UK)-stimulated adhesion to A549 cells was attenuated by a PAFR blocker and N-acetyl cysteine. Urban PM increases adhesion of S pneumoniae to human airway epithelial cells. PM-stimulated adhesion is mediated by oxidative stress and PAFR. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  20. Dysregulation of type 2 innate lymphoid cells and TH2 cells impairs pollutant-induced allergic airway responses.

    PubMed

    De Grove, Katrien C; Provoost, Sharen; Hendriks, Rudi W; McKenzie, Andrew N J; Seys, Leen J M; Kumar, Smitha; Maes, Tania; Brusselle, Guy G; Joos, Guy F

    2017-01-01

    Although the prominent role of TH2 cells in type 2 immune responses is well established, the newly identified type 2 innate lymphoid cells (ILC2s) can also contribute to orchestration of allergic responses. Several experimental and epidemiologic studies have provided evidence that allergen-induced airway responses can be further enhanced on exposure to environmental pollutants, such as diesel exhaust particles (DEPs). However, the components and pathways responsible remain incompletely known. We sought to investigate the relative contribution of ILC2 and adaptive TH2 cell responses in a murine model of DEP-enhanced allergic airway inflammation. Wild-type, Gata-3(+/nlslacZ) (Gata-3-haploinsufficient), RAR-related orphan receptor α (RORα)(fl/fl)IL7R(Cre) (ILC2-deficient), and recombination-activating gene (Rag) 2(-/-) mice were challenged with saline, DEPs, or house dust mite (HDM) or DEP+HDM. Airway hyperresponsiveness, as well as inflammation, and intracellular cytokine expression in ILC2s and TH2 cells in the bronchoalveolar lavage fluid and lung tissue were assessed. Concomitant DEP+HDM exposure significantly enhanced allergic airway inflammation, as characterized by increased airway eosinophilia, goblet cell metaplasia, accumulation of ILC2s and TH2 cells, type 2 cytokine production, and airway hyperresponsiveness compared with sole DEPs or HDM. Reduced Gata-3 expression decreased the number of functional ILC2s and TH2 cells in DEP+HDM-exposed mice, resulting in an impaired DEP-enhanced allergic airway inflammation. Interestingly, although the DEP-enhanced allergic inflammation was marginally reduced in ILC2-deficient mice that received combined DEP+HDM, it was abolished in DEP+HDM-exposed Rag2(-/-) mice. These data indicate that dysregulation of ILC2s and TH2 cells attenuates DEP-enhanced allergic airway inflammation. In addition, a crucial role for the adaptive immune system was shown on concomitant DEP+HDM exposure. Copyright © 2016 American Academy of

  1. Pulmonary Stress Induced by Hyperthermia: Role of Airway Sensory Nerves

    DTIC Science & Technology

    2012-10-01

    mild asthma. 15. SUBJECT TERMS- Hyperthermia, asthma, airway constriction, cough , dyspnea 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...mechanism. 3) To determine if thermal stress generated various airway dysfunctions (airway constriction, cough , etc) in healthy volunteers, and in...patients. Breathing HA, but not RA, also triggered coughs in these patients. In sharp contrast, the increases in SRaw to the same challenges were

  2. α-Galactosylceramide-induced airway eosinophilia is mediated through the activation of NKT cells.

    PubMed

    Chuang, Ya-Hui; Wang, Tzu-Chun; Jen, Hsiao-Yu; Yu, Alice L; Chiang, Bor-Luen

    2011-04-15

    Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.

  3. Airway cells after swimming outdoors or in the sea in nonasthmatic athletes.

    PubMed

    Bonsignore, Maria R; Morici, Giuseppe; Riccobono, Loredana; Profita, Mirella; Bonanno, Anna; Paternò, Alessandra; Di Giorgi, Rossana; Chimenti, Laura; Abate, Pietro; Mirabella, Franco; Maurizio Vignola, A; Bonsignore, Giovanni

    2003-07-01

    Marathon runners and elite swimmers showed increased inflammatory cells in the airways at baseline. Although airway neutrophils increase further after a marathon race, the airway response to swimming is unknown. The aim of this study was to assess the effects of swimming on airway cells. To avoid the concomitant effects of chronic exposure to chlorine, the study was conducted in seven nonasthmatic swimmers [mean age (SD): 23.3 +/- 7.7 yr, training: 32 +/- 15 km.wk-1] habitually training in an outdoor pool (OP), i.e., a low-chlorine environment. Spirometry, exhaled nitric oxide (NO), induced sputum, and peripheral blood samples were obtained at baseline, after a 5-km trial in OP, and after a 5-km race in the sea (S), i.e., hypertonic airway exposure. Airway neutrophil differential counts at baseline were higher in swimmers than in sedentary controls (N = 10), but cell counts, neutrophil elastase, and eosinophil cationic protein were unaffected by 5-km swimming. After swimming, L-selectin expression on airway cells decreased, suggesting exercise-induced cell mobilization into the airways and/or direct effects of hyperventilation on airway cells. After S, airway eosinophil differential counts increased slightly. Exhaled NO concentration was 19 +/- 6 ppb at baseline, 8 +/- 4 ppb after OP, and 21 +/- 7 ppb after S (P < 0.005 for OP vs baseline and S). In swimmers not chronically exposed to high chlorine concentrations, data obtained at baseline suggest a direct relationship between airway neutrophilia and endurance training. The low L-selectin expression by airway cells postexercise suggests hyperventilation-induced cell recruitment or modulation of cell function. Hypertonic exposure of airways during exercise may slightly increase airway eosinophils and exhaled NO. Overall, 5-km swimming exerted smaller effects on airway cells than running a marathon.

  4. Mast Cells Can Amplify Airway Reactivity and Features of Chronic Inflammation in an Asthma Model in Mice

    PubMed Central

    Williams, Cara M.M.; Galli, Stephen J.

    2000-01-01

    The importance of mast cells in the development of the allergen-induced airway hyperreactivity and inflammation associated with asthma remains controversial. We found that genetically mast cell–deficient WBB6F1-W/Wv mice that were sensitized to ovalbumin (OVA) without adjuvant, then challenged repetitively with antigen intranasally, exhibited much weaker responses in terms of bronchial hyperreactivity to aerosolized methacholine, lung tissue eosinophil infiltration, and numbers of proliferating cells within the airway epithelium than did identically treated WBB6F1-+/+ normal mice. However, W/Wv mice that had undergone selective reconstitution of tissue mast cells with in vitro–derived mast cells of congenic +/+ mouse origin exhibited airway responses that were very similar to those of the +/+ mice. By contrast, W/Wv mice that were sensitized with OVA emulsified in alum and challenged with aerosolized OVA exhibited levels of airway hyperreactivity and lung tissue eosinophil infiltration that were similar to those of the corresponding +/+ mice. Nevertheless, these W/Wv mice exhibited significantly fewer proliferating cells within the airway epithelium than did identically treated +/+ mice. These results show that, depending on the “asthma model” investigated, mast cells can either have a critical role in, or not be essential for, multiple features of allergic airway responses in mice. PMID:10934234

  5. Dendritic cell-nerve clusters are sites of T cell proliferation in allergic airway inflammation.

    PubMed

    Veres, Tibor Z; Shevchenko, Marina; Krasteva, Gabriela; Spies, Emma; Prenzler, Frauke; Rochlitzer, Sabine; Tschernig, Thomas; Krug, Norbert; Kummer, Wolfgang; Braun, Armin

    2009-03-01

    Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2'-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semi-automated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.

  6. Toll-like Receptors, Triggering Receptor Expressed on Myeloid Cells Family Members and Receptor for Advanced Glycation End-products in Allergic Airway Inflammation

    PubMed Central

    Hall, Sannette C.; Agrawal, Devendra K.

    2016-01-01

    Asthma is a chronic disorder of the airways characterized by cellular infiltration, airway hyper-responsive and airway inflammation. Innate immune cells are the first line of defense against endogenous and exogenous signals in the airways and as such possess a diverse array of pattern recognition receptors. Toll-like receptors are crucial sentinels which when activated, can either promote or ameliorate the inflammatory response in predisposed individuals. The recently discovered triggering receptor expressed on myeloid cells family members are emerging mediators of inflammation. These receptors are believed to modulate inflammatory responses by collaborating with classic PRRs. Endogenous signals like HMGB-1, signaling through the receptor for advanced glycation end products, also promotes inflammation, however, its contribution to inflammation in the airways is not well known. Here, we discuss the role of each receptor in airway inflammation and highlight potential synergistic mechanisms, which contribute to disease pathogenesis in allergic asthma. PMID:26678062

  7. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    PubMed Central

    Yaghi, Asma; Dolovich, Myrna B.

    2016-01-01

    Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations. PMID:27845721

  8. NK cells contribute to persistent airway inflammation and AHR during the later stage of RSV infection in mice.

    PubMed

    Long, Xiaoru; Xie, Jun; Zhao, Keting; Li, Wei; Tang, Wei; Chen, Sisi; Zang, Na; Ren, Luo; Deng, Yu; Xie, Xiaohong; Wang, Lijia; Fu, Zhou; Liu, Enmei

    2016-10-01

    RSV can lead to persistent airway inflammation and AHR and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. There are high numbers of NK cells in the lung, which not only play important roles in the acute stage of RSV infection, but also are pivotal in regulating the pathogenesis of asthma. Therefore, in this study, we assumed that NK cells might contribute to persistent airway disease during the later stage of RSV infection. Mice were killed at serial time points after RSV infection to collect samples. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. Cytokines were detected by ELISA, and NK cells were determined by flow cytometry. Rabbit anti-mouse asialo-GM-1 antibodies and resveratrol were used to deplete or suppress NK cells. Inflammatory cells in BALF, lung tissue damage and AHR were persistent for 60 days post-RSV infection. Type 2 cytokines and NK cells were significantly increased during the later stage of infection. When NK cells were decreased by the antibodies or resveratrol, type 2 cytokines, the persistent airway inflammation and AHR were all markedly reduced. NK cells can contribute to the RSV-associated persistent airway inflammation and AHR at least partially by promoting type 2 cytokines. Therefore, therapeutic targeting of NK cells may provide a novel approach to alleviating the recurrent wheezing subsequent to RSV infection.

  9. Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

    PubMed

    Van Maele, L; Fougeron, D; Janot, L; Didierlaurent, A; Cayet, D; Tabareau, J; Rumbo, M; Corvo-Chamaillard, S; Boulenouar, S; Jeffs, S; Vande Walle, L; Lamkanfi, M; Lemoine, Y; Erard, F; Hot, D; Hussell, T; Ryffel, B; Benecke, A G; Sirard, J-C

    2014-05-01

    Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

  10. Programmed cell death ligand 2 regulates TH9 differentiation and induction of chronic airway hyperreactivity

    PubMed Central

    Kerzerho, Jerome; Maazi, Hadi; Speak, Anneliese O.; Szely, Natacha; Lombardi, Vincent; Khoo, Bryant; Geryak, Stacey; Lam, Jonathan; Soroosh, Pejman; Van Snick, Jacques; Akbari, Omid

    2013-01-01

    Background Asthma is defined as a chronic inflammatory disease of the airways; however, the underlying physiologic and immunologic processes are not fully understood. Objective The aim of this study was to determine whether TH9 cells develop in vivo in a model of chronic airway hyperreactivity (AHR) and what factors control this development. Method We have developed a novel chronic allergen exposure model using the clinically relevant antigen Aspergillus fumigatus to determine the time kinetics of TH9 development in vivo. Results TH9 cells were detectable in the lungs after chronic allergen exposure. The number of TH9 cells directly correlated with the severity of AHR, and anti–IL-9 treatment decreased airway inflammation. Moreover, we have identified programmed cell death ligand (PD-L) 2 as a negative regulator of TH9 cell differentiation. Lack of PD-L2 was associated with significantly increased TGF-β and IL-1α levels in the lungs, enhanced pulmonary TH9 differentiation, and higher morbidity in the sensitized mice. Conclusion Our findings suggest that PD-L2 plays a pivotal role in the regulation of TH9 cell development in chronic AHR, providing novel strategies for modulating adaptive immunity during chronic allergic responses. PMID:23174661

  11. Bitter taste receptor agonists alter mitochondrial function and induce autophagy in airway smooth muscle cells.

    PubMed

    Pan, Shi; Sharma, Pawan; Shah, Sushrut D; Deshpande, Deepak A

    2017-07-01

    Airway remodeling, including increased airway smooth muscle (ASM) mass, is a hallmark feature of asthma and COPD. We previously identified the expression of bitter taste receptors (TAS2Rs) on human ASM cells and demonstrated that known TAS2R agonists could promote ASM relaxation and bronchodilation and inhibit mitogen-induced ASM growth. In this study, we explored cellular mechanisms mediating the antimitogenic effect of TAS2R agonists on human ASM cells. Pretreatment of ASM cells with TAS2R agonists chloroquine and quinine resulted in inhibition of cell survival, which was largely reversed by bafilomycin A1, an autophagy inhibitor. Transmission electron microscope studies demonstrated the presence of double-membrane autophagosomes and deformed mitochondria. In ASM cells, TAS2R agonists decreased mitochondrial membrane potential and increased mitochondrial ROS and mitochondrial fragmentation. Inhibiting dynamin-like protein 1 (DLP1) reversed TAS2R agonist-induced mitochondrial membrane potential change and attenuated mitochondrial fragmentation and cell death. Furthermore, the expression of mitochondrial protein BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) and mitochondrial localization of DLP1 were significantly upregulated by TAS2R agonists. More importantly, inhibiting Bnip3 mitochondrial localization by dominant-negative Bnip3 significantly attenuated cell death induced by TAS2R agonist. Collectively the TAS2R agonists chloroquine and quinine modulate mitochondrial structure and function, resulting in ASM cell death. Furthermore, Bnip3 plays a central role in TAS2R agonist-induced ASM functional changes via a mitochondrial pathway. These findings further establish the cellular mechanisms of antimitogenic effects of TAS2R agonists and identify a novel class of receptors and pathways that can be targeted to mitigate airway remodeling as well as bronchoconstriction in obstructive airway diseases. Copyright © 2017 the American Physiological

  12. Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

    PubMed Central

    Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C.; Hershenson, Marc B.

    2008-01-01

    Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function. PMID:18787220

  13. Autophagy plays a role in FSTL1-induced epithelial mesenchymal transition and airway remodeling in asthma.

    PubMed

    Liu, Tian; Liu, Yahui; Miller, Marina; Cao, Liuzhao; Zhao, Jiping; Wu, Jinxiang; Wang, Junfei; Liu, Lin; Li, Shuo; Zou, Minfang; Xu, Jiawei; Broide, David H; Dong, Liang

    2017-07-01

    Asthma is a chronic disease related to airway hyperresponsiveness and airway remodeling. Airway remodeling is the important reason of refractory asthma and is associated with differentiation of airway epithelia into myofibroblasts via epithelial-mesenchymal transition (EMT) to increase the process of subepithelial fibrosis. There is growing evidence that autophagy modulates remodeling. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we hypothesized that Follistatin-like 1 (FSTL1) promotes EMT and airway remodeling by intensifying autophagy. With the use of transmission electron microscopy (TEM), double-membrane autophagosomes were detected in the airways of patients and mice. More autophagosomes were in patients with asthma and OVA-challenged mice compared with healthy controls. The expression of FSTL1 and beclin-1 was upregulated in the airways of patients with asthma and OVA-challenged mice, accompanied by airway EMT and remodeling. In OVA-challenged Fstl1(+/-) mice, the degree of airway remodeling and autophagy was decreased compared with control mice. The effects of FSTL1 on autophagy and EMT were also tested in 16HBE cells in vitro. Additionally, inhibition of autophagy by using LY-294002 and siRNA-ATG5 reduced the FSTL1-induced EMT in 16HBE cells, as measured by E-cadherin, N-cadherin, and vimentin expression. In line herewith, administration of LY-294002 reduced the expression of autophagy, EMT, and airway remodeling markers in FSTL1-challenged WT mice. Taken together, our study suggests that FSTL1 may induce EMT and airway remodeling by activating autophagy. These findings may provide novel avenues for therapeutic research targeting the autophagy and FSTL1 pathway, which may be beneficial to patients with refractory asthma. Copyright © 2017 the American Physiological Society.

  14. Baicalin inhibits PDGF-induced proliferation and migration of airway smooth muscle cells.

    PubMed

    Yang, Guang; Li, Jian-Qiang; Bo, Jian-Ping; Wang, Bei; Tian, Xin-Rui; Liu, Tan-Zhen; Liu, Zhuo-La

    2015-01-01

    Airway smooth muscle (ASM) cell proliferation and migration play important roles in airway remodeling in asthma. In vitro platelet-derived growth factor (PDGF) induced ASM cell proliferation and migration. Baicalin is one of flavonoid extracts from Scutellaria baicalensis, which has an anti-asthma effect. However, little is known about its role in PDGF-induced proliferation and migration in rat ASM (RASM) cells. In this study, we aimed to investigate the effects of baicalin on PDGF-induced RASM cell proliferation and migration. We also identified the signaling pathway by which baicalin influences RASM cell proliferation and migration. In the current study, we demonstrated that baicalin suppressed PDGF-induced RASM cell proliferation, arrested PDGF-induced cell-cycle progression. It also suppressed PDGF-induced RASM cell migration. Furthermore, baicalin suppressed PDGF-induced expression of phosphorylated p38, ERK1/2 and JNK in RASM cells. In summary, our study is the first to show that baicalin pretreatment can significantly inhibit PDGF-induced RASM cell proliferation and migration by suppressing the MAPK signaling pathway, and baicalin may be a useful chemotherapeutic agent for asthma.

  15. Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

    PubMed Central

    Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

  16. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    PubMed

    Sherwood, Cara L; Liguori, Andrew E; Olsen, Colin E; Lantz, R Clark; Burgess, Jefferey L; Boitano, Scott

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  17. Airway responses towards allergens - from the airway epithelium to T cells.

    PubMed

    Papazian, D; Hansen, S; Würtzen, P A

    2015-08-01

    The prevalence of allergic diseases such as allergic rhinitis is increasing, affecting up to 30% of the human population worldwide. Allergic sensitization arises from complex interactions between environmental exposures and genetic susceptibility, resulting in inflammatory T helper 2 (Th2) cell-derived immune responses towards environmental allergens. Emerging evidence now suggests that an epithelial dysfunction, coupled with inherent properties of environmental allergens, can be responsible for the inflammatory responses towards allergens. Several epithelial-derived cytokines, such as thymic stromal lymphopoietin (TSLP), IL-25 and IL-33, influence tissue-resident dendritic cells (DCs) as well as Th2 effector cells. Exposure to environmental allergens does not elicit Th2 inflammatory responses or any clinical symptoms in nonatopic individuals, and recent findings suggest that a nondamaged, healthy epithelium lowers the DCs' ability to induce inflammatory T-cell responses towards allergens. The purpose of this review was to summarize the current knowledge on which signals from the airway epithelium, from first contact with inhaled allergens all the way to the ensuing Th2-cell responses, influence the pathology of allergic diseases.

  18. Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

    PubMed Central

    Eckerle, Isabella; Ehlen, Lukas; Kallies, René; Wollny, Robert; Corman, Victor M.; Cottontail, Veronika M.; Tschapka, Marco; Oppong, Samuel; Drosten, Christian; Müller, Marcel A.

    2014-01-01

    Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells. PMID:24454736

  19. Pulmonary Stress Induced by Hyperthermia: Role of Airway Sensory Nerves

    DTIC Science & Technology

    2011-10-01

    cough , bronchoconstriction, and other cardiopulmonary reflex responses (1). Recent studies conducted in our lab have established the first evidence...dyspnea, airway constriction, cough , etc) in healthy volunteers, and in patients with mild asthma, allergic rhinitis and post upper respiratory...cmH2O/L/sec (P>0.05). Furthermore, increasing airway temperature also consistently elicited bouts of cough in asthmatic patients, but not in healthy

  20. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium.

    PubMed

    Rawlins, Emma L; Okubo, Tadashi; Xue, Yan; Brass, David M; Auten, Richard L; Hasegawa, Hiroshi; Wang, Fan; Hogan, Brigid L M

    2009-06-05

    To directly test the contribution of Scgb1a1(+) Clara cells to postnatal growth, homeostasis, and repair of lung epithelium, we generated a Scgb1a1-CreER "knockin" mouse for lineage-tracing these cells. Under all conditions tested, the majority of Clara cells in the bronchioles both self-renews and generates ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung, it has been proposed that bronchioalveolar stem cells (BASCs) which coexpress Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis, or repair. Rather, our results support a model in which the trachea, bronchioles, and alveoli are maintained by distinct populations of epithelial progenitor cells.

  1. RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

    PubMed Central

    Liesman, Rachael M.; Buchholz, Ursula J.; Luongo, Cindy L.; Yang, Lijuan; Proia, Alan D.; DeVincenzo, John P.; Collins, Peter L.; Pickles, Raymond J.

    2014-01-01

    Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease. PMID:24713657

  2. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  3. Dust mite allergen, glutathione S-transferase, induces T cell immunoglobulin mucin domain-4 in dendritic cells to facilitate initiation of airway allergy.

    PubMed

    Mo, L-H; Yang, L-T; Zeng, L; Xu, L-Z; Zhang, H-P; Li, L-J; Liu, J-Q; Xiao, X-J; Zheng, P-Y; Liu, Z-G; Yang, P-C

    2017-02-01

    Allergens from dust mites play a critical role in the pathogenesis of airway allergy. The mechanism by which dust mite allergens induce allergic diseases is not fully understood yet. This study tests a hypothesis that the eighth subtypes of Dermatophagoides farina allergen (Derf8) play an important role in the induction of airway allergy. The protein of Derf8 was synthesized via molecular cloning approach. Dendritic cells (DC) were stimulated with Derf8 in the culture, and then, the expression of T cell immunoglobulin mucin domain 4 (TIM4) in dendritic cells (DC) was analysed. The role of Derf8 in the induction of airway allergy was evaluated with a mouse model. Exposure to Derf8 markedly induced the TIM4 expression in DCs by modulating the chromatin at the TIM4 promoter locus. Derf8 played a critical role in the expansion of the T helper 2 response in the mouse airway via inducing DCs to produce TIM4. Administration with Derf8-depleted dust mite extracts (DME) inhibited the allergic inflammation and induced regulatory T cells in mice with airway allergy. Derf8 plays an important role in the initiation of dust mite allergy. Vaccination with Derf8-deficient DME is more efficient to inhibit the dust mite allergic inflammation than using wild DME. © 2016 John Wiley & Sons Ltd.

  4. Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction.

    PubMed

    Tubby, Carolyn; Negm, Ola H; Harrison, Timothy; Tighe, Patrick J; Todd, Ian; Fairclough, Lucy C

    2017-06-01

    The three main types of killer cells - CD8(+) T cells, NK cells and NKT cells - have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. Peripheral CD8(+) T cells (CD8(+)CD3(+)CD56(-)), NK cells (CD56(+)CD3(-)) and NKT-like cells (CD56(+)CD3(+)) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.

  5. Chlamydia pneumoniae infection induced allergic airway sensitization is controlled by regulatory T-cells and plasmacytoid dendritic cells.

    PubMed

    Crother, Timothy R; Schröder, Nicolas W J; Karlin, Justin; Chen, Shuang; Shimada, Kenichi; Slepenkin, Anatoly; Alsabeh, Randa; Peterson, Ellena; Arditi, Moshe

    2011-01-01

    Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.

  6. Role of leukotrienes in airway hyperresponsiveness in guinea-pigs.

    PubMed Central

    Ishida, K.; Thomson, R. J.; Schellenberg, R. R.

    1993-01-01

    1. Repeated aerosolization of leukotriene C4 (LTC4) to guinea-pigs produced leftward shift in their pulmonary resistance (RL) dose-response curves to inhaled acetylcholine (ACh) without increasing the maximum responses. 2. Repeated LTC4 aerosolization did not increase airway eosinophils. 3. The 5-lipoxygenase-activating protein (FLAP) inhibitor, MK-886, prevented the leftward shift in RL dose-response curves to ACh following repeated antigen challenge in guinea-pigs. 4. MK-886 did not inhibit the increased maximal RL produced by repeated antigen challenge, nor inhibit the airway eosinophilia induced by repeated antigen challenge. 5. Our findings suggest that leukotrienes may account for the leftward shift in pulmonary resistance responses caused by antigen but do not cause the airway eosinophilia nor enhanced maximum broncho-constrictor response to antigen. PMID:8467358

  7. The innate immune function of airway epithelial cells in inflammatory lung disease

    PubMed Central

    Hiemstra, Pieter S.; McCray, Paul B.; Bals, Robert

    2016-01-01

    The airway epithelium is now considered central to the orchestration of pulmonary inflammatory and immune responses, and is also key to tissue remodelling. It acts as a first barrier in the defence against a wide range of inhaled challenges, and is critically involved in the regulation of both innate and adaptive immune responses to these challenges. Recent progress in our understanding of the developmental regulation of this tissue, the differentiation pathways, recognition of pathogens and antimicrobial responses is now exploited to help understand how epithelial cell function and dysfunction contributes to the pathogenesis of a variety of inflammatory lung diseases. In the review, advances in our knowledge of the biology of airway epithelium, as well as its role and (dys)function in asthma, COPD and cystic fibrosis, are discussed. PMID:25700381

  8. The role of the small airways in the pathophysiology of asthma and chronic obstructive pulmonary disease.

    PubMed

    Bonini, Matteo; Usmani, Omar S

    2015-12-01

    Chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), represent a major social and economic burden for worldwide health systems. During recent years, increasing attention has been directed to the role of small airways in respiratory diseases, and their exact contribution to the pathophysiology of asthma and COPD continues to be clarified. Indeed, it has been suggested that small airways play a distinct role in specific disease phenotypes. Besides providing information on small airways structure and diagnostic procedures, this review therefore aims to present updated and evidence-based findings on the role of small airways in the pathophysiology of asthma and COPD. Most of the available information derives from either pathological studies or review articles and there are few data on the natural history of small airways disease in the onset or progression of asthma and COPD. Comparisons between studies on the role of small airways are hard to draw because both asthma and COPD are highly heterogeneous conditions. Most studies have been performed in small population samples, and different techniques to characterize aspects of small airways function have been employed in order to assess inflammation and remodelling. Most methods of assessing small airways dysfunction have been largely confined to research purposes, but some data are encouraging, supporting the utilization of certain techniques into daily clinical practice, particularly for early-stage diseases, when subjects are often asymptomatic and routine pulmonary function tests may be within normal ranges. In this context further clinical trials and real-life feedback on large populations are desirable. © The Author(s), 2015.

  9. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  10. Allergens stimulate store-operated calcium entry and cytokine production in airway epithelial cells

    PubMed Central

    Jairaman, Amit; Maguire, Chelsea H.; Schleimer, Robert P.; Prakriya, Murali

    2016-01-01

    Aberrant immune responses to environmental allergens including insect allergens from house dust mites and cockroaches contribute to allergic inflammatory diseases such as asthma in susceptible individuals. Airway epithelial cells (AECs) play a critical role in this process by sensing the proteolytic activity of allergens via protease-activated receptors (PAR2) to initiate inflammatory and immune responses in the airway. Elevation of cytosolic Ca2+ is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca2+ elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca2+ elevations in AECs through the activation of Ca2+ release-activated Ca2+ (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca2+ entry then activates NFAT/calcineurin signaling to induce transcriptional production of the proinflammatory cytokines IL-6 and IL-8. These findings highlight a key role for CRAC channels as regulators of allergen induced inflammatory responses in the airway. PMID:27604412

  11. Fluticasone impact on airway dendritic cells in smokers: a randomized controlled trial

    PubMed Central

    2013-01-01

    Background Myeloid Dendritic cells are key drivers of inflammation in smoke-related lung diseases, whereas plasmacytoid DCs play a crucial role in the defense against infections. Effects of inhaled corticosteroids (ICS) on airway DCs in smokers are unknown. Methods In this randomized, double-blind, placebo-controlled clinical trial, 45 active cigarette smokers inhaled placebo, fluticasone or fluticasone plus salmeterol twice daily for 4 weeks. Bronchoalveolar lavage fluid DCs were analyzed using four-color flow cytometry before and after the inhalation period. In addition, fluticasone effects were tested on T-cell proliferation in co-cultures with blood myeloid DCs from smokers. Results Inhalation of fluticasone plus salmeterol, but not fluticasone alone or placebo, reduced endobronchial concentrations of myeloid DCs (median decrease: 24%), macrophages (median decrease: 26%) and neutrophils (median decrease: 76%). In contrast, fluticasone reduced plasmacytoid DC concentrations independently of salmeterol. There were no changes in the expression of function-associated surface molecules on myeloid DC (such as CD1a, Langerin, BDCA-1, CD83 or CCR5) in all groups after treatment. Fluticasone (either alone or in combination with salmeterol) suppressed T-cell proliferation in co-cultures with blood myeloid DCs from smokers. Conclusions Resistance to ICS monotherapy in smokers might in part be due to lacking effects on airway myeloid DCs, whereas the increased risk for infections during ICS therapy could be attributable to a reduction in plasmacytoid DCs. Combination therapy of fluticasone with salmeterol is associated with a reduction in airway myeloid DCs, but also airway macrophages and neutrophils. Trial registration Registered at ClinicalTrials.gov (identifier: NCT00908362) and the European Clinical Trial Database, EudraCT (identifier: 2009-009459-40). PMID:24168756

  12. Fluticasone impact on airway dendritic cells in smokers: a randomized controlled trial.

    PubMed

    Lommatzsch, Marek; Kraeft, Ulrike; Troebs, Laura; Garbe, Katharina; Bier, Andrea; Stoll, Paul; Klammt, Sebastian; Kuepper, Michael; Bratke, Kai; Virchow, Johann Christian

    2013-10-29

    Myeloid Dendritic cells are key drivers of inflammation in smoke-related lung diseases, whereas plasmacytoid DCs play a crucial role in the defense against infections. Effects of inhaled corticosteroids (ICS) on airway DCs in smokers are unknown. In this randomized, double-blind, placebo-controlled clinical trial, 45 active cigarette smokers inhaled placebo, fluticasone or fluticasone plus salmeterol twice daily for 4 weeks. Bronchoalveolar lavage fluid DCs were analyzed using four-color flow cytometry before and after the inhalation period. In addition, fluticasone effects were tested on T-cell proliferation in co-cultures with blood myeloid DCs from smokers. Inhalation of fluticasone plus salmeterol, but not fluticasone alone or placebo, reduced endobronchial concentrations of myeloid DCs (median decrease: 24%), macrophages (median decrease: 26%) and neutrophils (median decrease: 76%). In contrast, fluticasone reduced plasmacytoid DC concentrations independently of salmeterol. There were no changes in the expression of function-associated surface molecules on myeloid DC (such as CD1a, Langerin, BDCA-1, CD83 or CCR5) in all groups after treatment. Fluticasone (either alone or in combination with salmeterol) suppressed T-cell proliferation in co-cultures with blood myeloid DCs from smokers. Resistance to ICS monotherapy in smokers might in part be due to lacking effects on airway myeloid DCs, whereas the increased risk for infections during ICS therapy could be attributable to a reduction in plasmacytoid DCs. Combination therapy of fluticasone with salmeterol is associated with a reduction in airway myeloid DCs, but also airway macrophages and neutrophils. Registered at ClinicalTrials.gov (identifier: NCT00908362) and the European Clinical Trial Database, EudraCT (identifier: 2009-009459-40).

  13. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    PubMed Central

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

  14. Innate Lymphoid Cells Mediate Pulmonary Eosinophilic Inflammation, Airway Mucous Cell Metaplasia, and Type 2 Immunity in Mice Exposed to Ozone.

    PubMed

    Kumagai, Kazuyoshi; Lewandowski, Ryan P; Jackson-Humbles, Daven N; Buglak, Nicholas; Li, Ning; White, Kaylin; Van Dyken, Steven J; Wagner, James G; Harkema, Jack R

    2017-01-01

    Exposure to elevated levels of ambient ozone in photochemical smog is associated with eosinophilic airway inflammation and nonatopic asthma in children. In the present study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced nonatopic asthma by using lymphoid cell-sufficient C57BL/6 mice, ILC-sufficient Rag2(-/-) mice (devoid of T and B cells), and ILC-deficient Rag2(-/-)Il2rg(-/-) mice (depleted of all lymphoid cells including ILCs). Mice were exposed to 0 or 0.8 parts per million ozone for 1 day or 9 consecutive weekdays (4 hr/day). A single exposure to ozone caused neutrophilic inflammation, airway epithelial injury, and reparative DNA synthesis in all strains of mice, irrespective of the presence or absence of ILCs. In contrast, 9-day exposures induced eosinophilic inflammation and mucous cell metaplasia only in the lungs of ILC-sufficient mice. Repeated ozone exposures also elicited increased messenger RNA expression of transcripts associated with type 2 immunity and airway mucus production in ILC-sufficient mice. ILC-deficient mice repeatedly exposed to ozone had no pulmonary pathology or increased gene expression related to type 2 immunity. These results suggest a new paradigm for the biologic mechanisms underlying the development of a phenotype of childhood nonatopic asthma that has been linked to ambient ozone exposures.

  15. Apigenin inhibits TGF-β1-induced proliferation and migration of airway smooth muscle cells.

    PubMed

    Li, Li-Hua; Lu, Bin; Wu, Hong-Ke; Zhang, Hao; Yao, Fei-Fei

    2015-01-01

    It is well known that the proliferation and migration of ASM cells (ASMCs) plays an important role in the pathogenesis of airway remodeling in asthma. Previous studies reported that apigenin can inhibit airway remodeling in a mouse asthma model. However, its effects on the proliferation and migration of ASMCs in asthma remain unknown. Therefore, the aim of our present study was to investigate the effects of apigenin on ASMC proliferation and migration, and explore the possible molecular mechanism. We found that apigenin inhibited transforming growth factor-β1 (TGF-β1)-induced ASMC proliferation. The cell cycle was blocked at G1/S-interphase by apigenin. It also suppressed TGF-β1-induced ASMCs migration. Furthermore, apigenin inhibited TGF-β1-induced Smad 2 and Smad 3 phosphorylation in ASMCs. Taken together, these results suggested that apigenin inhibited the proliferation and migration of TGF-β1-stimulated ASMCs by inhibiting Smad signaling pathway. These data might provide useful information for treating asthma and show that apigenin has potential for attenuating airway remodeling.

  16. Apigenin inhibits TGF-β1-induced proliferation and migration of airway smooth muscle cells

    PubMed Central

    Li, Li-Hua; Lu, Bin; Wu, Hong-Ke; Zhang, Hao; Yao, Fei-Fei

    2015-01-01

    It is well known that the proliferation and migration of ASM cells (ASMCs) plays an important role in the pathogenesis of airway remodeling in asthma. Previous studies reported that apigenin can inhibit airway remodeling in a mouse asthma model. However, its effects on the proliferation and migration of ASMCs in asthma remain unknown. Therefore, the aim of our present study was to investigate the effects of apigenin on ASMC proliferation and migration, and explore the possible molecular mechanism. We found that apigenin inhibited transforming growth factor-β1 (TGF-β1)-induced ASMC proliferation. The cell cycle was blocked at G1/S-interphase by apigenin. It also suppressed TGF-β1-induced ASMCs migration. Furthermore, apigenin inhibited TGF-β1-induced Smad 2 and Smad 3 phosphorylation in ASMCs. Taken together, these results suggested that apigenin inhibited the proliferation and migration of TGF-β1-stimulated ASMCs by inhibiting Smad signaling pathway. These data might provide useful information for treating asthma and show that apigenin has potential for attenuating airway remodeling. PMID:26722444

  17. The role of contractile unit reorganization in force generation in airway smooth muscle.

    PubMed

    Brook, B S; Jensen, O E

    2014-06-01

    Airway smooth muscle (ASM) cells undergo remodelling and reside in a tissue structure that is subject to heterogenous stress distributions that change dynamically during the breathing cycle. In this paper, we develop a structural model of an ASM cell that consists of contractile units (actin and myosin filaments) in series and parallel, anchored to a nonlinearly elastic cytoskeleton. We mimic a typical experimental protocol that involves isometric force generation through triggering of the contractile machinery, followed by oscillatory length fluctuation of the cell. We use the model to predict the effect of a single instance of rearrangement of the contractile machinery, combined with strain-stiffening of the cytoskeleton, on the force generated by the sarcomeres, and the total force generated by the cell. By linking intra-cellular events to whole-cell behaviour, the model reveals mechanistic relationships between structural properties and cell-level force-length loops. We show how contractile force, shortening velocity and sarcomere operating lengths vary as the internal cell architecture is altered. Additionally, we show how interactions between the internal contractile machinery and cytoskeletal structure play a role in the regulation of force generation and hysteresis of the cell. © The Authors 2013.

  18. Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model

    PubMed Central

    Spaziano, Giuseppe; Piegari, Elena; Matteis, Maria; Cappetta, Donato; Esposito, Grazia; Russo, Rosa; Tartaglione, Gioia; De Palma, Raffaele; Rossi, Francesco; D’Agostino, Bruno

    2016-01-01

    Background The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs) can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce. Objectives To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model. Methods GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro. Results Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties. Conclusion Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide

  19. Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model.

    PubMed

    Urbanek, Konrad; De Angelis, Antonella; Spaziano, Giuseppe; Piegari, Elena; Matteis, Maria; Cappetta, Donato; Esposito, Grazia; Russo, Rosa; Tartaglione, Gioia; De Palma, Raffaele; Rossi, Francesco; D'Agostino, Bruno

    2016-01-01

    The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs) can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce. To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model. GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro. Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties. Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide system activation and tissue remodeling.

  20. Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration

    PubMed Central

    Moodley, Serisha; Derouet, Mathieu; Bai, Xiao Hui; Xu, Feng; Kapus, Andras; Yang, Burton B.; Liu, Mingyao

    2016-01-01

    Repair of airway epithelium after injury requires migration of neighboring epithelial cells to injured areas. However, the molecular mechanisms regulating airway epithelial cell migration is not well defined. We have previously shown that XB130, a scaffold protein, is required for airway epithelial repair and regeneration in vivo, and interaction between XB130 and another scaffold protein, Tks5, regulates cell proliferation and survival in human bronchial epithelial cells. The objective of the present study was to determine the role of XB130 and Tks5 interaction in airway epithelial cell migration. Interestingly, we found that XB130 only promotes lateral cell migration, whereas, Tks5 promotes cell migration/invasion via proteolysis of extracellular matrix. Upon stimulation with EGF, PKC activator phorbol 12, 13-dibutyrate or a nicotinic acetylcholine receptor ligand, XB130 and Tks5 translocated to the cell membrane in a stimulus-dependent manner. The translocation and distribution of XB130 is similar to lamellipodial marker, WAVE2; whereas Tks5 is similar to podosome marker, N-WASP. Over-expression of XB130 or Tks5 alone enhances cell migration, whereas co-expression of both XB130 and Tks5 inhibits cell migration processes and signaling. Furthermore, XB130 interacts with Rac1 whereas Tks5 interacts with Cdc42 to promote Rho GTPase activity. Our results suggest that dissociation between XB130 and Tks5 may facilitate lateral cell migration via XB130/Rac1, and vertical cell migration via Tks5/Cdc42. These molecular mechanisms will help our understanding of airway epithelial repair and regeneration. PMID:27835612

  1. The Anti-inflammatory Effect of Alpha-1 Antitrypsin in Rhinovirus-infected Human Airway Epithelial Cells

    PubMed Central

    Jiang, Di; Berman, Reena; Wu, Qun; Stevenson, Connor; Chu, Hong Wei

    2017-01-01

    Objective Excessive airway inflammation is seen in chronic obstructive pulmonary disease (COPD) patients experiencing acute exacerbations, which are often associated with human rhinovirus (HRV) infection. Alpha-1 antitrypsin (A1AT) has anti-inflammatory function in endothelial cells and monocytes, but its anti-inflammatory effect has not been investigated in COPD airway epithelial cells. We determined A1AT’s anti-inflammatory function in COPD airway epithelial cells and the underlying mechanisms such as the role of caspase-1. Methods Brushed bronchial epithelial cells from COPD and normal subjects were cultured at air-liquid interface and treated with A1AT or bovine serum albumin (BSA, control) two hours prior to whole cigarette smoke (WCS) or air exposure, followed by HRV-16 infection. After 24 hours of viral infection, cell supernatants were collected for measuring IL-8, and cells were examined for caspase-1. The in vivo anti-inflammatory function of A1AT was determined by infecting mice intranasally with HRV-1B followed by aerosolized A1AT or BSA. Results A1AT significantly reduced WCS and HRV-16-induced IL-8 production in normal and COPD airway epithelial cells. COPD cells are less sensitive to A1AT’s anti-inflammatory effect than normal cells. A1AT exerted the anti-inflammatory function in part via reducing caspase-1 in normal cells, but not in COPD cells. In mice, A1AT significantly reduced HRV-1B induced lung neutrophilic inflammation. Conclusions A1AT exerts an anti-inflammatory effect in cigarette smoke-exposed and HRV-infected human airway epithelial cells, which may be related to its inhibitory effect on caspase-1 activity. PMID:28191362

  2. Mast Cells Mediate Hyperoxia-Induced Airway Hyper-reactivity in Newborn Rats

    PubMed Central

    Schultz, Eric D.; Potts, Erin N.; Mason, Stanley N.; Foster, W. Michael; Auten, Richard L.

    2011-01-01

    Premature infants are at increased risk of developing airway hyper-reactivity following oxidative stress and inflammation. Mast cells contribute to airway hyper-reactivity partly by mediator release, so we sought to determine if blocking mast cell degranulation or recruitment prevents hyperoxia-induced airway hyper-reactivity, mast cell accumulation, and airway smooth muscle changes. Rats were exposed at birth to air or 60% O2 for 14 days, inducing significantly increased airway hyper-reactivity (AHR) in the latter group, induced by nebulized methacholine challenge, measured by forced oscillometry. Daily treatment (postnatal days 1-14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal days 5-14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways, and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell. PMID:20386143

  3. Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows

    PubMed Central

    Higuita-Castro, Natalia; Mihai, Cosmin; Hansford, Derek J.

    2014-01-01

    Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation. PMID:25213636

  4. The transcription factor Etv5 controls TH17 cell development and allergic airway inflammation

    PubMed Central

    Pham, Duy; Sehra, Sarita; Sun, Xin; Kaplan, Mark H.

    2014-01-01

    Background The differentiation of TH17 cells, which promote pulmonary inflammation, requires the cooperation of a network of transcription factors. Objectives We sought to define the role of Etv5, an Ets-family transcription factor, in TH17 cell development and function. Methods TH17 development was examined in primary mouse T cells wherein Etv5 expression was altered by retroviral transduction, small interfering RNA targeting a specific gene, and mice with a conditional deletion of Etv5 in T cells. The direct function of Etv5 on the Il17 locus was tested with chromatin immunoprecipitation and reporter assays. The house dust mite–induced allergic inflammation model was used to test the requirement for Etv5-dependent TH17 functions in vivo. Results We identify Etv5 as a signal transducer and activator of transcription 3–induced positive regulator of TH17 development. Etv5 controls TH17 differentiation by directly promoting 0a and Il17f expression. Etv5 recruits histone-modifying enzymes to the Il17a–Il17f locus, resulting in increased active histone marks and decreased repressive histone marks. In a model of allergic airway inflammation, mice with Etv5-deficient T cells have reduced airway inflammation and IL-17A/F production in the lung and bronchoalveolar lavage fluid compared with wild-type mice, without changes in TH2 cytokine production. Conclusions These data define signal transducer and activator of transcription 3–dependent feed-forward control of TH17 cytokine production and a novel role for Etv5 in promoting T cell–dependent airway inflammation. PMID:24486067

  5. Cell-contact dependent inhibition of monocytes by airway epithelial cells and reversion by infection with Respiratory Syncytial Virus.

    PubMed

    Oumouna, Mustapha; Weitnauer, Michael; Mijošek, Vedrana; Schmidt, Lotte M; Eigenbrod, Tatjana; Dalpke, Alexander H

    2015-11-01

    Airway epithelial cells (AEC) are the first line of defense against airborne infectious microbes and play an important role in regulating the local immune response. However, the interplay of epithelial cells and professional immune cells during both homeostasis and infection has only been partially studied. The present study was performed to determine how bronchial epithelial cells affect the activation of monocytes. Under healthy conditions, AECs were shown to inhibit reactivity of monocytes. We hypothesized that upon infection, monocytes might be released from inhibition by AECs. We report that direct contact of monocytes with unstimulated BEAS2B epithelial cells results in inhibition of TNF secretion by activated monocytes. In addition to the known soluble modulators, we show that cell contacts between epithelial cells and monocytes or macrophages also contribute to homeostatic inhibitory actions. We find AECs to express the inhibitory molecule PD-L1 and blockade of PD-L1 results in increased secretion of pro-inflammatory cytokines from monocytes. Contrary to the inhibitory activities during homeostasis, epithelial cells infected with Respiratory Syncitial Virus (RSV) induce a significant release of inhibition. However, release of inhibition was not due to modulation of PD-L1 expression in AECs. We conclude that airway epithelial cells control the reactivity of monocytes through direct and indirect interactions; however tonic inhibition can be reverted upon stimulation of AECs with RSV and thereof derived molecular patterns. The study confirms the important role of airway epithelial cells for local immune reactions. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Functional invariant NKT cells in pig lungs regulate the airway hyperreactivity: a potential animal model.

    PubMed

    Renukaradhya, Gourapura J; Manickam, Cordelia; Khatri, Mahesh; Rauf, Abdul; Li, Xiangming; Tsuji, Moriya; Rajashekara, Gireesh; Dwivedi, Varun

    2011-04-01

    Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4(+) cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.

  7. Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

    PubMed Central

    Maestre-Batlle, Danay; Pena, Olga M.; Hirota, Jeremy A.; Gunawan, Evelyn; Rider, Christopher F.; Sutherland, Darren; Alexis, Neil E.; Carlsten, Chris

    2017-01-01

    Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. PMID:28165060

  8. Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

    PubMed Central

    Zhang, Zhaolin; Leir, Shih-Hsing

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator gene (CFTR) expression in human airway epithelial cells involves the recruitment of distal cis-regulatory elements, which are associated with airway-selective DNase hypersensitive sites at −44 kb and −35 kb from the gene. The −35-kb site encompasses an enhancer that is regulated by the immune mediators interferon regulatory factor 1 and 2 and by nuclear factor Y. Here we investigate the −44-kb element, which also has enhancer activity in vitro in airway epithelial cells but is inactive in intestinal epithelial cells. This site contains an antioxidant response element (ARE) that plays a critical role in its function in airway cell lines and primary human bronchial epithelial cells. The natural antioxidant sulforaphane (SFN) induces nuclear translocation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcription factor that regulates genes with AREs in their promoters, many of which are involved in response to injury. Under normal conditions, the −44-kb ARE is occupied by the repressor BTB and CNC homology 1, basic leucine zipper transcription factor (Bach1), and v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) heterodimers. After 2 hours of SFN treatment, Nrf2 displaces these repressive factors and activates CFTR expression. Site-directed mutagenesis shows that both the ARE and an adjacent NF-κB binding site are required for activation of the –44-kb element in airway epithelial cells. Moreover, this element is functionally linked to the −35-kb enhancer in modulating CFTR expression in response to environmental stresses in the airway. PMID:25259561

  9. The role of airway mucus in pulmonary toxicology.

    PubMed Central

    Samet, J M; Cheng, P W

    1994-01-01

    Airway mucus is a complex airway secretion whose primary function as part of the mucociliary transport mechanism is to to serve as renewable and transportable barrier against inhaled particulates and toxic agents. The rheologic properties necessary for this function are imparted by glycoproteins, or mucins. Some respiratory disease states, e.g., asthma, cystic fibrosis, and bronchitis, are characterized by quantitative and qualitative changes in mucus biosynthesis that contribute to pulmonary pathology. Similar alterations in various aspects of mucin biochemistry and biophysics, leading to mucus hypersecretion and altered mucus rheology, result from inhalation of certain air pollutants, such as ozone, sulfur dioxide, nitrogen dioxide, and cigarette smoke. The consequences of these pollutant-induced alterations in mucus biology are discussed in the context of pulmonary pathophysiology and toxicology. PMID:7925190

  10. Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling.

    PubMed

    Paul, Manash K; Bisht, Bharti; Darmawan, Daphne O; Chiou, Richard; Ha, Vi L; Wallace, William D; Chon, Andrew T; Hegab, Ahmed E; Grogan, Tristan; Elashoff, David A; Alva-Ornelas, Jackelyn A; Gomperts, Brigitte N

    2014-08-07

    Airways are exposed to myriad environmental and damaging agents such as reactive oxygen species (ROS), which also have physiological roles as signaling molecules that regulate stem cell function. However, the functional significance of both steady and dynamically changing ROS levels in different stem cell populations, as well as downstream mechanisms that integrate ROS sensing into decisions regarding stem cell homeostasis, are unclear. Here, we show in mouse and human airway basal stem cells (ABSCs) that intracellular flux from low to moderate ROS levels is required for stem cell self-renewal and proliferation. Changing ROS levels activate Nrf2, which activates the Notch pathway to stimulate ABSC self-renewal and an antioxidant program that scavenges intracellular ROS, returning overall ROS levels to a low state to maintain homeostatic balance. This redox-mediated regulation of lung stem cell function has significant implications for stem cell biology, repair of lung injuries, and diseases such as cancer.

  11. Pulmonary Stress Induced by Hyperthermia: Role of Airway Sensory Nerves

    DTIC Science & Technology

    2013-10-01

    constriction, cough , dyspnea 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON...constriction, cough , etc.) in patients with allergic rhinitis. 3) To determine if thermal stress generated various airway dysfunctions in patients with...hyperventilation of humid warm air (WA) triggered cough and reflex bronchoconstriction in patients with mild asthma (Am. J. Resp. Crit. Care Med. 185:1190

  12. Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells.

    PubMed

    Wu, Qun; Jiang, Di; Minor, Maisha; Chu, Hong Wei

    2014-01-01

    The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection. We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.

  13. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

    PubMed

    Allahverdian, Sima; Harada, Norihiro; Singhera, Gurpreet K; Knight, Darryl A; Dorscheid, Delbert R

    2008-02-01

    Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

  14. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  15. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  16. Chibby functions to preserve normal ciliary morphology through the regulation of intraflagellar transport in airway ciliated cells.

    PubMed

    Siller, Saul S; Burke, Michael C; Li, Feng-Qian; Takemaru, Ken-Ichi

    2015-01-01

    Airway cilia provide the coordinated motive force for mucociliary transport, which prevents the accumulation of mucus, debris, pollutants, and bacteria in our respiratory tracts. As airway cilia are constantly exposed to the environment and, hence, are an integral component of the pathogenesis of several congenital and chronic pulmonary disorders, it is necessary to understand the molecular mechanisms that control ciliated cell differentiation and ciliogenesis. We have previously reported that loss of the basal body protein Chibby (Cby) results in chronic upper airway infection in mice due to a significant reduction in the number of airway cilia. In the present work, we demonstrate that Cby is required for normal ciliary structure and proper distribution of proteins involved in the bidirectional intraflagellar transport (IFT) system, which consists of 2 distinct sub-complexes, IFT-A and IFT-B, and is essential for ciliary biogenesis and maintenance. In fully differentiated ciliated cells, abnormal paddle-like cilia with dilated ciliary tips are observed in Cby-/- airways and primary cultures of mouse tracheal epithelial cells (MTECs). In addition, IFT88, an IFT-B sub-complex protein, robustly accumulates within the dilated tips of both multicilia in Cby-/- MTECs and primary cilia in Cby-/- mouse embryonic fibroblasts (MEFs). Furthermore, we show that only IFT-B components, including IFT20 and IFT57, but not IFT-A and Bardet-Biedl syndrome (BBS) proteins, amass with IFT88 in these distended tips in Cby-/- ciliated cells. Taken together, our findings suggest that Cby plays a role in the proper distribution of IFT particles to preserve normal ciliary morphology in airway ciliated cells.

  17. CGRP induction in cystic fibrosis airways alters the submucosal gland progenitor cell niche in mice

    PubMed Central

    Xie, Weiliang; Fisher, John T.; Lynch, Thomas J.; Luo, Meihui; Evans, Turan I.A.; Neff, Traci L.; Zhou, Weihong; Zhang, Yulong; Ou, Yi; Bunnett, Nigel W.; Russo, Andrew F.; Goodheart, Michael J.; Parekh, Kalpaj R.; Liu, Xiaoming; Engelhardt, John F.

    2011-01-01

    In cystic fibrosis (CF), a lack of functional CF transmembrane conductance regulator (CFTR) chloride channels causes defective secretion by submucosal glands (SMGs), leading to persistent bacterial infection that damages airways and necessitates tissue repair. SMGs are also important niches for slow-cycling progenitor cells (SCPCs) in the proximal airways, which may be involved in disease-related airway repair. Here, we report that calcitonin gene–related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. Since CGRP-expressing neuroendocrine cells reside in bronchiolar SCPC niches, we hypothesized that the glandular SCPC niche may be dysfunctional in CF. Consistent with this hypothesis, CFTR-deficient mice failed to maintain glandular SCPCs following airway injury. In wild-type mice, CGRP levels increased following airway injury and functioned as an injury-induced mitogen that stimulated SMG progenitor cell proliferation in vivo and altered the proliferative potential of airway progenitors in vitro. Components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation in a non-cell-autonomous manner. These findings demonstrate that CGRP-dependent pathways for CFTR activation are abnormally upregulated in CF SMGs and that this sustained mitogenic signal alters properties of the SMG progenitor cell niche in CF airways. This discovery may have important implications for injury/repair mechanisms in the CF airway. PMID:21765217

  18. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    PubMed Central

    Bauer, Rebecca N.; Müller, Loretta; Brighton, Luisa E.; Duncan, Kelly E.

    2015-01-01

    The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell–Mac coculture model to investigate how epithelial cell–derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell–Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell–derived signals are important determinants of Mac immunophenotype and response to O3. PMID:25054807

  19. Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma.

    PubMed

    Kan-o, Keiko; Matsunaga, Yuko; Fukuyama, Satoru; Moriwaki, Atsushi; Hirai-Kitajima, Hiroko; Yokomizo, Takehiko; Aritake, Kosuke; Urade, Yoshihiro; Nakanishi, Yoichi; Inoue, Hiromasa; Matsumoto, Koichiro

    2013-03-04

    Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13. The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit+/+ mice and -deficient KitW/KitW-v mice. The outcome of mast cell reconstitution was further investigated. Airway eosinophilia and IL-13 production were augmented by poly IC in Kit+/+ mice but not in KitW/KitW-v mice. When KitW/KitW-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in KitW/KitW-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B4 and prostaglandin D2 are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia. We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B4 or prostaglandin D2.

  20. Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma

    PubMed Central

    2013-01-01

    Background Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13. Methods The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit+/+ mice and -deficient KitW/KitW-v mice. The outcome of mast cell reconstitution was further investigated. Results Airway eosinophilia and IL-13 production were augmented by poly IC in Kit+/+ mice but not in KitW/KitW-v mice. When KitW/KitW-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in KitW/KitW-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B4 and prostaglandin D2 are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia. Conclusions We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B4 or prostaglandin D2. PMID:23452625

  1. NEUTROPHILS PLAY A CRITICAL ROLE IN THE DEVELOPMENT OF LPS-INDUCED AIRWAY DISEASE

    EPA Science Inventory

    ETD-02-045 (GAVETT) GPRA # 10108

    Neutrophils Play a Critical Role in the Development of LPS-Induced Airway Disease.
    Jordan D. Savov, Stephen H. Gavett*, David M. Brass, Daniel L. Costa*, and David A. Schwartz

    ABSTRACT
    We investigated the role of neutrophils...

  2. NEUTROPHILS PLAY A CRITICAL ROLE IN THE DEVELOPMENT OF LPS-INDUCED AIRWAY DISEASE

    EPA Science Inventory

    ETD-02-045 (GAVETT) GPRA # 10108

    Neutrophils Play a Critical Role in the Development of LPS-Induced Airway Disease.
    Jordan D. Savov, Stephen H. Gavett*, David M. Brass, Daniel L. Costa*, and David A. Schwartz

    ABSTRACT
    We investigated the role of neutrophils...

  3. Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

    PubMed Central

    2013-01-01

    Background As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype. Methods To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed. Results We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process. Conclusion Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents. PMID:24298994

  4. A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development. Evidence for stimulation of airway growth.

    PubMed Central

    Li, K; Nagalla, S R; Spindel, E R

    1994-01-01

    Gastrin-releasing peptide (GRP) is developmentally expressed in human fetal lung and is a growth factor for normal and neoplastic lung but its role in normal lung development has yet to be clearly defined. In this study we have characterized the expression of GRP and its receptor in fetal rhesus monkey lung and determined the effects of bombesin on fetal lung development in vitro. By RNA blot analysis, GRP mRNA was first detectable in fetal monkey lung at 63 days gestation, reached highest levels at 80 days gestation, and then declined to near adult levels by 120 days gestation; a pattern closely paralleling GRP expression in human fetal lung. As in human lung, in situ hybridization localized GRP mRNA to neuroendocrine cells though during the canalicular phase of development (between 63-80 days gestation) GRP mRNA was present not only in classic pulmonary neuroendocrine cells, but also in cells of budding airways. Immunohistochemistry showed that bombesin-like immunoreactivity was present in neuroendocrine cells, but not in budding airways, suggesting that in budding airways either the GRP mRNA is not translated, is rapidly secreted, or a related, but different RNA is present. RNase protection analysis using a probe to the monkey GRP receptor demonstrated that the time course of receptor RNA expression closely paralleled the time course of GRP RNA expression. In situ hybridization showed that GRP receptors were primarily expressed in epithelial cells of the developing airways. Thus GRP would appear to be secreted from neuroendocrine cells to act on target cells in developing airways. This hypothesis was confirmed by organ culture of fetal monkey lung in the presence of bombesin and bombesin antagonists. Bombesin treatment at 1 and 10 nM significantly increased DNA synthesis in airway epithelial cells and significantly increased the number and size of airways in cultured fetal lung. In fact, culturing 60 d fetal lung for 5 d with 10 nM bombesin increased airway size

  5. Role of mandibular displacement and airway size in improving breathing after rapid maxillary expansion

    PubMed Central

    2014-01-01

    Background Oral breathing and maxillary deficiency are often associated with steep mandibular plane angle, and retrognathic mandible compared with the faces of healthy controls. Some studies suggested that after rapid maxillary expansion, improvement in nasal breathing and repositioning of mandible with transitory increasing of facial height and, in some cases, spontaneous forward repositioning might occur. The abovementioned mandibular effects could contribute to enlarge oropharynx volume with repositioning of tongue and soft palate with an improvement of upper airway volume after treatment. The aim of this study was to investigate by cone beam computed tomography the role of oropharyngeal volume and mandibular position changes after rapid maxillary expansion in patients showing improved breathing pattern confirmed by polysomnography exam. Methods The final sample of this retrospective study comprised 14 Caucasian patients (mean age 7.6 years) who undergone rapid maxillary expansion with Haas-type expander banded on second deciduous upper molars. Cone beam computed tomography scans and polysomnography exams were collected before placing the appliance (T0) and after 12 months (T1). Mandibular landmarks localization and airway semiautomatic segmentation on cone beam computed tomography scans allowed airway volume computing and measurements. Results No significant differences were found between oropharyngeal airway changes and mandibular displacement after rapid maxillary expansion in growing patients. Conclusions The suggested improvement in upper airway and breathing after rapid maxillary expansion should be further related to different compartments of airway such as rhinopharynx and nasal cavity. PMID:24934328

  6. Infections causing central airway obstruction: role of bronchoscopy in diagnosis and management.

    PubMed

    Keshishyan, Sevak; DeLorenzo, Lawrence; Hammoud, Kassem; Avagyan, Arpine; Assallum, Hussein; Harris, Kassem

    2017-06-01

    Central airway obstructive infections (CAOI) are challenging medical conditions that may represent an advanced and complicated process of ongoing infections. The epidemiology of CAOI is unknown as well as the pathophysiology and the mechanism of development. This is due to sparse data in the literature that consists mainly of case reports and retrospective case series. CAOI can be caused by fungal, bacterial, parasitic and viral infections. Most patients with CAOI can be diagnosed clinically and with chest imaging, which demonstrate obstruction of the central airways. However, bronchoscopy is commonly used to confirm and obtain a specific diagnosis to guide specific therapy. In recent years, interventional pulmonology (IP) is becoming widely available and offer a minimally invasive approach for the management of central airway diseases such as cancers, benign strictures, and other conditions. Various bronchoscopic modalities are used to treat central airway obstruction (CAO), such as mechanical debulking, endobronchial laser therapy, electrocautery, argon plasma coagulation, cryotherapy, and airway stenting. In patients with CAOI, the role of therapeutic bronchoscopy is not clearly defined, but many isolated reports in the literature described bronchoscopic intervention in combination with medical therapy as the initial management approach. In this paper, we present cases of CAOI that underwent bronchoscopic intervention as part of their management. We described the infectious etiology, locations, bronchoscopic findings and bronchoscopic modalities for airway management.

  7. Increased numbers of activated group 2 innate lymphoid cells in the airways of patients with severe asthma and persistent airway eosinophilia.

    PubMed

    Smith, Steven G; Chen, Ruchong; Kjarsgaard, Melanie; Huang, Chynna; Oliveria, John-Paul; O'Byrne, Paul M; Gauvreau, Gail M; Boulet, Louis-Philippe; Lemiere, Catherine; Martin, James; Nair, Parameswaran; Sehmi, Roma

    2016-01-01

    In patients with severe eosinophilic asthma, local maturation rather than systemic recruitment of mature cells might contribute to persistent airway eosinophilia. Group 2 innate lymphoid cells (ILC2s) are a major source of type 2 cytokines (IL-5 and IL-13) and can facilitate eosinophilic inflammatory responses in mouse models of asthma in the absence of CD4+ lymphocytes. This study investigated the potential role of ILC2s in driving chronic airway eosinophilia in patients with severe asthma, despite regular high-dose oral corticosteroid therapy. In a cross-sectional study we enumerated blood and sputum ILC2s (lin(-)CD45(+)127(+)ST2(+)) and levels of intracellular IL-5 and IL-13 in patients with severe asthma (n = 25), patients with steroid-naive mild atopic asthma (n = 19), and nonatopic control subjects (n = 5). Results were compared with numbers of CD4+ lymphocytes, eosinophil lineage-committed progenitors (eosinophilopoietic progenitor cells [EoPs]), and mature eosinophils. Significantly greater numbers of total and type 2 cytokine-producing ILC2s were detected in blood and sputum of patients with severe asthma compared to mild asthmatics. In contrast, intracellular cytokine expression by CD4 cells and EoPs within the airways did not differ between the asthmatic groups. In patients with severe asthma, although sputum CD4+ cells were more abundant than ILC2s and EoPs, proportionally, ILC2s were the predominant source of type 2 cytokines. In addition, there were significantly greater numbers of sputum IL-5(+)IL-13(+) ILC2s in patients with severe asthma whose airway eosinophilia was greater than 3%, despite normal blood eosinophil numbers (<300/μL). Our findings suggest that ILC2s can promote the persistence of airway eosinophilia in patients with severe asthma through uncontrolled localized production of the type 2 cytokines IL-5 and IL-13, despite high-dose oral corticosteroid therapy. Copyright © 2015 American Academy of Allergy, Asthma & Immunology

  8. Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells.

    PubMed

    Takahara, Norihiro; Ito, Satoru; Furuya, Kishio; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-12-01

    Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca(2+)-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

  9. Critical role of aldehydes in cigarette smoke-induced acute airway inflammation.

    PubMed

    van der Toorn, Marco; Slebos, Dirk-Jan; de Bruin, Harold G; Gras, Renee; Rezayat, Delaram; Jorge, Lucie; Sandra, Koen; van Oosterhout, Antoon J M

    2013-04-17

    Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation. BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs. Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein. Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

  10. Critical role of aldehydes in cigarette smoke-induced acute airway inflammation

    PubMed Central

    2013-01-01

    Background Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation. Methods BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs. Results Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein. Conclusion Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation. PMID:23594194

  11. Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

    PubMed

    Zsembery, Akos; Boyce, Amanda T; Liang, Lihua; Peti-Peterdi, János; Bell, P Darwin; Schwiebert, Erik M

    2003-04-11

    Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

  12. Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review

    PubMed Central

    Croft, Carys A.; Culibrk, Luka; Moore, Margo M.; Tebbutt, Scott J.

    2016-01-01

    Aspergillus fumigatus is an environmental filamentous fungus that also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell (AEC). The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with AECs is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and AECs, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by AECs, the immune response of AECs, as well as the role of fungal virulence factors, and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. fumigatus could point to novel targets for therapy and prophylaxis. PMID:27092126

  13. deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

    PubMed

    Arason, Ari Jon; Jonsdottir, Hulda R; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K

    2014-01-01

    The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

  14. Injury induces direct lineage segregation of functionally distinct airway basal stem/progenitor cell subpopulations

    PubMed Central

    Pardo-Saganta, Ana; Law, Brandon M; Tata, Purushothama Rao; Villoria, Jorge; Saez, Borja; Mou, Hongmei; Zhao, Rui; Rajagopal, Jayaraj

    2015-01-01

    Summary Following injury, stem cells restore normal tissue architecture by producing the proper number and proportions of differentiated cells. Current models of airway epithelial regeneration propose that distinct cytokeratin 8-expressing progenitor cells, arising from p63+ basal stem cells, subsequently differentiate into secretory and ciliated cell lineages. We now show that immediately following injury, discrete subpopulations of p63+ airway basal stem/progenitor cells themselves express Notch pathway components associated with either secretory or ciliated cell fate commitment. One basal cell population displays intracellular Notch2 activation and directly generates secretory cells; the other expresses c-myb and directly yields ciliated cells. Furthermore, disrupting Notch ligand activity within the basal cell population at large disrupts the normal pattern of lineage segregation. These non-cell autonomous effects demonstrate that effective airway epithelial regeneration requires intercellular communication within the broader basal stem/progenitor cell population. These findings have broad implications for understanding epithelial regeneration and stem cell heterogeneity. PMID:25658372

  15. Dectin-2 promotes house dust mite-induced T helper type 2 and type 17 cell differentiation and allergic airway inflammation in mice.

    PubMed

    Norimoto, Ayako; Hirose, Koichi; Iwata, Arifumi; Tamachi, Tomohiro; Yokota, Masaya; Takahashi, Kentaro; Saijo, Shinobu; Iwakura, Yoichiro; Nakajima, Hiroshi

    2014-08-01

    The fact that sensitization against fungi is closely related to the severity of asthma suggests that immune systems recognizing fungi are involved in the pathogenesis of severe asthma. Recently, Dectin-2 (gene symbol, Clec4n), a C-type lectin receptor, has been shown to function as not only a major pattern-recognition receptor for fungi, but also a receptor for some components of house dust mite (HDM) extract, a major allergen for asthma. However, the roles of Dectin-2 in the induction of HDM-induced allergic airway inflammation remain largely unknown. Our objective was to determine the roles of Dectin-2 in HDM-induced allergic airway inflammation. We examined the roles of Dectin-2 in the induction of HDM-induced T helper (Th) 2 and Th17 cell differentiation and subsequent allergic airway inflammation by using Clec4n-deficient (Clec4n(-/-)) mice. We also investigated Dectin-2-expressing cells in the lung and their roles in HDM-induced allergic airway inflammation. Clec4n(-/-) mice showed significantly attenuated HDM-induced allergic airway inflammation and decreased Th2 and Th17 cell differentiation. Dectin-2 mRNA, together with Dectin-3 and Fc receptor-γ mRNAs, was expressed in CD11b(+) dendritic cells (DCs), but not in CD4(+) T cells or epithelial cells in the lung. CD11b(+) DCs isolated from Clec4n(-/-) mice expressed lower amounts of proinflammatory cytokines and costimulatory molecules, which could lead to Th2 and Th17 cell differentiation than those from wild-type mice. HDM-pulsed Clec4n(-/-) DCs were less efficient for the induction of allergic airway inflammation than HDM-pulsed wild-type DCs. In conclusion, Dectin-2 expressed on CD11b(+) DCs promotes HDM-induced Th2 and Th17 cell differentiation and allergic airway inflammation.

  16. The Role of Airway and Endobronchial Ultrasound in Perioperative Medicine

    PubMed Central

    Votruba, Jiri; Zemanová, Petra; Lambert, Lukas; Vesela, Michaela Michalkova

    2015-01-01

    Recent years have witnessed an increased use of ultrasound in evaluation of the airway and the lower parts of the respiratory system. Ultrasound examination is fast and reliable and can be performed at the bedside and does not carry the risk of exposure to ionizing radiation. Apart from use in diagnostics it may also provide safe guidance for invasive and semi-invasive procedures. Ultrasound examination of the oral cavity structures, epiglottis, vocal cords, and subglottic space may help in the prediction of difficult intubation. Preoperative ultrasound may diagnose vocal cord palsy or deviation or stenosis of the trachea. Ultrasonography can also be used for confirmation of endotracheal tube, double-lumen tube, or laryngeal mask placement. This can be achieved by direct examination of the tube inside the trachea or by indirect methods evaluating lung movements. Postoperative airway ultrasound may reveal laryngeal pathology or subglottic oedema. Conventional ultrasound is a reliable real-time navigational tool for emergency cricothyrotomy or percutaneous dilational tracheostomy. Endobronchial ultrasound is a combination of bronchoscopy and ultrasonography and is used for preoperative examination of lung cancer and solitary pulmonary nodules. The method is also useful for real-time navigated biopsies of such pathological structures. PMID:26788507

  17. The Role of Airway and Endobronchial Ultrasound in Perioperative Medicine.

    PubMed

    Votruba, Jiri; Zemanová, Petra; Lambert, Lukas; Vesela, Michaela Michalkova

    2015-01-01

    Recent years have witnessed an increased use of ultrasound in evaluation of the airway and the lower parts of the respiratory system. Ultrasound examination is fast and reliable and can be performed at the bedside and does not carry the risk of exposure to ionizing radiation. Apart from use in diagnostics it may also provide safe guidance for invasive and semi-invasive procedures. Ultrasound examination of the oral cavity structures, epiglottis, vocal cords, and subglottic space may help in the prediction of difficult intubation. Preoperative ultrasound may diagnose vocal cord palsy or deviation or stenosis of the trachea. Ultrasonography can also be used for confirmation of endotracheal tube, double-lumen tube, or laryngeal mask placement. This can be achieved by direct examination of the tube inside the trachea or by indirect methods evaluating lung movements. Postoperative airway ultrasound may reveal laryngeal pathology or subglottic oedema. Conventional ultrasound is a reliable real-time navigational tool for emergency cricothyrotomy or percutaneous dilational tracheostomy. Endobronchial ultrasound is a combination of bronchoscopy and ultrasonography and is used for preoperative examination of lung cancer and solitary pulmonary nodules. The method is also useful for real-time navigated biopsies of such pathological structures.

  18. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling.

    PubMed

    Salmon, M; Walsh, D A; Koto, H; Barnes, P J; Chung, K F

    1999-09-01

    Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.

  19. Protective Roles for RGS2 in a Mouse Model of House Dust Mite-Induced Airway Inflammation

    PubMed Central

    George, Tresa; Bell, Matthew; Chakraborty, Mainak; Siderovski, David P.; Giembycz, Mark A.

    2017-01-01

    The GTPase-accelerating protein, regulator of G-protein signalling 2 (RGS2) reduces signalling from G-protein-coupled receptors (GPCRs) that signal via Gαq. In humans, RGS2 expression is up-regulated by inhaled corticosteroids (ICSs) and long-acting β2-adrenoceptor agonists (LABAs) such that synergy is produced in combination. This may contribute to the superior clinical efficacy of ICS/LABA therapy in asthma relative to ICS alone. In a murine model of house dust mite (HDM)-induced airways inflammation, three weeks of intranasal HDM (25 μg, 3×/week) reduced lung function and induced granulocytic airways inflammation. Compared to wild type animals, Rgs2-/- mice showed airways hyperresponsiveness (increased airways resistance and reduced compliance). While HDM increased pulmonary inflammation observed on hematoxylin and eosin-stained sections, there was no difference between wild type and Rgs2-/- animals. HDM-induced mucus hypersecretion was also unaffected by RGS2 deficiency. However, inflammatory cell counts in the bronchoalveolar lavage fluid of Rgs2-/- animals were significantly increased (57%) compared to wild type animals and this correlated with increased granulocyte (neutrophil and eosinophil) numbers. Likewise, cytokine and chemokine (IL4, IL17, IL5, LIF, IL6, CSF3, CXCLl, CXCL10 and CXCL11) release was increased by HDM exposure. Compared to wild type, Rgs2-/- animals showed a trend towards increased expression for many cytokines/chemokines, with CCL3, CCL11, CXCL9 and CXCL10 being significantly enhanced. As RGS2 expression was unaffected by HDM exposure, these data indicate that RGS2 exerts tonic bronchoprotection in HDM-induced airways inflammation. Modest anti-inflammatory and anti-remodelling roles for RGS2 are also suggested. If translatable to humans, therapies that maximize RGS2 expression may prove advantageous. PMID:28107494

  20. MicroRNA 4423 is a primate-specific regulator of airway epithelial cell differentiation and lung carcinogenesis

    PubMed Central

    Perdomo, Catalina; Campbell, Joshua D.; Gerrein, Joseph; Tellez, Carmen S.; Garrison, Carly B.; Walser, Tonya C.; Drizik, Eduard; Si, Huiqing; Gower, Adam C.; Vick, Jessica; Anderlind, Christina; Jackson, George R.; Mankus, Courtney; Schembri, Frank; O’Hara, Carl; Gomperts, Brigitte N.; Dubinett, Steven M.; Hayden, Patrick; Belinsky, Steven A.; Lenburg, Marc E.; Spira, Avrum

    2013-01-01

    Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. Although microRNAs are regulators of many airway gene-expression changes induced by smoking, their role in modulating changes associated with lung cancer in these cells remains unknown. Here, we use next-generation sequencing of small RNAs in the airway to identify microRNA 4423 (miR-4423) as a primate-specific microRNA associated with lung cancer and expressed primarily in mucociliary epithelium. The endogenous expression of miR-4423 increases as bronchial epithelial cells undergo differentiation into mucociliary epithelium in vitro, and its overexpression during this process causes an increase in the number of ciliated cells. Furthermore, expression of miR-4423 is reduced in most lung tumors and in cytologically normal epithelium of the mainstem bronchus of smokers with lung cancer. In addition, ectopic expression of miR-4423 in a subset of lung cancer cell lines reduces their anchorage-independent growth and significantly decreases the size of the tumors formed in a mouse xenograft model. Consistent with these phenotypes, overexpression of miR-4423 induces a differentiated-like pattern of airway epithelium gene expression and reverses the expression of many genes that are altered in lung cancer. Together, our results indicate that miR-4423 is a regulator of airway epithelium differentiation and that the abrogation of its function contributes to lung carcinogenesis. PMID:24158479

  1. Polystyrene nanoparticles activate ion transport in human airway epithelial cells

    PubMed Central

    McCarthy, J; Gong, X; Nahirney, D; Duszyk, M; Radomski, MW

    2011-01-01

    Background Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function. Methods Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR) and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Clchannels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches. Results Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl− channel blocker. Polystyrene nanoparticles activated basolateral K+ channels and affected Cl− and HCO3 − secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl− channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl− channels by the nanoparticles. Conclusion This is the first study to identify the activation of ion channels in airway cells after exposure to polystyrene-based nanomaterials. Thus, polystyrene nanoparticles cannot be considered as a simple neutral vehicle for drug delivery for the treatment of lung diseases, due to the fact

  2. Defining an olfactory receptor function in airway smooth muscle cells

    PubMed Central

    Aisenberg, William H.; Huang, Jessie; Zhu, Wanqu; Rajkumar, Premraj; Cruz, Randy; Santhanam, Lakshmi; Natarajan, Niranjana; Yong, Hwan Mee; De Santiago, Breann; Oh, Jung Jin; Yoon, A-Rum; Panettieri, Reynold A.; Homann, Oliver; Sullivan, John K.; Liggett, Stephen B.; Pluznick, Jennifer L.; An, Steven S.

    2016-01-01

    Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma. PMID:27905542

  3. Defining an olfactory receptor function in airway smooth muscle cells.

    PubMed

    Aisenberg, William H; Huang, Jessie; Zhu, Wanqu; Rajkumar, Premraj; Cruz, Randy; Santhanam, Lakshmi; Natarajan, Niranjana; Yong, Hwan Mee; De Santiago, Breann; Oh, Jung Jin; Yoon, A-Rum; Panettieri, Reynold A; Homann, Oliver; Sullivan, John K; Liggett, Stephen B; Pluznick, Jennifer L; An, Steven S

    2016-12-01

    Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.

  4. Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases.

    PubMed Central

    Oishi, K; Sonoda, F; Kobayashi, S; Iwagaki, A; Nagatake, T; Matsushima, K; Matsumoto, K

    1994-01-01

    To evaluate of the role of interleukin-8 (IL-8), a chemotactic cytokine, in the continuous neutrophil accumulation in the airways of patients with chronic airway disease (CAD) and persistent Pseudomonas aeruginosa infection, we investigated the cell population, IL-8 levels, IL-1 beta levels, tumor necrosis factor (TNF) activities, and neutrophil elastase (NE) activities of bronchoalveolar lavage (BAL) fluids in 17 CAD patients (with P. aeruginosa infections [CAD+PA], n = 9; without any bacterial infections [CAD-PA], n = 8) and 8 normal volunteers. We found significant elevations of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from CAD patients, in the following rank order: CAD+PA > CAD-PA > normal volunteers. IL-1 beta/albumin ratios were elevated only in CAD+PA, while no TNF bioactivity was detected in BAL fluids. The neutrophil numbers correlated significantly with the IL-8/albumin ratios and NE/albumin ratios in the BAL fluids of CAD patients. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in BAL fluids, the mean reduction rate of NCF activities in CAD+PA patients was significantly higher than that in CAD-PA patients. We also evaluated the effects of low-dose, long-term erythromycin therapy in BAL fluids from three CAD+PA and two CAD-PA patients. Treatment with erythromycin caused significant reductions of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from these patients. To elucidate the mechanism of erythromycin therapy, we also examined whether erythromycin suppressed IL-8 production by human alveolar macrophages and neutrophils in vitro. We demonstrated a moderate inhibitory effect of erythromycin on IL-8 production in Pseudomonas-stimulated neutrophils but not in alveolar macrophages. Our data support the view that persistent P. aeruginosa infection enhances IL-8 production and IL-8-derived NCF activity, causing neutrophil

  5. PM10-stimulated airway epithelial cells activate primary human dendritic cells independent of uric acid: application of an in vitro model system exposing dendritic cells to airway epithelial cell-conditioned media.

    PubMed

    Hirota, Jeremy A; Alexis, Neil E; Pui, Mandy; Wong, SzeWing; Fung, Elkie; Hansbro, Phillip; Knight, Darryl A; Sin, Don D; Carlsten, Chris

    2014-08-01

    Airway epithelial cells represent the first line of defence against inhaled insults, including air pollution. Air pollution can activate innate immune signalling in airway epithelial cells leading to the production of soluble mediators that can influence downstream inflammatory cells. Our objective was to develop and validate a model of dendritic cell exposure to airway epithelial cell-conditioned media. After establishing the model, we explored how soluble mediators released from airway epithelial cells in response to air pollution influenced the phenotype of dendritic cells. Human airway epithelial cells were cultured under control and urban particulate matter (PM10) exposure conditions with or without pharmacological inhibitors of the uric acid pathway. Culture supernatants were collected for conditioned media experiments with peripheral blood mononuclear cell-derived dendritic cells analysed by flow cytometry. Monocytes derived from peripheral blood mononuclear cells cultured in interleukin-4 and granulocyte macrophage colony stimulating factor differentiated into immature dendritic cells that phenotypically differentiated into mature dendritic cells in response to conditioned media from phorbol myristate acetate-activated THP-1 monocytes. Exposure of immature dendritic cells to conditioned media from airway epithelial cells exposed to PM10 resulted in dendritic cell maturation that was independent of uric acid. We present a conditioned media model useful for interrogating the contribution of soluble mediators produced by airway epithelial cells to dendritic cell phenotype and function. Furthermore, we demonstrate that PM10 exposure induces airway epithelial cell production of soluble mediators that induce maturation of dendritic cells independent of uric acid. © 2014 Asian Pacific Society of Respirology.

  6. Airway Basal Cells. The “Smoking Gun” of Chronic Obstructive Pulmonary Disease

    PubMed Central

    2014-01-01

    The earliest abnormality in the lung associated with smoking is hyperplasia of airway basal cells, the stem/progenitor cells of the ciliated and secretory cells that are central to pulmonary host defense. Using cell biology and ’omics technologies to assess basal cells isolated from bronchoscopic brushings of nonsmokers, smokers, and smokers with chronic obstructive pulmonary disease (COPD), compelling evidence has been provided in support of the concept that airway basal cells are central to the pathogenesis of smoking-associated lung diseases. When confronted by the chronic stress of smoking, airway basal cells become disorderly, regress to a more primitive state, behave as dictated by their inheritance, are susceptible to acquired changes in their genome, lose the capacity to regenerate the epithelium, are responsible for the major changes in the airway that characterize COPD, and, with persistent stress, can undergo malignant transformation. Together, these observations led to the conclusion that accelerated loss of lung function in susceptible individuals begins with disordered airway basal cell biology (i.e., that airway basal cells are the “smoking gun” of COPD, a potential target for the development of therapies to prevent smoking-related lung disorders). PMID:25354273

  7. EGF-Amphiregulin Interplay in Airway Stem/Progenitor Cells Links the Pathogenesis of Smoking-Induced Lesions in the Human Airway Epithelium

    PubMed Central

    Zuo, Wu-Lin; Yang, Jing; Gomi, Kazunori; Chao, IonWa; Crystal, Ronald G.; Shaykhiev, Renat

    2017-01-01

    The airway epithelium of cigarette smokers undergoes dramatic remodeling with hyperplasia of basal cells (BC) and mucus-producing cells, squamous metaplasia, altered ciliated cell differentiation and decreased junctional barrier integrity, relevant to chronic obstructive pulmonary disease and lung cancer. In this study, we show that epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) is induced by smoking in human airway epithelium as a result of epidermal growth factor (EGF)-driven squamous differentiation of airway BC stem/progenitor cells. In turn, AREG induced a unique EGFR activation pattern in human airway BC, distinct from that evoked by EGF, leading to BC- and mucous hyperplasia, altered ciliated cell differentiation and impaired barrier integrity. Further, AREG promoted its own expression and suppressed expression of EGF, establishing an autonomous self-amplifying signaling loop in airway BC relevant for promotion of EGF-independent hyperplastic phenotypes. Thus, EGF-AREG interplay in airway BC stem/progenitor cells is one of the mechanisms that mediates the interconnected pathogenesis of all major smoking-induced lesions in the human airway epithelium. PMID:27709733

  8. Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells

    PubMed Central

    Crother, Timothy R.; Schröder, Nicolas W. J.; Karlin, Justin; Chen, Shuang; Shimada, Kenichi; Slepenkin, Anatoly; Alsabeh, Randa; Peterson, Ellena; Arditi, Moshe

    2011-01-01

    Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs. PMID:21695198

  9. Monocyte/macrophage-derived microparticles up-regulate inflammatory mediator synthesis by human airway epithelial cells.

    PubMed

    Cerri, Chiara; Chimenti, Daniele; Conti, Ilaria; Neri, Tommaso; Paggiaro, Pierluigi; Celi, Alessandro

    2006-08-01

    Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

  10. Epidermal growth factor receptor signalling regulates granulocyte-macrophage colony-stimulating factor production by airway epithelial cells and established allergic airway disease.

    PubMed

    Acciani, T H; Suzuki, T; Trapnell, B C; Le Cras, T D

    2016-02-01

    Airway epithelial cells (AEC) are increasingly recognized as a major signalling centre in the pathogenesis of allergic asthma. A previous study demonstrated that epithelial growth factor receptor (EGFR) signalling in AEC regulated key features of allergic airway disease. However, it is unclear what mediators are regulated by EGFR signalling in AEC, although the production of the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is EGFR dependent in keratinocytes. To determine whether EGFR signalling regulates GM-CSF production by human AEC downstream of the clinically relevant mediators house dust mite (HDM) and interleukin (IL)-17A and in a mouse model of established allergic asthma. EGFR inhibitors were used to determine whether EGFR signalling regulates GM-CSF production by cultured human AEC in response to HDM and IL-17A. The roles of EGFR ligands, p38 mitogen-activated protein kinase (MAPK) and tumour necrosis factor-alpha (TNF-α) converting enzyme (TACE) were also assessed. To determine whether EGFR regulates GM-CSF as well as key asthma characteristics in vivo, mice were chronically exposed to HDM to establish allergic airway disease and then treated with the EGFR inhibitor Erlotinib. EGFR inhibition reduced HDM and IL-17A induced GM-CSF production in a dose-dependent manner in cultured human AEC. GM-CSF production also required amphiregulin, p38 MAPK signalling and protease/TACE activity. In mice with established allergic airway disease, EGFR inhibition reduced levels of GM-CSF and TNF-α, as well as airway hyperreactivity, cellular inflammation, smooth muscle thickening and goblet cell metaplasia without changes in IgE and Th1, Th2 and Th17 cytokines. Results link HDM, IL-17A, amphiregulin, EGFR and GM-CSF in a mechanistic pathway in AEC and demonstrate that EGFR regulates GM-CSF production and the severity of established disease in a clinically relevant asthma model. These results identify the EGFR→GM-CSF axis as a

  11. Epidermal growth factor receptor signaling regulates granulocyte-macrophage colony-stimulating factor production by airway epithelial cells and established allergic airway disease

    PubMed Central

    Acciani, Thomas H.; Suzuki, Takuji; Trapnell, Bruce C.; Cras, Timothy D. Le

    2015-01-01

    Background Airway epithelial cells (AEC) are increasingly recognized as a major signaling center in the pathogenesis of allergic asthma. A previous study demonstrated that epithelial growth factor receptor (EGFR) signaling in AEC regulated key features of allergic airway disease. However, it is unclear what mediators are regulated by EGFR signaling in AEC, although the production of the pro-inflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is EGFR-dependent in keratinocytes. Objectives To determine if EGFR signaling regulates GM-CSF production by human AEC downstream of the clinically relevant mediators house dust mite (HDM) and interleukin (IL)-17A and in a mouse model of established allergic asthma. Methods EGFR inhibitors were used to determine whether EGFR signaling regulates GM-CSF production by cultured human AEC in response to HDM and IL-17A. The roles of EGFR ligands, p38 mitogen activated protein kinase (MAPK), and tumor necrosis factor alpha (TNFα) converting enzyme (TACE) were also assessed. To determine if EGFR regulates GM-CSF as well as key asthma characteristics in vivo, mice were chronically exposed to HDM to establish allergic airway disease and then treated with the EGFR inhibitor Erlotinib. Results EGFR inhibition reduced HDM and IL-17A induced GM-CSF production in a dose-dependent manner in cultured human AEC. GM-CSF production also required amphiregulin, p38 MAPK signaling, and protease/TACE activity. In mice with established allergic airway disease, EGFR inhibition reduced levels of GM-CSF and TNFα, as well as airway hyperreactivity, cellular inflammation, smooth muscle thickening, and goblet cell metaplasia without changes in IgE and Th1, Th2, and Th17 cytokines. Conclusions and Clinical Relevance Results link HDM, IL-17A, amphiregulin, EGFR and GM-CSF in a mechanistic pathway in AEC, and demonstrate that EGFR regulates GM-CSF production and the severity of established disease in a clinically relevant asthma

  12. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells.

    PubMed

    Huff, Ryan D; Hsu, Alan C-Y; Nichol, Kristy S; Jones, Bernadette; Knight, Darryl A; Wark, Peter A B; Hansbro, Philip M; Hirota, Jeremy A

    2017-01-01

    The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines.

  13. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    EPA Science Inventory

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  14. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    EPA Science Inventory

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  15. Adoptive transfer of dendritic cells isolated from helminth-infected mice enhanced T regulatory cell responses in airway allergic inflammation.

    PubMed

    Liu, J-Y; Li, L-Y; Yang, X-Z; Li, J; Zhong, G; Wang, J; Li, L-J; Ji, B; Wu, Z-Q; Liu, H; Yang, X; Liu, P-M

    2011-10-01

    Our and others' previous studies have shown that Schistosoma japonicum (SJ) infection can inhibit allergic reactions. Moreover, we found that adoptive transfer of dendritic cells (DCs) from inhibited mice showed a similar inhibitory effect on allergy, suggesting a critical role of DCs in SJ-infected mediated inhibition of allergy. In this study, we further examined the mechanism by which DCs contribute to inhibition of allergy. Our results showed that DCs from SJ-infected mice (SJDCs) produced significantly higher levels of IL-10 compared to those from naive control mice (NDCs). Adoptive transfer of SJDCs, unlike NDCs, significantly increased CD4+CD25+Foxp3+ T cells and CD4+CD25+IL-10+ T cells regulatory T-cell responses in vivo. This was correlated with significantly reduced production of IL-4 and IL-5 by CD4+ T cells, eotaxin in lung tissues and reduced airway allergic inflammation in the SJDC recipients following allergen sensitization and challenge. These data suggest that helminth infection may induce tolerogenic DCs that can inhibit the development of airway allergic inflammation through enhancing T regulatory cell responses.

  16. Reduction of DNA mismatch repair protein expression in airway epithelial cells of premenopausal women chronically exposed to biomass smoke.

    PubMed

    Mukherjee, Bidisha; Dutta, Anindita; Chowdhury, Saswati; Roychoudhury, Sanghita; Ray, Manas Ranjan

    2014-02-01

    Biomass burning is a major source of indoor air pollution in rural India. This study examined whether chronic inhalation of biomass smoke causes change in the DNA mismatch repair (MMR) pathway in the airway cells. For this, airway cells exfoliated in sputum were collected from 72 premenopausal nonsmoking rural women (median age 34 years) who cooked with biomass (wood, dung, crop residues) and 68 control women who cooked with cleaner fuel liquefied petroleum gas (LPG) for the past 5 years or more. The levels of particulate matters with diameters less than 10 and 2.5 μm (PM10 and PM2.5) in indoor air were measured by real-time aerosol monitor. Benzene exposure was monitored by measuring trans,trans-muconic acid (t,t-MA) in urine by high-performance liquid chromatography with ultraviolet detector. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in airway cells were measured by flow cytometry and spectrophotometry, respectively. Immunocytochemical assay revealed lower percentage of airway epithelial cells expressing MMR proteins mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) in biomass-using women compared to LPG-using controls. Women who cooked with biomass had 6.7 times higher level of urinary t,t-MA, twofold increase in ROS generation, and 31 % depletion of SOD. Indoor air of biomass-using households had three times more particulate matters than that of controls. ROS, urinary t,t-MA, and particulate pollution in biomass-using kitchen had negative correlation, while SOD showed positive correlation with MSH2 and MLH1 expression. It appears that chronic exposure to biomass smoke reduces MMR response in airway epithelial cells, and oxidative stress plays an important role in the process.

  17. Role of Neprilysin in Airway Inflammation Induced by Diesel Exhaust Emissions

    PubMed Central

    Wong, Simon S.; Sun, Nina N.; Fastje, Cynthia D.; Witten, Mark L.; Lantz, R. Clark; Lu, Bao; Sherrill, Duane L.; Gerard, Craig J.; Burgess, Jefferey L.

    2016-01-01

    In this study, we examined the role of neprilysin (NEP*), a key membrane-bound endopeptidase, in the inflammatory response induced by diesel exhaust emissions (DEE) in the airways through a number of approaches: in vitro, animal, and controlled human exposure. Our specific aims were (1) to examine the role of NEP in inflammatory injury induced by diesel exhaust particles (DEP) using Nep-intact (wild-type) and Nep-null mice; (2) to examine which components of DEP are associated with NEP downregulation in vitro; (3) to determine the molecular impact of DEP exposure and decreased NEP expression on airway epithelial cells’ gene expression in vitro, using a combination of RNA interference (RNAi) and microarray approaches; and (4) to evaluate the effects on NEP activity of human exposure to DEE. We report four main results: First, we found that exposure of normal mice to DEP consisting of standard reference material (SRM) 2975 via intratracheal installation can downregulate NEP expression in a concentration-dependent manner. The changes were accompanied by increases in the number of macrophages and epithelial cells, as well as proinflammatory cytokines, examined in bronchoalveolar lavage (BAL) fluid and cells. Nep-null mice displayed increased and/or additional inflammatory responses when compared with wild-type mice, especially in response to exposure to the higher dose of DEP that we used. These in vivo findings suggest that loss of NEP in mice could cause increased susceptibility to injury or exacerbate inflammatory responses after DEP exposure via release of specific cytokines from the lungs. Second, we found evidence, using in vitro studies, that downregulation of NEP by DEP in cultured human epithelial BEAS-2B cells was mostly attributable to DEP-adsorbed organic compounds, whereas the carbonaceous core and transition metal components of DEP had little or no effect on NEP messenger RNA (mRNA) expression. This NEP downregulation was not a specific response to DEP

  18. Restoration of Chloride Efflux by Azithromycin in Airway Epithelial Cells of Cystic Fibrosis Patients▿

    PubMed Central

    Saint-Criq, Vinciane; Rebeyrol, Carine; Ruffin, Manon; Roque, Telma; Guillot, Loïc; Jacquot, Jacky; Clement, Annick; Tabary, Olivier

    2011-01-01

    Azithromycin (AZM) has shown promising anti-inflammatory properties in chronic obstructive pulmonary diseases, and clinical studies have presented an improvement in the respiratory condition of cystic fibrosis (CF) patients. The aim of this study was to investigate, in human airway cells, the mechanism by which AZM has beneficial effects in CF. We demonstrated that AZM did not have any anti-inflammatory effect on CF airway cells but restored Cl− efflux. PMID:21220528

  19. Airway cell composition at rest and after an all-out test in competitive rowers.

    PubMed

    Morici, Giuseppe; Bonsignore, Maria R; Zangla, Daniele; Riccobono, Loredana; Profita, Mirella; Bonanno, Anna; Paternò, Alessandra; Di Giorgi, Rossana; Mirabella, Franco; Chimenti, Laura; Benigno, Arcangelo; Vignola, Antonio M; Bellia, Vincenzo; Amato, Giuseppe; Bonsignore, Giovanni

    2004-10-01

    This study was designed to assess: a) whether rowing affects airway cell composition, and b) the possible relationship between the degree of ventilation during exercise and airway cells. In nine young, nonasthmatic competitive rowers (mean age +/- SD: 16.2 +/- 1.0 yr), induced sputum samples were obtained at rest and shortly after an all-out rowing test over 1000 m (mean duration: 200 +/- 14 s), during which ventilatory and metabolic variables were recorded breath-by-breath (Cosmed K4b, Italy). At rest, induced sputum showed prevalence of neutrophils (60%) over macrophages (40%); after exercise, total cell and bronchial epithelial cell (BEC) counts tended to increase. In the last minute of exercise, mean VE was 158.0 +/- 41.5 L x min(-1), and VO2 x kg(-1) 62 +/- 11 mL x min(-1). Exercise VE correlated directly with postexercise total cell (Spearman rho: 0.75, P < 0.05) an dmacrophage (rho: 0.82, P < 0.05) counts. A similar trend was observed for exercise VE and changes in BEC counts from baseline to postexercise (rho: 0.64, P = 0.11). Exercise VE did not correlate with airway neutrophil counts at rest or after exercise. Expression of adhesion molecules by airway neutrophils, macrophages, and eosinophils decreased after the all-out test. Similar to endurance athletes, nonasthmatic competitive rowers showed increased neutrophils in induced sputum compared with values found in sedentary subjects. The trend toward increased BEC postexercise possibly reflected the effects of high airflows on airway epithelium. Airway macrophages postexercise were highest in rowers showing tile most intense exercise hyperpnea, suggesting early involvement of these cells during exercise. However, the low expression of adhesion molecules by all airway cell types suggests that intense short-lived exercise may be associated with a blunted response of airway cells in nonasthmatic well-trained rowers.

  20. Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation

    SciTech Connect

    Svensson Holm, Ann-Charlotte B.; Bengtsson, Torbjoern; Grenegard, Magnus; Lindstroem, Eva G.

    2012-03-10

    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.

  1. T-bet inhibits innate lymphoid cell-mediated eosinophilic airway inflammation by suppressing IL-9 production.

    PubMed

    Matsuki, Ayako; Takatori, Hiroaki; Makita, Sohei; Yokota, Masaya; Tamachi, Tomohiro; Suto, Akira; Suzuki, Kotaro; Hirose, Koichi; Nakajima, Hiroshi

    2017-04-01

    Innate lymphoid cells (ILCs) are emerging subsets of immune cells that produce large amounts of cytokines upon cytokine and/or alarmin stimulation. Recent studies have shown that T-bet plays pivotal roles in the development of ILC3s and type 1 ILCs; however, the roles of T-bet in lung type 2 innate lymphoid cells (ILC2s) remain unknown. We sought to determine the role of T-bet in ILC2-mediated airway inflammation. The expression of T-bet in lung ILCs (defined as Thy1.2(+) Lin(-) cells) was examined. The roles of T-bet in the development of lung ILC2s and airway inflammation induced by IL-33 administration were examined by using T-bet-deficient (T-bet(-/-)) mice. Gene expression profiles of T-bet(-/-) lung ILCs were analyzed by RNA sequencing. T-bet was expressed in lung ILC2s (defined as Thy1.2(+) Lin(-) cells expressing ST2 or CD25) and IFN-γ enhanced its expression. Although the development of lung ILC2s at steady-state conditions was normal in T-bet(-/-) mice, IL-33-induced accumulation of lung ILC2s and eosinophilic airway inflammation were exacerbated in T-bet(-/-) mice. The exacerbated accumulation of ILC2s and eosinophilic airway inflammation by the absence of T-bet were evident even in a RAG2(-/-) background, suggesting that T-bet expressed in non-T/non-B population is involved in the suppression of IL-33-induced eosinophilic airway inflammation. Transcriptome analysis revealed that IL-9 expression in IL-33-stimulated lung ILCs was upregulated in T-bet(-/-) mice compared with that in wild-type mice. Importantly, neutralization of IL-9 markedly attenuated IL-33-induced accumulation of lung ILC2s and eosinophilic inflammation in T-bet(-/-) mice. T-bet suppresses IL-9 production from lung ILC2s and thereby inhibits IL-33-induced eosinophilic airway inflammation. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  2. Toluene diisocyanate exposure induces airway inflammation of bronchial epithelial cells via the activation of transient receptor potential melastatin 8

    PubMed Central

    Kim, Joo-Hee; Jang, Young-Sook; Jang, Seung-Hun; Jung, Ki-Suck; Kim, Seung-Hyun; Ye, Young-Min; Park, Hae-Sim

    2017-01-01

    Toluene diisocyanate (TDI) is the most important cause of occupational asthma (OA), and various pathogenic mechanisms have been suggested. Of these mechanisms, neurogenic inflammation is an important inducer of airway inflammation. Transient receptor potential melastatin 8 (TRPM8) is a well-established cold-sensing cation channel that is expressed in both neuronal cells and bronchial epithelial cells. A recent genome-wide association study of TDI-exposed workers found a significant association between the phenotype of TDI-induced OA and the single-nucleotide polymorphism rs10803666, which has been mapped to the TRPM8 gene. We hypothesized that TRPM8 located in airway epithelial cells may be involved in the pathogenic mechanisms of TDI-induced OA and investigated its role. Bronchial epithelial cells were treated with TDI in a dose- and time-dependent manner. The expression levels of TRPM8 mRNA and protein were determined by quantitative real-time polymerase chain reaction and western blotting. TDI-induced morphological changes in the cells were evaluated by immunocytochemistry. Alterations in the transcripts of inflammatory cytokines were examined in accordance with TRPM8 activation by TDI. TRPM8 expression at both the mRNA and protein levels was enhanced by TDI in airway epithelial cells. TRPM8 activation by TDI led to significant increases in the mRNA of interleukin (IL)-4, IL-13, IL-25 and IL-33. The increased expression of the cytokine genes by TDI was partly attenuated after treatment with a TRPM8 antagonist. TDI exposure induces increased expression of TRPM8 mRNA in airway epithelial cells coupled with enhanced expression of inflammatory cytokines, suggesting a novel role of TRPM8 in the pathogenesis of TDI-induced OA. PMID:28255167

  3. Targeting drugs to the airways: The role of spacer devices.

    PubMed

    Lavorini, Federico; Fontana, Giovanni A

    2009-01-01

    Spacer devices are inhalation aids of varying dimension and complexity, specifically designed to overcome problems with the use of pressurised metered dose inhalers (pMDIs). The aim of this review is to examine the current understanding about these inhalation devices and discuss their advantages and disadvantages. The pertinent literature concerning the characteristics and effects of spacers on delivery and lung deposition of inhaled medications, as well as their clinical efficacy in patients with reversible airway obstruction, is examined. Spacers minimise problems of poor inhalation technique with pMDI, reduce oropharyngeal deposition and increase lung deposition. Spacers improve the clinical effect of inhaled medications, especially in patients unable to use a pMDI properly. Compared to both pMDIs and dry-powder inhalers, spacers may increase the response to beta-adrenergic bronchodilators, even in patients with correct inhalation technique. A pMDI plus spacer has proven to be viable lower cost alternative to the use of a nebuliser for delivering large bronchodilator doses in patients with severe acute asthma or chronic obstructive pulmonary disease. The use of large-volume spacers is recommended for delivering high doses of inhaled corticosteroids, and may permit a lower maintenance dose to be used. pMDIs may be routinely fitted with a spacer, especially in situations where correct pMDI use is unlikely.

  4. Rhinovirus Delays Cell Repolarization in a Model of Injured/Regenerating Human Airway Epithelium

    PubMed Central

    Faris, Andrea N.; Ganesan, Shyamala; Chattoraj, Asamanja; Chattoraj, Sangbrita S.; Comstock, Adam T.; Unger, Benjamin L.; Hershenson, Marc B.

    2016-01-01

    Rhinovirus (RV), which causes exacerbation in patients with chronic airway diseases, readily infects injured airway epithelium and has been reported to delay wound closure. In this study, we examined the effects of RV on cell repolarization and differentiation in a model of injured/regenerating airway epithelium (polarized, undifferentiated cells). RV causes only a transient barrier disruption in a model of normal (mucociliary-differentiated) airway epithelium. However, in the injury/regeneration model, RV prolongs barrier dysfunction and alters the differentiation of cells. The prolonged barrier dysfunction caused by RV was not a result of excessive cell death but was instead associated with epithelial-to-mesenchymal transition (EMT)-like features, such as reduced expression of the apicolateral junction and polarity complex proteins, E-cadherin, occludin, ZO-1, claudins 1 and 4, and Crumbs3 and increased expression of vimentin, a mesenchymal cell marker. The expression of Snail, a transcriptional repressor of tight and adherence junctions, was also up-regulated in RV-infected injured/regenerating airway epithelium, and inhibition of Snail reversed RV-induced EMT-like features. In addition, compared with sham-infected cells, the RV-infected injured/regenerating airway epithelium showed more goblet cells and fewer ciliated cells. Inhibition of epithelial growth factor receptor promoted repolarization of cells by inhibiting Snail and enhancing expression of E-cadherin, occludin, and Crumbs3 proteins, reduced the number of goblet cells, and increased the number of ciliated cells. Together, these results suggest that RV not only disrupts barrier function, but also interferes with normal renewal of injured/regenerating airway epithelium by inducing EMT-like features and subsequent goblet cell hyperplasia. PMID:27119973

  5. Innate immune response of human pluripotent stem cell-derived airway epithelium.

    PubMed

    McIntyre, Brendan A S; Kushwah, Rahul; Mechael, Rami; Shapovalova, Zoya; Alev, Cantas; Bhatia, Mickie

    2015-07-01

    The acquisition of innate immune response is requisite to having bona fide differentiation of airway epithelium. Procedures developed to differentiate lung airway from human pluripotent stem cells (hPSCs) have demonstrated anecdotal evidence for innate immune response, but an in-depth exploration of response levels is lacking. Herein, using an established method of airway epithelial generation from hPSCs, we show that hPSC-derived epithelial cells are able to up-regulate expression of TNFα, IL8 and IL1β in response to challenge with bacterial endotoxin LPS, but lack response from genes associated with innate immune response in other cell types. Further, stimulation of cells with TNF-α resulted in auto-induction of TNFα transcript, as well as cytokine responses of IL8 and IL1β. The demonstration of innate immune induction in hPSC-derived airway epithelia gives further strength to the functionality of in vitro protocols aimed at generating differentiated airway cells that can potentially be used in a translational setting. Finally, we propose that innate immune challenge of airway epithelium from human pluripotent stem cell sources be used as a robust validation of functional in vitro differentiation.

  6. Kalanchoe pinnata inhibits mast cell activation and prevents allergic airway disease.

    PubMed

    Cruz, E A; Reuter, S; Martin, H; Dehzad, N; Muzitano, M F; Costa, S S; Rossi-Bergmann, B; Buhl, R; Stassen, M; Taube, C

    2012-01-15

    Aqueous extract of Kalanchoe pinnata (Kp) have been found effective in models to reduce acute anaphylactic reactions. In the present study, we investigate the effect of Kp and the flavonoid quercetin (QE) and quercitrin (QI) on mast cell activation in vitro and in a model of allergic airway disease in vivo. Treatment with Kp and QE in vitro inhibited degranulation and cytokine production of bone marrow-derived mast cells following IgE/FcɛRI crosslinking, whereas treatment with QI had no effect. Similarly, in vivo treatment with Kp and QE decreased development of airway hyperresponsiveness, airway inflammation, goblet cell metaplasia and production of IL-5, IL-13 and TNF. In contrast, treatment with QI had no effect on these parameters. These findings demonstrate that treatment with Kp or QE is effective in treatment of allergic airway disease, providing new insights to the immunomodulatory functions of this plant.

  7. Curcumin and anthocyanin inhibit pepsin-mediated cell damage and carcinogenic changes in airway epithelial cells.

    PubMed

    Samuels, Tina L; Pearson, Amy C S; Wells, Clive W; Stoner, Gary D; Johnston, Nikki

    2013-10-01

    Laryngopharyngeal reflux (LPR) is associated with inflammatory and neoplastic airway diseases. Gastric pepsin internalized by airway epithelial cells during reflux contributes to oxidative stress, inflammation, and carcinogenesis. Several plant extracts and compounds inhibit digestive enzymes and inflammatory or neoplastic changes to the esophagus in models of gastroesophageal reflux. This study examined the potential of chemoprotective phytochemicals to inhibit peptic activity and mitigate pepsin-mediated damage of airway epithelial cells. Cultured human laryngeal and hypopharyngeal epithelial cells were pretreated with curcumin (10 micromol/L), ecabet sodium (125 microg/mL), and anthocyanin-enriched black-raspberry extract (100 microg/mL) 30 minutes before treatment with pepsin (0.1 mg/mL; 1 hour; pH 7). Controls were treated with media pH 7 or pepsin pH 7 without phytochemicals. Cell damage and proliferative changes were assessed by electron microscopy, cell count, thymidine analog incorporation, and real-time polymerase chain reaction array. Pepsin inhibition was determined by in vitro kinetic assay. Micromolar concentrations of curcumin, ecabet sodium, and black-raspberry extract inhibited peptic activity and pepsin-induced mitochondrial damage and hyperproliferation. Curcumin abrogated pepsin-mediated depression of tumor suppressor gene expression and altered the subcellular localization of pepsin following endocytosis. Several phytochemicals inhibit the pepsin-mediated cell damage underlying inflammatory or neoplastic manifestations of LPR. Dietary supplementation or adjunctive therapy with phytochemicals may represent novel preventive or therapeutic strategies for LPR-attributed disease.

  8. Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

    PubMed

    Ghosh, Moumita; Ahmad, Shama; White, Carl W; Reynolds, Susan D

    2017-01-01

    Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

  9. Equine Airway Mast Cells are Sensitive to Cell Death Induced by Lysosomotropic Agents.

    PubMed

    Wernersson, S; Riihimäki, M; Pejler, G; Waern, I

    2017-01-01

    Mast cells are known for their detrimental effects in various inflammatory conditions. Regimens that induce selective mast cell apoptosis may therefore be of therapeutic significance. Earlier studies have demonstrated that murine- and human-cultured mast cells are highly sensitive to apoptosis induced by the lysosomotropic agent LeuLeuOMe (LLME). However, the efficacy of lysosomotropic agents for inducing apoptosis of in vivo-derived airway mast cells and the impact on mast cells in other species have not been assessed. Here we addressed whether lysosomotropic agents can induce cell death of equine in vivo-derived mast cells. Bronchoalveolar lavage (BAL) fluids from horses were incubated with LLME at 15-100 μm for up to 48 h. The overall cell viability was unaffected by 15 μm LLME up to 48 h, whereas a relatively modest drop in total cell counts (~30%) was seen at the highest LLME dose used. In contrast to the relatively low effect on total cell counts, LLME efficiently and dose dependently reduced the number of mast cells in BAL fluids, with an almost complete depletion (96%) of mast cells after 24 h of incubation with 100 μm LLME. A significant but less dramatic reduction (up to ~45%) of lymphocytes was also seen, whereas macrophages and neutrophils were essentially resistant. The appearance of apoptotic bodies suggested a mechanism involving apoptosis rather than necrosis. These findings suggest that equine airway mast cells are highly sensitive to lysosomotropic agents. Possibly, lysosomotropic agents could be of therapeutic value to treat disorders involving harmful accumulation of mast cells in the airways.

  10. Grepafloxacin inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in human airway epithelial cells.

    PubMed

    Hashimoto, S; Matsumoto, K; Gon, Y; Maruoka, S; Hayashi, S; Asai, Y; Machino, T; Horie, T

    2000-01-01

    We examined the effect of grepafloxacin (GPFX), a new fluoroquinolone antimicrobial agent, on interleukin-8 (IL-8) expression in tumor necrosis factor-alpha (TNF-alpha)-stimulated human airway epithelial cells (AEC). GPFX inhibited IL-8 protein production as well as mRNA expression in a concentration-dependent manner (2.5 - 25 micro g/ml), but the inhibition of IL-8 expression by corresponding concentrations of GPFX to serum and airway lining fluids was not complete. We discuss the modulatory effect of GPFX on IL-8 production in the context of its efficacy on controlling chronic airway inflammatory diseases.

  11. Basal cells as stem cells of the mouse trachea and human airway epithelium

    PubMed Central

    Rock, Jason R.; Onaitis, Mark W.; Rawlins, Emma L.; Lu, Yun; Clark, Cheryl P.; Xue, Yan; Randell, Scott H.; Hogan, Brigid L. M.

    2009-01-01

    The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreERT2 transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay. PMID:19625615

  12. [Role and mechanism of heat shock protein 70 in airway hypersecretion].

    PubMed

    Zhou, Zhiyu; Zhang, Ting; Zhou, Xiangdong; Li, Qi; Liu, Chunyi

    2015-03-03

    To explore the role and mechanism of heat shock protein 70 (HSP70) in airway hypersecretion. After a stimulation of 8% cigarette smoke extract (CSE), airway cells A549 were treated with HSP70 antibody and c-Jun N-terminal kinase (JNK) specific inhibitor SP600125 respectively. And the cells were divided into 4 groups of blank contrast (serum-free medium), CSE stimulation (8% CSE for 24 h), HSP70 antibody (30-min pre-treatment of HSP70 antibody and culturing in 8% CSE for 24 h) and SP600125 (30-min pre-treatment of SP600125 30 µmol/L and culturing in 8% CSE for 24 h). The relative expression levels of MUC5AC protein in various groups were determined by enzyme-linked immunosorbent assay (ELISA). And the relative transcription level of MUC5AC mRNA was detected by reverse transcription-polymerase chain reaction (PCR) while the synthesized levels of HS70 as well as the phosphorylation levels of JNK and activated protein-1 (mostly c-Jun) were measured by Western blot. As compared with those in blank contrast group (0.26 ± 0.10, 0.28 ± 0.06, 0.30 ± 0.05, 0.30 ± 0.08, 0.36 ± 0.08), HSP70 antibody group (0.30 ± 0.12, 0.29 ± 0.09, 0.34 ± 0.06, 0.47 ± 0.19, 0.39 ± 0.13) and SP600125 group (0.38 ± 0.06, 0.31 ± 0.14, 0.39 ± 0.04, 0.44 ± 0.12, 0.48 ± 0.11), the relative expression levels of MUC5AC protein and mRNA, phosphorylation JNK (p-JNK), c-Jun and p-c-Jun (0.52 ± 0.07, 0.64 ± 0.11, 0.73 ± 0.06, 0.67 ± 0.10, 0.67 ± 0.09) significantly increased in CSE stimulation group (all P < 0.05). And the synthesis levels of HSP70 (0.75 ± 0.09) in CSE stimulation group increased than blank contrast group (0.29 ± 0.03) and HSP70 antibody group (0.40 ± 0.11) (all P < 0.05). HSP70 may enhance the expression of MUC5AC in bronchial epithelial A549 cells via a signaling pathway of JNK/AP-1..

  13. The role of transforming growth factor-β1 in airway inflammation of childhood asthma.

    PubMed

    Gagliardo, R; Chanez, P; Gjomarkaj, M; La Grutta, S; Bonanno, A; Montalbano, A M; Di Sano, C; Albano, G D; Gras, D; Anzalone, G; Riccobono, L; Profita, M

    2013-01-01

    TGF-beta-targeting structural and inflammatory cells has been implicated in the mechanisms leading to the inflammatory and restructuring processes in asthma, suggesting an impact of TGF-beta1 signaling on the development and persistency of this disease. We investigated the potential early involvement of TGF-beta1 activity in the immunological and molecular mechanisms underlying progression of inflammation in childhood asthma. We evaluated the levels of TGF-beta1 in induced sputum supernatants (ISSs) and the expression of small mother cell against decapentaplegic (Smad) 2 and Smad7 proteins in induced sputum cells (ISCs) from children with intermittent asthma (IA), moderate asthma (MA) and control subjects (C). Furthermore, we investigated the regulatory role of TGF-beta1 activity on eosinophil and neutrophil adhesion to epithelial cells using adhesion assay, and on the granulocyte expression of adhesion molecule CD11b/CD18 Macrophage-1 antigen (MAC-1), by flow cytometry. We found that the levels of TGF-beta1 are increased in ISSs of IA and MA in comparison to C, concomitantly to the activation of intracellular signaling TGFbeta/Smads pathway in ISCs. In MA, TGF-beta1 levels correlated with the number of sputum eosinophils and neutrophils. Furthermore, we showed the ability of sputum TGF-beta1 to promote eosinophil and neutrophil adhesion to epithelial cells, and to increase the expression of MAC-1 on the granulocyte surface. This study shows the activation of TGFbeta/Smad signaling pathway in the airways of children with IA and, despite the regular ICS treatment, in children with MA, and provides evidence for the contribution of TGF-beta1 in the regulation of granulocyte activation and trafficking.

  14. T cell chemotaxis and chemokine release after Staphylococcus aureus interaction with polarized airway epithelium.

    PubMed

    Escotte, Sandie; Al Alam, Denise; Le Naour, Richard; Puchelle, Edith; Guenounou, Moncef; Gangloff, Sophie C

    2006-03-01

    In response to bacterial infection, airway epithelium releases inflammatory mediators including cytokines and chemokines that lead to immune cell efflux and could stimulate the adaptive T cell immune response. The aim of our study was to analyze, in a double chamber culture, the chemokine changes in response to Staphylococcus aureus and their consequences for T cells. Our data show that S. aureus stimulates basolateral and apical release of IL-8 and eotaxin by airway epithelial cells. We also observed increased chemokine receptor expression on CD8+ and CD4+ T cells and enhanced chemotaxis of CD4+ T cells toward apical supernatant. Our data strongly suggest that S. aureus interaction with airway epithelium contributes to specific migration of T cells to inflamed sites.

  15. Expression of IL-4/IL-13 receptors in differentiating human airway epithelial cells

    PubMed Central

    Martin, Linda D.; Stern, Randi; Laxman, Bharathi; Marroquin, Bertha A.

    2010-01-01

    IL-4 and IL-13 elicit several important responses in airway epithelium including chemokine secretion and mucous secretion that may contribute to airway inflammation, cell migration, and differentiation. These cytokines have overlapping but not identical effector profiles likely due to shared subunits in their receptor complexes. These receptors are variably described in epithelial cells, and the relative expression, localization, and function of these receptors in differentiated and repairing epithelial cells are not clear. We examined IL-4/IL-13 receptor expression and localization in primary airway epithelial cells collected from normal human lungs and grown under conditions yielding both undifferentiated and differentiated cells inclusive of basal, goblet, and ciliated cell phenotypes. Gene expression of the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2 receptor subunits increased with differentiation, but different patterns of localization and protein abundance were seen for each subunit based on both differentiation and the cell subtypes present. Increased expression of receptor subunits observed in more differentiated cells was associated with more substantial functional responses to IL-4 stimulation including increased eotaxin-3 expression and accelerated migration after injury. We demonstrate substantial differences in IL-4/IL-13 receptor subunit expression and responsiveness to IL-4 based on the extent of airway epithelial cell differentiation and suggest that these differences may have functional consequences in airway inflammation. PMID:20729386

  16. Unjamming and cell shape in the asthmatic airway epithelium

    NASA Astrophysics Data System (ADS)

    Park, Jin-Ah; Kim, Jae Hun; Bi, Dapeng; Mitchel, Jennifer A.; Qazvini, Nader Taheri; Tantisira, Kelan; Park, Chan Young; McGill, Maureen; Kim, Sae-Hoon; Gweon, Bomi; Notbohm, Jacob; Steward, Robert, Jr.; Burger, Stephanie; Randell, Scott H.; Kho, Alvin T.; Tambe, Dhananjay T.; Hardin, Corey; Shore, Stephanie A.; Israel, Elliot; Weitz, David A.; Tschumperlin, Daniel J.; Henske, Elizabeth P.; Weiss, Scott T.; Manning, M. Lisa; Butler, James P.; Drazen, Jeffrey M.; Fredberg, Jeffrey J.

    2015-10-01

    From coffee beans flowing in a chute to cells remodelling in a living tissue, a wide variety of close-packed collective systems--both inert and living--have the potential to jam. The collective can sometimes flow like a fluid or jam and rigidify like a solid. The unjammed-to-jammed transition remains poorly understood, however, and structural properties characterizing these phases remain unknown. Using primary human bronchial epithelial cells, we show that the jamming transition in asthma is linked to cell shape, thus establishing in that system a structural criterion for cell jamming. Surprisingly, the collapse of critical scaling predicts a counter-intuitive relationship between jamming, cell shape and cell-cell adhesive stresses that is borne out by direct experimental observations. Cell shape thus provides a rigorous structural signature for classification and investigation of bronchial epithelial layer jamming in asthma, and potentially in any process in disease or development in which epithelial dynamics play a prominent role.

  17. TRPC3-mediated Ca(2+) entry contributes to mouse airway smooth muscle cell proliferation induced by lipopolysaccharide.

    PubMed

    Chen, Xiao-Xu; Zhang, Jia-Hua; Pan, Bin-Hua; Ren, Hui-Li; Feng, Xiu-Ling; Wang, Jia-Ling; Xiao, Jun-Hua

    2016-10-01

    Airway remodeling is a histopathological hallmark of chronic respiratory diseases that includes airway smooth muscle cell (ASMC) proliferation. Canonical transient receptor potential channel-3 (TRPC3)-encoded nonselective cation channels (NSCCs) are important native constitutively active channels that play significant roles in physiological and pathological conditions in ASMCs. Lipopolysaccharides (LPSs), known as lipoglycans and endotoxin, have been proven to be inducers of airway remodeling, though the mechanisms remain unclear. We hypothesized that TRPC3 is important in LPS-induced airway remodeling by regulating ASMC proliferation. To test this hypothesis, mouse ASMCs were cultured with or without LPS for 48h. Cell viability, TRPC3 protein expression, NSCC currents and changes in intracellular calcium concentration ([Ca(2+)]i) were then analyzed using an MTT assay, western blotting, whole-cell patch clamp and calcium imaging, respectively. The results showed that LPS treatment significantly induced ASMC proliferation, up-regulation of TRPC3 protein expression and enhancement of NSCC currents, resting [Ca(2+)]i and ACh-elicited changes in [Ca(2+)]i. TRPC3 blocker Gd(3+), TRPC3 blocking antibody or TRPC3 gene silencing by siRNA significantly inhibited LPS-induced up-regulation of TRPC3 protein, enhancement of NSCC currents, resting [Ca(2+)]i and ACh-elicited changes in [Ca(2+)]i, eventually inhibiting LPS-induced ASMCproliferation. These results demonstrated that TRPC3-mediated Ca(2+) entry contributed to LPS-induced ASMC proliferation and identified TRPC3 as a possible key target in airway remodeling intervention. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Vitamin D attenuates cytokine-induced remodeling in human fetal airway smooth muscle cells.

    PubMed

    Britt, Rodney D; Faksh, Arij; Vogel, Elizabeth R; Thompson, Michael A; Chu, Vivian; Pandya, Hitesh C; Amrani, Yassine; Martin, Richard J; Pabelick, Christina M; Prakash, Y S

    2015-06-01

    Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25-dihydroxyvitamin D3 ; 1,25(OH)2 D3 ), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro-inflammatory cytokines. Cells were pre-treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP-9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ-induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood.

  19. Static and dynamic upper airway obstruction in sleep apnea: role of the breathing gas properties.

    PubMed

    Farré, Ramon; Rigau, Jordi; Montserrat, Josep M; Buscemi, Lara; Ballester, Eugeni; Navajas, Daniel

    2003-09-15

    Increased upper airway collapsibility in the sleep apnea/hypopnea syndrome (SAHS) is usually interpreted by a collapsible resistor model characterized by a critical pressure (Pcrit) and an upstream resistance (Rup). To investigate the role played by the upstream segment of the upper airway, we tested the hypothesis that breathing different gases would modify Rup but not Pcrit. The study was performed on 10 patients with severe SAHS (apnea-hypopnea index: 59 +/- 14 events/hour) when breathing air and helium-oxygen (He-O2) during non-REM sleep. The continuous positive airway pressure that normalized flow (CPAPopt) was measured. Rup and Pcrit were determined from the linear relationship between maximal inspiratory flow VImax and nasal pressure (PN):VImax = (PN - Pcrit)/Rup. Changing the breathing gas selectively modified the severity of dynamic (CPAPopt, Rup) and static (Pcrit) obstructions. CPAPopt was significantly (p = 0.0013) lower when breathing He-O2 (8.44 +/- 1.66 cm H2O; mean +/- SD) than air (10.18 +/- 2.34 cm H2O). Rup was markedly lower (p = 0.0001) when breathing He-O2 (9.21 +/- 3.93 cm H2O x s/L) than air (15.92 +/- 6.27 cm H2O x s/L). Pcrit was similar (p = 0.039) when breathing He-O2 (4.89 +/- 2.37 cm H2O) and air (4.19 +/- 2.93 cm H2O). The data demonstrate the role played by the upstream segment of the upper airway and suggest that different mechanisms determine static (Pcrit) and dynamic (Rup) upper airway obstructions in SAHS.

  20. Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells.

    PubMed

    Kolesnikova, Larissa; Heck, Sonja; Matrosovich, Tatyana; Klenk, Hans-Dieter; Becker, Stephan; Matrosovich, Mikhail

    2013-05-01

    The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy. Budding of spherical and filamentous virions was observed on the apical surfaces of cells with no association with cilia and secretory granules. Quantitative analysis of the distribution of viral buds on the cell surface indicated that the tips of the microvilli represented a prominent site of influenza virus budding in the human airway epithelium. As the microvilli of differentiated cells are involved in many fundamental cell functions, these data will prompt further studies on the biological significance of microvilli-associated budding for virus replication, transmission and pathogenicity.

  1. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering.

    PubMed

    Butler, Colin R; Hynds, Robert E; Gowers, Kate H C; Lee, Dani Do Hyang; Brown, James M; Crowley, Claire; Teixeira, Vitor H; Smith, Claire M; Urbani, Luca; Hamilton, Nicholas J; Thakrar, Ricky M; Booth, Helen L; Birchall, Martin A; De Coppi, Paolo; Giangreco, Adam; O'Callaghan, Christopher; Janes, Sam M

    2016-07-15

    Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

  2. Culture of Airway Epithelial Cells from Neonates Sampled within 48-Hours of Birth

    PubMed Central

    Miller, David; Turner, Steve W.; Spiteri-Cornish, Daniella; McInnes, Neil; Scaife, Alison; Danielian, Peter J.; Devereux, Graham; Walsh, Garry M.

    2013-01-01

    Introduction Little is known about how neonatal airway epithelial cell phenotype impacts on respiratory disease in later life. This study aimed to establish a methodology to culture and characterise neonatal nasal epithelial cells sampled from healthy, non-sedated infants within 48 hours of delivery. Methods Nasal epithelial cells were sampled by brushing both nostrils with an interdental brush, grown to confluence and sub-cultured. Cultured cells were characterised morphologically by light and electron microscopy and by immunocytochemistry. As an exemplar pro-inflammatory chemokine, IL-8 concentrations were measured in supernatants from unstimulated monolayers and after exposure to IL-1β/TNF-α or house dust mite extract. Results Primary cultures were successfully established in 135 (91%) of 149 neonatal samples seeded, with 79% (n  =  117) successfully cultured to passage 3. The epithelial lineage of the cells was confirmed by morphological analysis and immunostaining. Constitutive IL-8 secretion was observed and was upregulated by IL-1β/TNF-α or house dust mite extract in a dose dependent manner. Conclusion We describe a safe, minimally invasive method of culturing nasal epithelial cells from neonates suitable for functional cell analysis offering an opportunity to study “naïve” cells that may prove useful in elucidating the role of the epithelium in the early origins of asthma and/or allergic rhinitis. PMID:24223790

  3. Ambient particulate matter induces an exacerbation of airway inflammation in experimental asthma: role of interleukin-33.

    PubMed

    Shadie, A M; Herbert, C; Kumar, R K

    2014-08-01

    High levels of ambient environmental particulate matter (PM10 i.e. < 10 μm median aerodynamic diameter) have been linked to acute exacerbations of asthma. We examined the effects of delivering a single dose of Sydney PM10 by intranasal instillation to BALB/c mice that had been sensitized to ovalbumin and challenged repeatedly with a low (≈3 mg/m(3)) mass concentration of aerosolized ovalbumin for 4 weeks. Responses were compared to animals administered carbon black as a negative control, or a moderate (≈30 mg/m(3)) concentration of ovalbumin to simulate an allergen-induced acute exacerbation of airway inflammation. Delivery of PM10 to mice, in which experimental mild chronic asthma had previously been established, elicited characteristic features of enhanced allergic inflammation of the airways, including eosinophil and neutrophil recruitment, similar to that in the allergen-induced exacerbation. In parallel, there was increased expression of mRNA for interleukin (IL)-33 in airway tissues and an increased concentration of IL-33 in bronchoalveolar lavage fluid. Administration of a monoclonal neutralizing anti-mouse IL-33 antibody prior to delivery of particulates significantly suppressed the inflammatory response induced by Sydney PM10, as well as the levels of associated proinflammatory cytokines in lavage fluid. We conclude that IL-33 plays a key role in driving airway inflammation in this novel experimental model of an acute exacerbation of chronic allergic asthma induced by exposure to PM10.

  4. Role of the parasympathetic nervous system in airway hyperresponsiveness after ozone inhalation

    SciTech Connect

    Jones, G.L.; Lane, C.G.; Manning, P.J.; O'Byrne, P.M.

    1987-09-01

    Airway hyperresponsiveness develops in dogs after ozone inhalation. This study examined the role of the parasympathetic nervous system in ozone-induced airway hyperresponsiveness in dogs. Dose-response curves to acetylcholine (n = 8) and histamine (n = 4) were measured before and after exposure to ozone (3 ppm for 30 min). The provocative concentration of each agonist was measured on two randomly assigned days separated by at least 1 wk. On one day a control experiment was performed, and on the other day the dogs were pretreated with the ganglionic blocker hexamethonium bromide in doses that block ganglionic transmission. The acetylcholine provocative concentration decreased on the control day from 5.5 mg/ml (%SE 1.8) before ozone to 0.5 mg/ml (%SE 2.0) after ozone (P less than 0.0001). After pretreatment with hexamethonium the acetylcholine provocative concentration decreased from 9.0 mg/ml (%SE 1.8) before ozone to 1.0 mg/ml (%SE 2.0) after ozone (P = 0.002). The results were similar when histamine was used as the agonist. Therefore, ganglionic blockade does not prevent airway hyperresponsiveness after ozone inhalation, and a parasympathetic reflex mechanism is not responsible for airway hyperresponsiveness after ozone inhalation in dogs.

  5. Is there a role for airway clearance techniques in chronic obstructive pulmonary disease?

    PubMed

    Holland, Anne E; Button, Brenda M

    2006-01-01

    Chronic cough and sputum production are common features of chronic obstructive pulmonary disease (COPD) and have a significant impact on exacerbation frequency and quality of life. Despite this, techniques to assist with removal of sputum from the airway do not have a well-defined role in COPD management. Clinical trials of airway clearance techniques (ACTs) in COPD have shown mixed results with little evidence of long-term benefit. However many studies have failed to account for the heterogeneity of COPD lung disease, particularly with regard to the presence of bronchiectasis. Analysis of short-term studies suggests that there may be a cohort of patients who will benefit from prescription of a sputum clearance regimen. This review proposes a physiological rationale for the use of ACTs in COPD, taking into consideration the presence of bronchiectasis, the amount of sputum produced, the degree of airflow obstruction and the presence of decreased lung elastic recoil. The selection of an optimal ACT for individuals with COPD should take into account its effects on lung vloumes, expiratory flow and dynamic airway compression. Care should be taken to avoid airway collapse during forced expirations in patients with reduced lung recoil pressure; positive expiratory pressure therapy or autogenic drainage may prove effective in this patient group. The acceptability of ACTs to patients should also be considered, especially where long-term adherence is required. Future research should focus on more appropriate matching of the physiological effects of individual ACTs to the pathophysiology of lung disease in COPD.

  6. Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts

    PubMed Central

    Lama, Vibha N.; Harada, Hiroaki; Badri, Linda N.; Flint, Andrew; Hogaboam, Cory M.; McKenzie, Andrew; Martinez, Fernando J.; Toews, Galen B.; Moore, Bethany B.; Pinsky, David J.

    2006-01-01

    The pathogenesis of bronchiolitis obliterans (BO), a common and devastating obliterative disorder of small airways following lung transplantation, remains poorly understood. Lesions are characterized in their early stages by lymphocyte influx that evolves into dense fibrotic infiltrates. Airway specimens taken from patients with histological BO revealed infiltrating myofibroblasts, which strongly expressed the signaling chain of the high affinity interleukin-13 (IL-13) receptor IL-13Rα1. Because IL-13 has proinflammatory and profibrotic actions, a contributory role for IL-13 in BO development was examined using murine models of orthotopic and heterotopic tracheal transplantation. Compared with airway isografts, allografts exhibited a significant increase in relative IL-13 mRNA and protein levels. Allogeneic tracheas transplanted into IL-13-deficient mice were protected from BO in both transplant models. Flow cytometric analysis of orthotopic transplant tissue digests revealed markedly fewer infiltrating mononuclear phagocytes and CD3+ T lymphocytes in IL-13-deficient recipients. Furthermore, protection from luminal obliteration, collagen deposition, and myofibroblast infiltration was observed in heterotopic airways transplanted into the IL-13−/− recipients. Transforming growth factor-β1 expression was significantly decreased in tracheal allografts into IL-13−/− recipients, compared to wild-type counterparts. These human and murine data implicate IL-13 as a critical effector cytokine driving cellular recruitment and subsequent fibrosis in clinical and ex-perimental BO. PMID:16816360

  7. Ambient particulate matter induces an exacerbation of airway inflammation in experimental asthma: role of interleukin-33

    PubMed Central

    Shadie, A M; Herbert, C; Kumar, R K

    2014-01-01

    High levels of ambient environmental particulate matter (PM10 i.e. < 10 μm median aerodynamic diameter) have been linked to acute exacerbations of asthma. We examined the effects of delivering a single dose of Sydney PM10 by intranasal instillation to BALB/c mice that had been sensitized to ovalbumin and challenged repeatedly with a low (≈3 mg/m3) mass concentration of aerosolized ovalbumin for 4 weeks. Responses were compared to animals administered carbon black as a negative control, or a moderate (≈30 mg/m3) concentration of ovalbumin to simulate an allergen-induced acute exacerbation of airway inflammation. Delivery of PM10 to mice, in which experimental mild chronic asthma had previously been established, elicited characteristic features of enhanced allergic inflammation of the airways, including eosinophil and neutrophil recruitment, similar to that in the allergen-induced exacerbation. In parallel, there was increased expression of mRNA for interleukin (IL)-33 in airway tissues and an increased concentration of IL-33 in bronchoalveolar lavage fluid. Administration of a monoclonal neutralizing anti-mouse IL-33 antibody prior to delivery of particulates significantly suppressed the inflammatory response induced by Sydney PM10, as well as the levels of associated proinflammatory cytokines in lavage fluid. We conclude that IL-33 plays a key role in driving airway inflammation in this novel experimental model of an acute exacerbation of chronic allergic asthma induced by exposure to PM10. PMID:24730559

  8. IL-22 Is Produced by Innate Lymphoid Cells and Limits Inflammation in Allergic Airway Disease

    PubMed Central

    Gyülveszi, Gabor; Dehzad, Nina; Kreymborg, Katharina; Schneeweiss, Kristin; Michel, Erich; Reuter, Sebastian; Renauld, Jean-Christophe; Arnold-Schild, Danielle; Schild, Hansjörg; Buhl, Roland; Becher, Burkhard

    2011-01-01

    Interleukin (IL)-22 is an effector cytokine, which acts primarily on epithelial cells in the skin, gut, liver and lung. Both pro- and anti-inflammatory properties have been reported for IL-22 depending on the tissue and disease model. In a murine model of allergic airway inflammation, we found that IL-22 is predominantly produced by innate lymphoid cells in the inflamed lungs, rather than TH cells. To determine the impact of IL-22 on airway inflammation, we used allergen-sensitized IL-22-deficient mice and found that they suffer from significantly higher airway hyperreactivity upon airway challenge. IL-22-deficiency led to increased eosinophil infiltration lymphocyte invasion and production of CCL17 (TARC), IL-5 and IL-13 in the lung. Mice treated with IL-22 before antigen challenge displayed reduced expression of CCL17 and IL-13 and significant amelioration of airway constriction and inflammation. We conclude that innate IL-22 limits airway inflammation, tissue damage and clinical decline in allergic lung disease. PMID:21789181

  9. Airway Hyperresponsiveness in Children With Sickle Cell Anemia

    PubMed Central

    Field, Joshua J.; Stocks, Janet; Kirkham, Fenella J.; Rosen, Carol L.; Dietzen, Dennis J.; Semon, Trisha; Kirkby, Jane; Bates, Pamela; Seicean, Sinziana; DeBaun, Michael R.; Redline, Susan

    2011-01-01

    Background: The high prevalence of airway hyperresponsiveness (AHR) among children with sickle cell anemia (SCA) remains unexplained. Methods: To determine the relationship between AHR, features of asthma, and clinical characteristics of SCA, we conducted a multicenter, prospective cohort study of children with SCA. Dose response slope (DRS) was calculated to describe methacholine responsiveness, because 30% of participants did not achieve a 20% decrease in FEV1 after inhalation of the highest methacholine concentration, 25 mg/mL. Multiple linear regression analysis was done to identify independent predictors of DRS. Results: Methacholine challenge was performed in 99 children with SCA aged 5.6 to 19.9 years (median, 12.8 years). Fifty-four (55%) children had a provocative concentration of methacholine producing a 20% decrease in FEV1 < 4 mg/mL. In a multivariate analysis, independent associations were found between increased methacholine responsiveness and age (P < .001), IgE (P = .009), and lactate dehydrogenase (LDH) levels (P = .005). There was no association between methacholine responsiveness and a parent report of a doctor diagnosis of asthma (P = .986). Other characteristics of asthma were not associated with methacholine responsiveness, including positive skin tests to aeroallergens, exhaled nitric oxide, peripheral blood eosinophil count, and pulmonary function measures indicating airflow obstruction. Conclusions: In children with SCA, AHR to methacholine is prevalent. Younger age, serum IgE concentration, and LDH level, a marker of hemolysis, are associated with AHR. With the exception of serum IgE, no signs or symptoms of an allergic diathesis are associated with AHR. Although the relationship between methacholine responsiveness and LDH suggests that factors related to SCA may contribute to AHR, these results will need to be validated in future studies. PMID:20724735

  10. Immunostimulatory DNA mediates inhibition of eosinophilic inflammation and airway hyperreactivity independent of natural killer cells in vivo.

    PubMed

    Broide, D H; Stachnick, G; Castaneda, D; Nayar, J; Miller, M; Cho, J; Rodriquez, M; Roman, M; Raz, E

    2001-11-01

    Immunostimulatory DNA sequences (ISS) inhibit eosinophilic inflammation and airway hyperreactivity in mouse models of asthma. In vitro ISS activate natural killer (NK) cells to secrete IFN-gamma, and this cytokine is hypothesized to contribute to the antiallergic effect of ISS in vivo. We investigated whether ISS activation of NK cells is important in mediating the reduction in airway hyperreactivity and the antieosinophilic effect of ISS in vivo. We assessed whether ISS modulated the development of eosinophilic airway inflammation and airway hyperreactivity to methacholine in ovalbumin (OVA)-sensitized and OVA allergen-challenged mice pretreated with an antibody to deplete NK cells. Mice sensitized and challenged with OVA had significant bronchoalveolar lavage and lung eosinophilia, as well as airway hyperresponsiveness. ISS induced significant inhibition of bronchoalveolar lavage and lung eosinophilia, as well as airway hyperresponsiveness, in OVA-sensitized mice pretreated before OVA challenge with an NK cell-depleting antibody (NK(-) mice), as well as in mice pretreated with a control non-NK cell-depleting antibody (NK(+) mice). The NK cell-depleting antibody inhibited ISS-induced IFN-gamma production by spleen cells. These studies demonstrate that depletion of NK cells has no significant effect on ISS-mediated inhibition of airway eosinophilia and airway hyperresponsiveness in vivo, suggesting that non-NK cells and cytokines other than IFN-gamma derived from NK cells mediate the majority of the ISS-inhibitory effect on eosinophilic inflammation and airway hyperresponsiveness in vivo.

  11. Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung

    PubMed Central

    Thornton, Emily E.; Looney, Mark R.; Bose, Oishee; Sen, Debasish; Sheppard, Dean; Locksley, Richard; Huang, Xiaozhu

    2012-01-01

    Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b+ dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung. PMID:22585735

  12. Airway Epithelial Cell Integrity Protects from Cytotoxicity of Pseudomonas aeruginosa Quorum-Sensing Signals.

    PubMed

    Losa, Davide; Köhler, Thilo; Bacchetta, Marc; Saab, Joanna Bou; Frieden, Maud; van Delden, Christian; Chanson, Marc

    2015-08-01

    Cell-to-cell communication via gap junctions regulates airway epithelial cell homeostasis and maintains the epithelium host defense. Quorum-sensing molecules produced by Pseudomonas aeruginosa coordinate the expression of virulence factors by this respiratory pathogen. These bacterial signals may also incidentally modulate mammalian airway epithelial cell responses to the pathogen, a process called interkingdom signaling. We investigated the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). C12 degradation and its effects on cells were monitored in various airway epithelial cell models grown under nonpolarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. C12 rapidly altered epithelial integrity and decreased GJIC in nonpolarized airway epithelial cells, whereas other quorum-sensing molecules had no effect. The effects of C12 were dependent on [Ca(2+)]i and could be prevented by inhibitors of Src tyrosine family and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12 except when undergoing repair after wounding. In vivo during colonization of intubated patients, C12 did not accumulate, but it paralleled bacterial densities. In vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in nonpolarized cells, whereas PON2 expression was increased during epithelial polarization. The cytotoxicity of C12 on nonpolarized epithelial cells, combined with its impaired degradation allowing its accumulation, provides an additional pathogenic mechanism for P. aeruginosa infections.

  13. RhoA orchestrates glycolysis for TH2 cell differentiation and allergic airway inflammation.

    PubMed

    Yang, Jun-Qi; Kalim, Khalid W; Li, Yuan; Zhang, Shuangmin; Hinge, Ashwini; Filippi, Marie-Dominique; Zheng, Yi; Guo, Fukun

    2016-01-01

    Mitochondrial metabolism is known to be important for T-cell activation. However, its involvement in effector T-cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T-cell activation and effector cell differentiation and function remains largely unknown. We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for TH2 cell differentiation and TH2-mediated allergic airway inflammation. Conditional RhoA-deficient mice were generated by crossing RhoA(flox/flox) mice with CD2-Cre transgenic mice. Effects of RhoA on TH2 differentiation were evaluated based on in vitro TH2-polarized culture conditions and in vivo in ovalbumin-induced allergic airway inflammation. Cytokine levels were measured by using intracellular staining and ELISA. T-cell metabolism was measured by using the Seahorse XF24 Analyzer and flow cytometry. Disruption of RhoA inhibited T-cell activation and TH2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo, with no effect on TH1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways, such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to TH2 cell differentiation and allergic airway inflammation through regulating IL-4 receptor mRNA expression and TH2-specific signaling events. Finally, inhibition of Rho-associated protein kinase, an immediate downstream effector of RhoA, blocked TH2 differentiation and allergic airway inflammation. RhoA is a key component of the signaling cascades leading to TH2 differentiation and allergic airway inflammation at least in part through control of T-cell metabolism and the Rho-associated protein kinase pathway. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  14. Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

    PubMed Central

    Gowers, Kate H. C.; Lee, Dani Do Hyang; Brown, James M.; Crowley, Claire; Teixeira, Vitor H.; Smith, Claire M.; Urbani, Luca; Hamilton, Nicholas J.; Thakrar, Ricky M.; Booth, Helen L.; Birchall, Martin A.; De Coppi, Paolo; Giangreco, Adam; O’Callaghan, Christopher

    2016-01-01

    Rationale: Stem cell–based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell–seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. Objectives: To define a scalable cell culture system to deliver airway epithelium to clinical grafts. Methods: Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air–liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air–liquid interface cultures. Measurements and Main Results: 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. Conclusions: Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical

  15. Hydrogen sulfide inhalation ameliorates allergen induced airway hypereactivity by modulating mast cell activation.

    PubMed

    Roviezzo, Fiorentina; Bertolino, Antonio; Sorrentino, Rosalinda; Terlizzi, Michela; Matteis, Maria; Calderone, Vincenzo; Mattera, Valentina; Martelli, Alma; Spaziano, Giuseppe; Pinto, Aldo; D'Agostino, Bruno; Cirino, Giuseppe

    2015-10-01

    Compelling evidence suggests that hydrogen sulfide represents an important gaseous transmitter in the mammalian respiratory system. In the present study, we have evaluated the role of mast cells in hydrogen sulfide-induced effects on airways in a mouse model of asthma. Mice were sensitized to ovalbumin and received aerosol of a hydrogen sulfide donor (NaHS; 100 ppm) starting at day 7 after ovalbumin challenge. Exposure to hydrogen sulfide abrogated ovalbumin-induced bronchial hypereactivity as well as the increase in lung resistance. Concomitantly, hydrogen sulfide prevented mast cell activity as well as FGF-2 and IL-13 upregulation. Conversely, pulmonary inflammation and the increase in plasmatic IgE levels were not affected by hydrogen sulfide. A lack of hydrogen sulfide effects in mast cell deficient mice occurred. Primary fibroblasts harvested from ovalbumin-sensitized mice showed an increased proliferation rate that was inhibited by hydrogen sulfide aerosol. Furthermore, ovalbumin-induced transdifferentiation of pulmonary fibroblasts into myofibroblasts was reversed. Finally, hydrogen sulfide did abrogate in vitro the degranulation of the mast cell-like RBL-2H3 cell line. Similarly to the in vivo experiments the inhibitory effect was present only when the cells were activated by antigen exposure. In conclusion, inhaled hydrogen sulfide improves lung function and inhibits bronchial hyper-reactivity by modulating mast cells and in turn fibroblast activation.

  16. Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response.

    PubMed

    Cao, Yuan; Zhang, Ejuan; Yang, Jingyi; Yang, Yi; Yu, Jie; Xiao, Yang; Li, Wei; Zhou, Dihan; Li, Yaoming; Zhao, Bali; Yan, Hu; Lu, Mengji; Zhong, Maohua; Yan, Huimin

    2017-09-01

    Flagellin, as a TLR5 agonist, is an established mucosal adjuvant for enhancing mucosal IgA responses by i.n. immunization. Nasal epithelial cells (NECs) are the first sentinel cells to be exposed to antigen and adjuvant in i.n. immunization, and it is suggested that they play an important role in the mucosal adjuvant activity of flagellin. However, the molecular mechanism leading to modulation and the response by flagellin-activated NECs remain unknown. We aimed to identify the soluble mediator(s) from flagellin-activated NECs that modulate the functions of airway dendritic cells (DCs) and enhance subsequent IgA response. In vitro studies showed that compared with the TLR4 agonist LPS, flagellin directly triggered slight up-regulation of CD80 on airway DCs but was insufficient to affect CD86 expression and DC-mediated IgA response. With the use of an in vitro system for culturing mouse primary NECs (mNECs), we demonstrated that flagellin-activated mNECs could functionally modulate airway DCs, which manifested as significant up-regulation of CD80/CD86 and enhancement of IgA production. The functional modulation of airway DCs was dependent on TLR5 activation of mNECs rather than direct TLR5 activation of airway DCs. With the use of cytokine array and antibody-blocking assays, we further identified that GM-CSF, a cytokine secreted from TLR5-activated mNECs, contributes to the activation of mNECs to airway DCs and subsequent IgA enhancement. In vivo blocking experiments confirmed that GM-CSF is an important factor in recombinant flagellin derived from Salmonella typhi (FliC)-induced airway DC activation and antigen-specific IgA enhancement. Our data directly demonstrate that nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway DCs and a subsequent IgA response. © Society for Leukocyte Biology.

  17. Successful establishment of primary small airway cell cultures in human lung transplantation

    PubMed Central

    2009-01-01

    Background The study of small airway diseases such as post-transplant bronchiolitis obliterans syndrome (BOS) is hampered by the difficulty in assessing peripheral airway function either physiologically or directly. Our aims were to develop robust methods for sampling small airway epithelial cells (SAEC) and to establish submerged SAEC cultures for downstream experimentation. Methods SAEC were obtained at 62 post-transplant bronchoscopies in 26 patients using radiologically guided bronchial brushings. Submerged cell cultures were established and SAEC lineage was confirmed using expression of clara cell secretory protein (CCSP). Results The cell yield for SAEC (0.956 ± 0.063 × 106) was lower than for large airway cells (1.306 ± 0.077 × 106) but did not significantly impact on the culture establishment rate (79.0 ± 5.2% vs. 83.8 ± 4.7% p = 0.49). The presence of BOS significantly compromised culture success (independent of cell yield) for SAEC (odds ratio (95%CI) 0.067 (0.01-0.40)) but not LAEC (0.3 (0.05-1.9)). Established cultures were successfully passaged and expanded. Conclusion Primary SAEC can be successfully obtained from human lung transplant recipients and maintained in culture for downstream experimentation. This technique will facilitate the development of primary in vitro models for BOS and other diseases with a small airway component such as asthma, cystic fibrosis and COPD. PMID:19857270

  18. Asthmatic airway epithelial cells differentially regulate fibroblast expression of extracellular matrix components

    PubMed Central

    Reeves, Stephen R.; Kolstad, Tessa; Lien, Tin-Yu; Elliott, Molly; Ziegler, Steven F.; Wight, Thomas N.; Debley, Jason S.

    2014-01-01

    Background Airway remodeling may explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling may regulate HLF ECM expression. Objectives Determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. Methods Primary AECs were obtained from well-characterized atopic-asthmatic (N=10) and healthy children (N=10) intubated under anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface (ALI) for 3 weeks, then co-cultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1), collagen III (COL3A1), hyaluronan synthase 2 (HAS2), and fibronectin (FNDC) expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs was assessed by RT-PCR. TGFb1&2 concentrations in media were measured by ELISA. Results COL1A1 and COL3A1 expression by HLFs co-cultured with asthmatic AECs was greater than HLFs co-cultured with healthy AECs (2.2 fold, p<0.02; 10.8 fold, p<0.02). HAS2 expression by HLFs co-cultured with asthmatic AECs was 2.5-fold higher than by HLFs co-cultured with healthy AECs (p<0.002). FNDC expression by HLFs co-cultured with asthmatic AECs was significantly greater than by HLFs alone. TGFb2 activity was elevated in asthmatic AEC-HLF co-cultures (p<0.05) while PGES2 was down regulated in AEC-HLF co-cultures (2.2 fold, p<0.006). Conclusions HLFs co-cultured with asthmatic AECs showed differential expression of ECM constituents COL1A1 & COL3A1, and HAS2 compared to HLFs co-cultured with healthy AECs. These findings support a role for altered ECM production in asthmatic airway remodeling, possibly regulated by unbalanced AEC signaling. PMID:24875618

  19. Type 2 innate lymphoid cells-new members of the "type 2 franchise" that mediate allergic airway inflammation.

    PubMed

    Mjösberg, Jenny; Spits, Hergen

    2012-05-01

    Type 2 innate lymphoid cells (ILC2s) are members of an ILC family, which contains NK cells and Rorγt(+) ILCs, the latter including lymphoid tissue inducer (LTi) cells and ILCs producing IL-17 and IL-22. ILC2s are dedicated to the production of IL-5 and IL-13 and, as such, ILC2s provide an early and important source of type 2 cytokines critical for helminth expulsion in the gut. Several studies have also demonstrated a role for ILC2s in airway inflammation. In this issue of the European Journal of Immunology, Klein Wolterink et al. [Eur. J. Immunol. 2012. 42: 1106-1116] show that ILC2s are instrumental in several models of experimental asthma where they significantly contribute to production of IL-5 and IL-13, key cytokines in airway inflammation. This study sheds light over the relative contribution of ILC2s versus T helper type 2 cells (Th2) in type 2 mediated allergen-specific inflammation in the airways as discussed in this commentary.

  20. IL13 activates autophagy to regulate secretion in airway epithelial cells.

    PubMed

    Dickinson, John D; Alevy, Yael; Malvin, Nicole P; Patel, Khushbu K; Gunsten, Sean P; Holtzman, Michael J; Stappenbeck, Thaddeus S; Brody, Steven L

    2016-01-01

    Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

  1. Tbet Deficiency Causes T Helper Cell Dependent Airways Eosinophilia and Mucus Hypersecretion in Response to Rhinovirus Infection

    PubMed Central

    Glanville, Nicholas; Schröder, Armin; Walton, Ross P.; Johnston, Sebastian L.

    2016-01-01

    Current understanding of adaptive immune, particularly T cell, responses to human rhinoviruses (RV) is limited. Memory T cells are thought to be of a primarily T helper 1 type, but both T helper 1 and T helper 2 memory cells have been described, and heightened T helper 2/ lessened T helper 1 responses have been associated with increased RV-induced asthma exacerbation severity. We examined the contribution of T helper 1 cells to RV-induced airways inflammation using mice deficient in the transcription factor T-Box Expressed In T Cells (Tbet), a critical controller of T helper 1 cell differentiation. Using flow cytometry we showed that Tbet deficient mice lacked the T helper 1 response of wild type mice and instead developed mixed T helper 2/T helper 17 responses to RV infection, evidenced by increased numbers of GATA binding protein 3 (GATA-3) and RAR-related orphan receptor gamma t (RORγt), and interleukin-13 and interleukin-17A expressing CD4+ T cells in the lung. Forkhead box P3 (FOXP3) and interleukin-10 expressing T cell numbers were unaffected. Tbet deficient mice also displayed deficiencies in lung Natural Killer, Natural Killer T cell and γδT cell responses, and serum neutralising antibody responses. Tbet deficient mice exhibited pronounced airways eosinophilia and mucus production in response to RV infection that, by utilising a CD4+ cell depleting antibody, were found to be T helper cell dependent. RV induction of T helper 2 and T helper 17 responses may therefore have an important role in directly driving features of allergic airways disease such as eosinophilia and mucus hypersecretion during asthma exacerbations. PMID:27683080

  2. Evidence and Role for Bacterial Mucin Degradation in Cystic Fibrosis Airway Disease

    PubMed Central

    Flynn, Jeffrey M.; Niccum, David; Dunitz, Jordan M.

    2016-01-01

    Chronic lung infections in cystic fibrosis (CF) patients are composed of complex microbial communities that incite persistent inflammation and airway damage. Despite the high density of bacteria that colonize the lower airways, nutrient sources that sustain bacterial growth in vivo, and how those nutrients are derived, are not well characterized. In this study, we examined the possibility that mucins serve as an important carbon reservoir for the CF lung microbiota. While Pseudomonas aeruginosa was unable to efficiently utilize mucins in isolation, we found that anaerobic, mucin-fermenting bacteria could stimulate the robust growth of CF pathogens when provided intact mucins as a sole carbon source. 16S rRNA sequencing and enrichment culturing of sputum also identified that mucin-degrading anaerobes are ubiquitous in the airways of CF patients. The collective fermentative metabolism of these mucin-degrading communities in vitro generated amino acids and short chain fatty acids (propionate and acetate) during growth on mucin, and the same metabolites were also found in abundance within expectorated sputum. The significance of these findings was supported by in vivo P. aeruginosa gene expression, which revealed a heightened expression of genes required for the catabolism of propionate. Given that propionate is exclusively derived from bacterial fermentation, these data provide evidence for an important role of mucin fermenting bacteria in the carbon flux of the lower airways. More specifically, microorganisms typically defined as commensals may contribute to airway disease by degrading mucins, in turn providing nutrients for pathogens otherwise unable to efficiently obtain carbon in the lung. PMID:27548479

  3. Role of Transient Receptor Potential Vanilloid 1 (TRPV1) in the Modulation of Airway Smooth Muscle Tone and Calcium Handling.

    PubMed

    Yocum, Gene T; Chen, Jun; Choi, Christine H; Townsend, Elizabeth A; Zhang, Yi; Xu, Dingbang; Fu, Xiao Wen; Sanderson, Michael J; Emala, Charles W

    2017-03-23

    Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel expressed on airway nerve fibers that modulates afferent signals resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+ ATPase 2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium-channel inhibitor bupivicaine (N=4, p<0.05) and the NK-2 receptor antagonist GR 159897 (N=4, p<0.05), suggesting that this contraction is neurally-mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (N=4, p<0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (N=4-5, p<0.05). Although capsazepine did not inhibit store-operated calicum entry in mouse ASM cells in PCLS (N=4-7, p=NS), it did inhibit calcium oscillations (N=3, p<0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillation.

  4. Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

    SciTech Connect

    Chen, Ming; Lv, Zhiqiang; Huang, Linjie; Zhang, Wei; Lin, Xiaoling; Shi, Jianting; Zhang, Wei; Liang, Ruiyun; Jiang, Shanping

    2015-02-15

    Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolide significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.

  5. The beta-agonist isoproterenol attenuates EGF-stimulated wound closure in human airway epithelial cells.

    PubMed

    Schnackenberg, Bradley J; Jones, Stacie M; Pate, Crystal; Shank, Brian; Sessions, Laura; Pittman, Laura M; Cornett, Lawrence E; Kurten, Richard C

    2006-03-01

    Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although beta-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that beta-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the beta-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing beta2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways.

  6. Cockroach protease allergen induces allergic airway inflammation via epithelial cell activation

    PubMed Central

    Kale, Sagar L.; Agrawal, Komal; Gaur, Shailendra Nath; Arora, Naveen

    2017-01-01

    Protease allergens are known to enhance allergic inflammation but their exact role in initiation of allergic reactions at mucosal surfaces still remains elusive. This study was aimed at deciphering the role of serine protease activity of Per a 10, a major cockroach allergen in initiation of allergic inflammation at mucosal surfaces. We demonstrate that Per a 10 increases epithelial permeability by disruption of tight junction proteins, ZO-1 and occludin, and enhances the migration of Monocyte derived dendritic cell precursors towards epithelial layer as exhibited by trans-well studies. Per a 10 exposure also leads to secretion of IL-33, TSLP and intracellular Ca2+ dependent increase in ATP levels. Further, in vivo experiments revealed that Per a 10 administration in mice elevated allergic inflammatory parameters along with high levels of IL-33, TSLP, IL-1α and uric acid in the mice lungs. We next demonstrated that Per a 10 cleaves CD23 (low affinity IgE receptor) from the surface of PBMCs and purified B cells and CD25 (IL-2 receptor) from the surface of PBMCs and purified T cells in an activity dependent manner, which might favour Th2 responses. In conclusion, protease activity of Per a 10 plays a significant role in initiation of allergic airway inflammation at the mucosal surfaces. PMID:28198394

  7. Myeloid cell HIF-1α regulates asthma airway resistance and eosinophil function

    PubMed Central

    Akong-Moore, Kathryn; Feldstein, Stephanie; Johansson, Per; Nyugen, Anh; McEachern, Elisa K.; Nicatia, Shari; Cowburn, Andrew S.; Olson, Joshua; Cho, Jae Youn; Isaacs, Hart; Johnson, Randall S.; Broide, David H.; Nizet, Victor

    2013-01-01

    Hypoxia-inducible factor (HIF)-1α is a master regulator of inflammatory activities of myeloid cells, including neutrophils and macrophages. These studies examine the role of myeloid cell HIF-1α in regulating asthma induction and pathogenesis, and for the first time, evaluate the roles of HIF-1α and HIF-2α in the chemotactic properties of eosinophils, the myeloid cells most associated with asthma. Wild-type (WT) and myeloid cell-specific HIF-1α knockout (KO) C57BL/6 mice were studied in an ovalbumin (OVA) model of asthma. Administration of the pharmacological HIF-1α antagonist YC-1 was used to corroborate findings from the genetic model. WT, HIF-1α, and HIF-2α KO eosinophils underwent in vitro chemotaxis assays. We found that deletion of HIF-1α in myeloid cells and systemic treatment with YC-1 during asthma induction decreased airway hyperresponsiveness (AHR). Deletion of HIF-1α in myeloid cells in OVA-induced asthma also reduced eosinophil infiltration, goblet cell hyperplasia, and levels 34 of cytokines IL-4, IL-5, and IL-13 in the lung. HIF-1α inhibition with YC-1 during asthma induction decreased eosinophilia in bronchoalveolar lavage, lung parenchyma, and blood, as well as decreased total lung inflammation, IL-5, and serum OVA-specific IgE levels. Deletion of HIF-1α in eosinophils decreased their chemotaxis, while deletion of the isoform HIF-2α led to increased chemotaxis. This work demonstrates that HIF-1α in myeloid cells plays a role in asthma pathogenesis, particularly in AHR development. Additionally, treatment with HIF-1α inhibitors during asthma induction decreases AHR and eosinophilia. Finally, we show that HIF- 1α and HIF-2α regulate eosinophil migration in opposing ways. PMID:23250618

  8. Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

    PubMed Central

    Salmon, Michael; Walsh, David A; Huang, Tung-Jung; Barnes, Peter J; Leonard, Thomas B; Hay, Douglas W P; Chung, K Fan

    1999-01-01

    Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin-exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline-exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5-lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen-induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure. PMID:10455261

  9. Oxidative stress in Nipah virus-infected human small airway epithelial cells

    PubMed Central

    Escaffre, Olivier; Halliday, Hailey; Borisevich, Viktoriya; Casola, Antonella

    2015-01-01

    Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development. PMID:26297489

  10. Epithelial mesenchymal transition (EMT) and non-small cell lung cancer (NSCLC): a mutual association with airway disease.

    PubMed

    Mahmood, Malik Quasir; Ward, Chris; Muller, Hans Konrad; Sohal, Sukhwinder Singh; Walters, Eugene Haydn

    2017-03-01

    NSCLC is a leading cause of morbidity and mortality worldwide. It includes adeno- and squamous cell carcinoma. In the background, COPD and smoking play a vital role in development of NSCLC. Local progression and metastasis of NSCLC has been associated with various mechanisms, but in particular by a process called epithelial mesenchymal transition (EMT), which is implicated in COPD pathogenesis. In this study, we have investigated whether expression of EGFR (activation marker) and S100A4, vimentin and N-cadherin (as EMT) is different both in central and leading edge of NSCLC and to what extent related to EMT activity of both small and large airways, stage and differentiation of NSCLC. We have investigated EMT biomarkers (S100A4, vimentin, and N-cadherin), an epithelial activation marker (EGFR) and a vascularity marker (Type-IV collagen) in surgically resected tissue from patients with NSCLC (adeno- and squamous cell carcinoma), and compared them with expression in the corresponding non-tumorous airways. EGFR, S100A4, vimentin, N-cadherin expression was higher in tumor cells located at the peripheral leading edge of NSCLC when compared with centrally located tumor cells of same subjects (P < 0.01). Type-IV collagen-expressing blood vessels were also more at the leading edge in comparison with central parts of NSCLC. EGFR and S100A4 expression was related to differentiation status (P < 0.05) and TNM stage (P < 0.05) of NSCLC. Moreover, EMT markers in the leading edge were significantly related to airway EMT activity, while peripheral edge vascularity of squamous cell carcinoma only was significantly related to large airway Rbm vascularity (P < 0.05). EGFR- and EMT-related protein expression was markedly high in the peripheral leading edge of NSCLCs and related to tumor characteristics associated with poor prognosis. The relationships between EMT-related tumor biomarker expression and those in the airway epithelium and Rbm provide a background for utility of

  11. Strain-dependent activation of NF-κB in the airway epithelium and its role in allergic airway inflammation

    PubMed Central

    Alcorn, John F.; Ckless, Karina; Brown, Amy L.; Guala, Amy S.; Kolls, Jay K.; Poynter, Matthew E.; Irvin, Charles G.; van der Vliet, Albert

    2010-01-01

    NF-κB activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003–7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-κB activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of IκBα in the airway epithelium (CC10-IκBαSR) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-IκBαSR transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-κB was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-α in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-α by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-α in regulating epithelial NF-κB activation in allergic airway disease. PMID:19897746

  12. Pulmonary Stress Induced by Hyperthermia: Role of Airway Sensory Nerves

    DTIC Science & Technology

    2016-01-01

    protection from oxidative stress (20), the regulation of insulin release (49), and the facilitation of bladder contraction (35). Recently, H2S has... insulin -secreting cells and the underlying mechanisms. J Physiol 569: 519–531, 2005. 50. Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G...they can be easily identified and recorded due to their regular discharge patterns and large amplitude of action potentials. During eupneic breathing

  13. Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

    SciTech Connect

    Bao, X.; Sinha, M. |; Liu, T.; Hong, C.; Luxon, B.A. |; Garofalo, R.P. ||; Casola, A. ||

    2008-04-25

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

  14. Airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain.

    PubMed

    Fairbank, Nigel J; Connolly, Sarah C; Mackinnon, James D; Wehry, Kathrin; Deng, Linhong; Maksym, Geoffrey N

    2008-09-01

    Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and "relaxability" (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness.

  15. Innate lymphoid cells contribute to allergic airway disease exacerbation by obesity.

    PubMed

    Everaere, Laetitia; Ait-Yahia, Saliha; Molendi-Coste, Olivier; Vorng, Han; Quemener, Sandrine; LeVu, Pauline; Fleury, Sebastien; Bouchaert, Emmanuel; Fan, Ying; Duez, Catherine; de Nadai, Patricia; Staels, Bart; Dombrowicz, David; Tsicopoulos, Anne

    2016-11-01

    Epidemiologic and clinical observations identify obesity as an important risk factor for asthma exacerbation, but the underlying mechanisms remain poorly understood. Type 2 innate lymphoid cells (ILC2s) and type 3 innate lymphoid cells (ILC3s) have been implicated, respectively, in asthma and adipose tissue homeostasis and in obesity-associated airway hyperresponsiveness (AHR). We sought to determine the potential involvement of innate lymphoid cells (ILCs) in allergic airway disease exacerbation caused by high-fat diet (HFD)-induced obesity. Obesity was induced by means of HFD feeding, and allergic airway inflammation was subsequently induced by means of intranasal administration of house dust mite (HDM) extract. AHR, lung and visceral adipose tissue inflammation, humoral response, cytokines, and innate and adaptive lymphoid populations were analyzed in the presence or absence of ILCs. HFD feeding exacerbated allergic airway disease features, including humoral response, airway and tissue eosinophilia, AHR, and TH2 and TH17 pulmonary profiles. Notably, nonsensitized obese mice already exhibited increased lung ILC counts and tissue eosinophil infiltration compared with values in lean mice in the absence of AHR. The numbers of total and cytokine-expressing lung ILC2s and ILC3s further increased in HDM-challenged obese mice compared with those in HDM-challenged lean mice, and this was accompanied by high IL-33 and IL-1β levels and decreased ILC markers in visceral adipose tissue. Furthermore, depletion of ILCs with an anti-CD90 antibody, followed by T-cell reconstitution, led to a profound decrease in allergic airway inflammatory features in obese mice, including TH2 and TH17 infiltration. These results indicate that HFD-induced obesity might exacerbate allergic airway inflammation through mechanisms involving ILC2s and ILC3s. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  16. Smoking-Associated Disordering of the Airway Basal Stem/Progenitor Cell Metabotype

    PubMed Central

    Deeb, Ruba S.; Walters, Matthew S.; Strulovici-Barel, Yael; Chen, Qiuying; Gross, Steven S.

    2016-01-01

    The airway epithelium is a complex pseudostratified multicellular layer lining the tracheobronchial tree, functioning as the primary defense against inhaled environmental contaminants. The major cell types of the airway epithelium include basal, intermediate columnar, ciliated, and secretory. Basal cells (BCs) are the proliferating stem/progenitor population that differentiate into the other specialized cell types of the airway epithelium during normal turnover and repair. Given that cigarette smoke delivers thousands of xenobiotics and high levels of reactive molecules to the lung epithelial surface, we hypothesized that cigarette smoke broadly perturbs BC metabolism. To test this hypothesis, primary airway BCs were isolated from healthy nonsmokers (n = 11) and healthy smokers (n = 7) and assessed by global metabolic profiling by liquid chromatography–mass spectrometry. The analysis identified 52 significant metabolites in BCs differentially expressed between smokers and nonsmokers (P < 0.05). These changes included metabolites associated with redox pathways, energy production, and inflammatory processes. Notably, BCs from smokers exhibited altered levels of the key enzyme cofactors/substrates nicotinamide adenine dinucleotide, flavin adenine dinucleotide, acetyl coenzyme A, and membrane phospholipid levels. Consistent with the high burden of oxidants in cigarette smoke, glutathione levels were diminished, whereas 3-nitrotyrosine levels were increased, suggesting that protection of airway epithelial cells against oxidative and nitrosative stress is significantly compromised in smoker BCs. It is likely that this altered metabotype is a reflection of, and likely contributes to, the disordered biology of airway BCs consequent to the stress cigarette smoking puts on the airway epithelium. PMID:26161876

  17. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration.

    PubMed

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping; Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (KATP) channels have been identified in ASMCs. Mount evidence has suggested that KATP channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K(+) channels triggers K(+) efflux, which leading to membrane hyperpolarization, preventing Ca(2+)entry through closing voltage-operated Ca(2+) channels. Intracellular Ca(2+) is the most important regulator of muscle contraction, cell proliferation and migration. K(+) efflux decreases Ca(2+) influx, which consequently influences ASMCs proliferation and migration. As a KATP channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca(2+)/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective KATP channel antagonist. These findings provide a strong evidence to support that Ipt antagonize the proliferating and migrating effects of PDGF-BB on

  18. Interactive effect of cigarette smoke extract and world trade center dust particles on airway cell cytotoxicity.

    PubMed

    Xu, Alice; Prophete, Colette; Chen, Lung-chi; Emala, Charles W; Cohen, Mitchell D

    2011-01-01

    Rescue workers and residents exposed to the environment surrounding the collapse of the World Trade Center (WTC) on September 11, 2001, have suffered a disproportionate incidence of chronic lung disease attributed to the inhalation of airborne dust. To date, the pathophysiology of this lung disease is poorly understood. The aim of this study was to examine whether airborne dust contaminants recovered from the surrounding area 24-48 h after the collapse of the WTC demonstrate direct cytotoxicity to two airway cell types that were most directly exposed to inhaled dust, airway epithelial and smooth muscle cells. It was also of interest to determine whether the presence of these dusts could modulate the effects of cigarette smoke on these cell types in that some of the individuals who responded to the collapse site were also smokers. Human cultured airway epithelial (BEAS-2B) cells were exposed to 10% cigarette smoke extract (CSE), WTC dust particles (10-53 μm; 0.01-0.5 μg/μl), or a combination of the two for 2-24 h. Cell viability was measured by determining mitochondrial integrity (MTT assays) and apoptosis (poly-ADP-ribose polymerase [PARP] immunoblotting). Conditioned cell culture media recovered from the CSE- and/or WTC dust-exposed BEAS-2B cells were then applied to cultured human airway smooth muscle cells that were subsequently assayed for mitochondrial integrity and their ability to synthesize cyclic AMP (a regulator of airway smooth muscle constriction). BEAS-2B cells underwent necrotic cell death following exposure to WTC dust or CSE for 2-24 h without evidence of apoptosis. Smooth muscle cells demonstrated cellular toxicity and enhanced cyclic AMP synthesis following exposure to conditioned media from WTC- or CSE-exposed epithelial cells. These acute toxicity assays of WTC dust and CSE offer insights into lung cell toxicity that may contribute to the pathophysiology of chronic lung disease in workers and residents exposed to WTC dust. These studies

  19. Role of Mechanical Stress in Regulating Airway Surface Hydration and Mucus Clearance Rates

    PubMed Central

    Button, Brian; Boucher, Richard C.

    2008-01-01

    Effective clearance of mucus is a critical innate airway defense mechanism, and under appropriate conditions, can be stimulated to enhance clearance of inhaled pathogens. It has become increasingly clear that extracellular nucleotides (ATP and UTP) and nucleosides (adenosine) are important regulators of mucus clearance in the airways as a result of their ability to stimulate fluid secretion, mucus hydration, and cilia beat frequency (CBF). One ubiquitous mechanism to stimulate ATP release is through external mechanical stress. This article addresses the role of physiologically-relevant mechanical forces in the lung and their effects on regulating mucociliary clearance (MCC). The effects of mechanical forces on the stimulating ATP release, fluid secretion, CBF, and MCC are discussed. Also discussed is evidence suggesting that airway hydration and stimulation of MCC by stress-mediated ATP release may play a role in several therapeutic strategies directed at improving mucus clearance in patients with obstructive lung diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). PMID:18585484

  20. Kv7 potassium channels in airway smooth muscle cells: signal transduction intermediates and pharmacological targets for bronchodilator therapy

    PubMed Central

    Brueggemann, Lioubov I.; Kakad, Priyanka P.; Love, Robert B.; Solway, Julian; Dowell, Maria L.; Cribbs, Leanne L.

    2012-01-01

    Expression and function of Kv7 (KCNQ) voltage-activated potassium channels in guinea pig and human airway smooth muscle cells (ASMCs) were investigated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), patch-clamp electrophysiology, and precision-cut lung slices. qRT-PCR revealed expression of multiple KCNQ genes in both guinea pig and human ASMCs. Currents with electrophysiological and pharmacological characteristics of Kv7 currents were measured in freshly isolated guinea pig and human ASMCs. In guinea pig ASMCs, Kv7 currents were significantly suppressed by application of the bronchoconstrictor agonists methacholine (100 nM) or histamine (30 μM), but current amplitudes were restored by addition of a Kv7 channel activator, flupirtine (10 μM). Kv7 currents in guinea pig ASMCs were also significantly enhanced by another Kv7.2–7.5 channel activator, retigabine, and by celecoxib and 2,5-dimethyl celecoxib. In precision-cut human lung slices, constriction of airways by histamine was significantly reduced in the presence of flupirtine. Kv7 currents in both guinea pig and human ASMCs were inhibited by the Kv7 channel blocker XE991. In human lung slices, XE991 induced robust airway constriction, which was completely reversed by addition of the calcium channel blocker verapamil. These findings suggest that Kv7 channels in ASMCs play an essential role in the regulation of airway diameter and may be targeted pharmacologically to relieve airway hyperconstriction induced by elevated concentrations of bronchoconstrictor agonists. PMID:21964407

  1. Impact and management of airway obstruction in patients with squamous cell carcinoma of the larynx.

    PubMed

    Chu, Pen-Yuan; Lee, Tsung-Lun; Chang, Shyue-Yih

    2011-01-01

    We compared postoperative complications and oncologic results after laryngectomy of patients with laryngeal squamous cell carcinoma (SCC), with and without airway obstruction. We retrospectively reviewed the medical records of 544 patients with laryngeal SCC between 1990 and 2000. Of 175 advanced cases receiving total laryngectomy, 32 initially presented with upper airway obstruction. Postoperative complications after laryngectomy did not differ significantly between patients with and without airway obstruction (36% vs 28%; p = .353). Although patients with airway obstruction had more T4 (81% vs 42%; p < .001) and stage IV disease (82% vs 48%; p = .0004), tumor recurrence rates did not increase (21% vs 29%; p = .374). Five-year overall (52% vs 60%; p = .251), disease-specific (73% vs 70%; p = .982), and relapse-free (72% vs 68%; p = .982) survival did not differ significantly between groups. After appropriate management of airway obstruction, the postoperative complications and oncologic results were similar to those without airway obstruction.  © 2010 Wiley Periodicals, Inc. Head Neck, 2011.

  2. TGF-β-dependent dendritic cell chemokinesis in murine models of airway disease

    PubMed Central

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Ma, Royce; Murray, Lynne A.; Tsui, Ping; Lou, Jianlong; Marks, James D.; Baron, Jody L.; Krummel, Matthew F.; Nishimura, Stephen L.

    2015-01-01

    Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of antigen surveillance and antigen presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1β) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 μm from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin αvβ8, a critical activator of TGF-β αvβ8-mediated TGF-β activation is known to enhance IL-1β-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which αvβ8, ccl20 and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli. PMID:26109638

  3. TGF-β-Dependent Dendritic Cell Chemokinesis in Murine Models of Airway Disease.

    PubMed

    Hashimoto, Mitsuo; Yanagisawa, Haruhiko; Minagawa, Shunsuke; Sen, Debasish; Ma, Royce; Murray, Lynne A; Tsui, Ping; Lou, Jianlong; Marks, James D; Baron, Jody L; Krummel, Matthew F; Nishimura, Stephen L

    2015-08-01

    Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of Ag surveillance and Ag presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1β) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 μm from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin αvβ8, a critical activator of TGF-β. αvβ8-Mediated TGF-β activation is known to enhance IL-1β-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which αvβ8, ccl20, and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli. Copyright © 2015 by The American Association of Immunologists, Inc.

  4. Cadmium Regulates the Expression of the CFTR Chloride Channel in Human Airway Epithelial Cells

    PubMed Central

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N.; Davis, Ian C.; Knoell, Daren L.; Parinandi, Narasimham L.; Cormet-Boyaka, Estelle

    2010-01-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function. PMID:20363832

  5. Cadmium regulates the expression of the CFTR chloride channel in human airway epithelial cells.

    PubMed

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N; Davis, Ian C; Knoell, Daren L; Parinandi, Narasimham L; Cormet-Boyaka, Estelle

    2010-07-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.

  6. Single-Cell Analysis of Mast Cell Degranulation Induced by Airway Smooth Muscle-Secreted Chemokines

    PubMed Central

    Manning, Benjamin M.; Meyer, Audrey F.; Gruba, Sarah M.; Haynes, Christy L.

    2015-01-01

    Background Asthma is a chronic inflammatory disease characterized by narrowed airways, bronchial hyper-responsiveness, mucus hyper-secretion, and airway remodeling. Mast cell (MC) infiltration into airway smooth muscle (ASM) is a defining feature of asthma, and ASM regulates the inflammatory response by secreting chemokines, including CXCL10 and CCL5. Single cell analysis offers a unique approach to study specific cellular signaling interactions within large and complex signaling networks such as the inflammatory microenvironment in asthma. Methods Carbon fiber microelectrode amperometry was used to study the effects of ASM–secreted chemokines on mouse peritoneal MC degranulation. Results MC degranulation in response to CXCL10 and CCL5 was monitored at the single cell level. Relative to IgE-mediated degranulation, CXCL10- and CCL5-stimulated MCs released a decreased amount of serotonin per granule with fewer release events per cell. Decreased serotonin released per granule was correlated with increased spike half-width and rise-time values. Conclusions MCs are directly activated with ASM-associated chemokines. CXCL10 and CCL5 induce less robust MC degranulation compared to IgE- and A23187-stimulation. The kinetics of MC degranulation are signaling pathway-dependent, suggesting a biophysical mechanism of regulated degranulation that incorporates control over granule trafficking, transport, and docking machinery. General Significance The biophysical mechanisms, including variations in number of exocytotic release events, serotonin released per granule, and the membrane kinetics of exocytosis that underlie MC degranulation in response to CXCL10 and CCL5 were characterized at the single cell level. These findings clarify the function of ASM-derived chemokines as instigators of MC degranulation relative to classical mechanisms of MC stimulation. PMID:25986989

  7. Whole transcriptome analysis reveals a role for OGG1-initiated DNA repair signaling in airway remodeling

    PubMed Central

    Aguilera-Aguirre, Leopoldo; Hosoki, Koa; Bacsi, Attila; Radák, Zsolt; Sur, Sanjiv; Hegde, Muralidhar L.; Tian, Bing; Saavedra-Molina, Alfredo; Brasier, Allan R.; Ba, Xueqing; Boldogh, Istvan

    2016-01-01

    Reactive oxygen species (ROS) generated by environmental exposures, and endogenously as by-products of respiration, oxidatively modify biomolecules including DNA. Accumulation of ROS-induced DNA damage has been implicated in various diseases that involve inflammatory processes, and efficient DNA repair is considered critical in preventing such diseases. One of the most abundant DNA base lesions is 7,8-dihydro-8-oxoguanine (8-oxoG), which is repaired by the 8-oxoguanine DNA glycosylase 1 (OGG1)-initiated base-excision repair (OGG1-BER) pathway. Recent studies have shown that the OGG1-BER byproduct 8-oxoG base forms a complex with cytosolic OGG1, activating small GTPases and downstream cell signaling in cultured cells and lungs. This implies that persistent OGG1-BER could result in signaling leading to histological changes in airways. To test this, we mimicked OGG1-BER by repeatedly challenging airways with its repair product 8-oxoG base. Gene expression was analyzed by RNA sequencing (RNA-Seq) and qRT-PCR, and datasets were evaluated by gene ontology and statistical tools. RNA-Seq analysis identified 3252 differentially expressed transcripts (2435 up- and 817 downregulated, Z3-fold change). Among the upregulated transcripts, 2080 mRNAs were identified whose encoded protein products were involved in modulation of the actin family cytoskeleton, extracellular matrix, cell adhesion, cadherin, and cell junctions, affecting biological processes such as tissue development, cell-to-cell adhesion, cell communication, and the immune system. These data are supported by histological observations showing epithelial alterations, subepithelial fibrosis, and collagen deposits in the lungs. These data imply that continuous challenge by the environment and consequent OGG1-BER-driven signaling trigger gene expression consistent with airway remodeling. PMID:26187872

  8. Multiple dosing of simvastatin inhibits airway mucus production of epithelial cells: implications in the treatment of chronic obstructive airway pathologies.

    PubMed

    Marin, Laura; Traini, Daniela; Bebawy, Mary; Colombo, Paolo; Buttini, Francesca; Haghi, Mehra; Ong, Hui Xin; Young, Paul

    2013-08-01

    Chronic obstructive pulmonary disease (COPD) is characterised by mucus hyper-production. This pathology, together with other inflammatory contributions, leads to airway obstruction and breathing complications. Newer therapeutic approaches are of increased interest, including the use of HMG-CoA reductase inhibitors. Retrospective studies have shown that statins are effective in reducing patient mortality and blood cytokines levels. These findings suggest statins may also provide a new therapeutic approach in COPD treatment. The aim of the present work was to study the transport of simvastatin (SV) across Calu-3 epithelial cells and to investigate its pharmacological action with respect to reduction in mucus production. Calu-3 cells were grown under liquid covered culture (LCC) conditions for transport studies in order to demonstrate the ability of SV to transport across the monolayer. For mucus detection, cells were grown under air interface culture (AIC) conditions. Samples collected for microscope analysis were stained with alcian blue; images of the stained cell surface were acquired and the mucus was quantified as the RGBB ratio. SV was transported through the cell monolayer and 'retained' inside the Calu-3 cells. Colour analysis of stained Calu-3 monolayers microscope-images showed that chronic administration of SV for 14 days caused a significant inhibition in mucus production. These findings suggest that local delivery of SV directly to the lungs may provide a promising treatment and potential disease management approach of COPD, with significant effects on mucus reduction. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

    SciTech Connect

    Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon; Heo, Won Il; Kim, Kyung Won; Kim, Kyu Earn; Sohn, Myung Hyun

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally

  10. Role of EP2 and EP4 receptors in airway microvascular leak induced by prostaglandin E2.

    PubMed

    Jones, Victoria C; Birrell, Mark A; Maher, Sarah A; Griffiths, Mark; Grace, Megan; O'Donnell, Valerie B; Clark, Stephen R; Belvisi, Maria G

    2016-03-01

    Airway microvascular leak (MVL) involves the extravasation of proteins from post-capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. PGE2, a product of COX-mediated metabolism of arachidonic acid, binds to four receptors, termed EP1–4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Evans Blue dye was used as a marker of airway MVL, and selective EP receptor agonists and antagonists were used alongside EP receptor-deficient mice to define the receptor subtype involved. PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2-induced MVL was demonstrated in Ptger2−/− and Ptger4−/− mice and in wild-type mice pretreated simultaneously with EP2 (PF-04418948) and EP4 (ER-819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2−/− and Ptger4−/− mice. PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2.

  11. Role of EP2 and EP4 receptors in airway microvascular leak induced by prostaglandin E2

    PubMed Central

    Jones, Victoria C; Birrell, Mark A; Maher, Sarah A; Griffiths, Mark; Grace, Megan; O'Donnell, Valerie B; Clark, Stephen R

    2016-01-01

    Background and Purpose Airway microvascular leak (MVL) involves the extravasation of proteins from post‐capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. PGE2, a product of COX‐mediated metabolism of arachidonic acid, binds to four receptors, termed EP1–4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Experimental Approach Evans Blue dye was used as a marker of airway MVL, and selective EP receptor agonists and antagonists were used alongside EP receptor‐deficient mice to define the receptor subtype involved. Key Results PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2‐induced MVL was demonstrated in Ptger2 −/− and Ptger4 −/− mice and in wild‐type mice pretreated simultaneously with EP2 (PF‐04418948) and EP4 (ER‐819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2 −/− and Ptger4 −/− mice. Conclusions and Implications PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2. PMID:26639895

  12. Mechanisms of inflammation-mediated airway smooth muscle plasticity and airways remodeling in asthma.

    PubMed

    Halayko, Andrew J; Amrani, Yassine

    2003-09-16

    Recent evidence points to progressive structural change in the airway wall, driven by chronic local inflammation, as a fundamental component for development of irreversible airway hyperresponsiveness. Acute and chronic inflammation is orchestrated by cytokines from recruited inflammatory cells, airway myofibroblasts and myocytes. Airway myocytes exhibit functional plasticity in their capacity for contraction, proliferation, and synthesis of matrix protein and cytokines. This confers a principal role in driving different components of the airway remodeling process, and mediating constrictor hyperresponsiveness. Functional plasticity of airway smooth muscle (ASM) is regulated by an array of environmental cues, including cytokines, which mediate their effects through receptors and a number of intracellular signaling pathways. Despite numerous studies of the cellular effects of cytokines on cultured airway myocytes, few have identified how intracellular signaling pathways modulate or induce these cellular responses. This review summarizes current understanding of these concepts and presents a model for the effects of inflammatory mediators on functional plasticity of ASM in asthma.

  13. Detection of a novel stem cell probably involved in normal turnover of the lung airway epithelium.

    PubMed

    Ortega-Martínez, Marta; Rodríguez-Flores, Laura E; de-la-Garza-González, Carlos; Ancer-Rodríguez, Jesús; Jaramillo-Rangel, Gilberto

    2015-11-01

    Regeneration of the lung airway epithelium after injury has been extensively studied. In contrast, analysis of its turnover in healthy adulthood has received little attention. In the classical view, this epithelium is maintained in the steady-state by the infrequent proliferation of basal or Clara cells. The intermediate filament protein nestin was initially identified as a marker for neural stem cells, but its expression has also been detected in other stem cells. Lungs from CD1 mice at the age of 2, 6, 12, 18 or 24 months were fixed in neutral-buffered formalin and paraffin-embedded. Nestin expression was examined by an immunohistochemical peroxidase-based method. Nestin-positive cells were detected in perivascular areas and in connective tissue that were in close proximity of the airway epithelium. Also, nestin-positive cells were found among the cells lining the airway epithelium. These findings suggest that nestin-positive stem cells circulate in the bloodstream, transmigrate through blood vessels and localize in the lung airway epithelium to participate in its turnover. We previously reported the existence of similar cells able to differentiate into lung chondrocytes. Thus, the stem cell reported here might be a bone marrow-derived mesenchymal stem cell (BMDMSC) able to generate several types of lung tissues. In conclusion, our findings indicate that there exist a BMDMSC in healthy adulthood that participates in the turnover of the lung airway epithelium. These findings may improve our knowledge about the lung stem cell biology and also provide novel approaches to therapy for devastating pulmonary diseases.

  14. Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells.

    PubMed

    Liu, Chi; Liu, Hui-jun; Xiang, Yang; Tan, Yu-rong; Zhu, Xiao-lin; Qin, Xiao-qun

    2010-08-01

    Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.

  15. Cholesterol depletion in cell membranes of human airway epithelial cells suppresses MUC5AC gene expression.

    PubMed

    Song, Kee Jae; Kim, Na Hyun; Lee, Gi Bong; Kim, Ji Hoon; Kwon, Jin Ho; Kim, Kyung-Su

    2013-05-01

    If cholesterol in the cell membrane is depleted by treating cells with methyl-β-cyclodextrin (MβCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1β is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1β showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. After NCI-H292 cells were pretreated with 1% MβCD before adding IL-1β for 24 hours, MUC5AC mRNA expression was determined by reverse transcription- polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MβCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. Cholesterol in the cell membrane was significantly depleted by treatment with MβCD on cells. IL-1β-induced MUC5AC mRNA expression was decreased by MβCD and this decrease occurred IL-1-receptor- specifically. Moreover, we have shown that MβCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1β. This result suggests that MβCD-mediated suppression of IL-1β-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.

  16. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.

    PubMed

    Kam, Yiu-Wing; Okumura, Yuushi; Kido, Hiroshi; Ng, Lisa F P; Bruzzone, Roberto; Altmeyer, Ralf

    2009-11-17

    Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases. PURIFIED TRISPIKE PROTEINS WERE READILY CLEAVED IN VITRO BY THREE DIFFERENT AIRWAY PROTEASES: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.

  17. Using Optical Tweezers to Study Cell Mechanics during Airway Reopening

    NASA Astrophysics Data System (ADS)

    Yalcin, Huseyin; Wang, Jing; Ghadiali, Samir; Ou-Yang, H. Daniel

    2006-03-01

    Patients suffering from the acute respiratory distress syndrome (ARDS) must be mechanically ventilated in order to survive. However, these ventilation protocols may generate injurious hydrodynamic stresses especially during low tidal volume (VT) ventilation when the flow of micron-sized air bubbles displace the surrounding liquid. In-vitro studies in our lab revealed that microbubble flows can severally damage lung epithelial cells (EC). The degree of injury was elevated for sub-confluent monolayers in small channel heights. Under these conditions, the micromechanics of individual EC may influence the degree of cellular injury. To investigate the role of cell mechanics, we used an oscillating Optical Tweezers (OT) technique to measure the intrinsic mechanical properties of EC before and after the flow of microbubbles. Knowledge of how the EC's micromechanical properties influence cell viability may lead to the development of novel treatment therapies that enhance the EC's ability to withstand injurious hydrodynamic stresses during ventilation treatment.

  18. Cultured airway epithelium responses to mineral particles: role of the oxidative stress.

    PubMed

    Guilianelli, C; Baeza-Squiban, A; Lapart, E; Marano, F

    1996-11-01

    We tested the hypothesis according to which mineral particles containing iron would be able to produce cytotoxic-ROS. We approached this problem in vitro using primary cultures of rabbit tracheal epithelial cells. The oxidizing power of three mineral particles, i.e. nemalite, chrysotile and hematite, has been evaluated for their capacity to induce lipid peroxidation, and to activate intra-cellular anti-oxidant enzymes. The results show that nemalite and chrysotile which contain Fe2+ have a strong oxidizing power, inducing an oxidative stress on airway epithelial cells, whereas hematite, the Fe3+ containing particles, is without effect.

  19. Do airway secretions play an underappreciated role in acute respiratory distress syndrome?

    PubMed

    Graf, Jerónimo; Marini, John J

    2008-02-01

    We review the evidence that airway secretions may have an underappreciated role in acute respiratory distress syndrome, contributing to physiologic disarrangements, ventilator dependence and perhaps to injury generation. As common manipulations of ventilator settings, position and fluid status have the potential to influence these problems, explorations into the secretion dynamics of acute lung injury may be fertile ground for developing therapeutic advances. Principles that govern the interaction of airflow and airway fluids suggest that mobile fluids and secretions are pumped by well-selected ventilatory patterns toward the airway opening. Conversely, other selections may inhibit these fluids from clearance or encourage their translocation between lung regions. Recent laboratory work demonstrates that choices for tidal volume and positive end-expiratory pressure may localize or disperse proteinaceous lung edema or bacteria. Gravitational factors may interact with ventilatory pattern for benefit or harm. Capability of ventilation and positioning to mobilize secretions implies the potential for clearance or containment of inflammatory mediators and infection. Ventilatory and positional prescriptions could be designed to meet one of either conflicting targets. Additional experimental and clinical investigations are required before adopting these proposed therapeutic principles into practice.

  20. Role of the nitric oxide-soluble guanylyl cyclase pathway in obstructive airway diseases.

    PubMed

    Dupont, Lisa L; Glynos, Constantinos; Bracke, Ken R; Brouckaert, Peter; Brusselle, Guy G

    2014-10-01

    Nitric oxide (NO) is a gaseotransmitter, which is involved in many signaling processes in health and disease. Three enzymes generate NO from l-arginine, with citrulline formed as a by-product: neuronal NO synthase (nNOS or NOS1), endothelial NOS (eNOS or NOS3) and inducible NOS (iNOS or NOS2). NO is a ligand of soluble guanylyl cyclase (sGC), an intracellular heterodimer enzyme that catalyzes the conversion of guanosine triphosphate (GTP) to cyclic GMP (cGMP). cGMP further activates protein kinase G that eventually reduces the smooth muscle tone in bronchi or vessels. Phosphodiesterase 5 (PDE5) degrades cGMP to GMP. However, NO reacts with superoxide anion (O2(-)), leading to formation of the pro-inflammatory molecule peroxynitrite. Under physiological conditions, NO plays a homeostatic bronchoprotective role in healthy subjects. In obstructive airway diseases, NO can be beneficial by its bronchodilating effect, but could also be detrimental by the formation of peroxynitrite. Since asthma and COPD are associated with increased levels of exhaled NO, chronic inflammation and increased airway smooth muscle tone, the NO/sGC/cGMP pathway could be involved in these highly prevalent obstructive airway diseases. Here we review the involvement of NO, NO synthases, guanylyl cyclases, cGMP and phophodiesterase-5 in asthma and COPD and potential therapeutic approaches to modulate this pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Eosinophilic airway inflammation: role in asthma and chronic obstructive pulmonary disease

    PubMed Central

    George, Leena; Brightling, Christopher E.

    2016-01-01

    The chronic lung diseases, asthma and chronic obstructive pulmonary disease (COPD), are common affecting over 500 million people worldwide and causing substantial morbidity and mortality. Asthma is typically associated with Th2-mediated eosinophilic airway inflammation, in contrast to neutrophilic inflammation observed commonly in COPD. However, there is increasing evidence that the eosinophil might play an important role in 10–40% of patients with COPD. Consistently in both asthma and COPD a sputum eosinophilia is associated with a good response to corticosteroid therapy and tailored strategies aimed to normalize sputum eosinophils reduce exacerbation frequency and severity. Advances in our understanding of the multistep paradigm of eosinophil recruitment to the airway, and the consequence of eosinophilic inflammation, has led to the development of new therapies to target these molecular pathways. In this article we discuss the mechanisms of eosinophilic trafficking, the tools to assess eosinophilic airway inflammation in asthma and COPD during stable disease and exacerbations and review current and novel anti-eosinophilic treatments. PMID:26770668

  2. THE EFFECTS OF COMBINATORIAL EXPOSURE OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES ON AIRWAY EPITHELIAL CELL RELEASE OF CHEMOTACTIC MEDIATORS

    EPA Science Inventory

    Asthma is a chronic inflammatory disorder of the airways affecting nearly 15 million individuals nationally. Within the inflamed asthmatic airway there exist complex interactions between many cells and the cytokines they release, in particular mast cells, eosinophils, T-lymphocy...

  3. THE EFFECTS OF COMBINATORIAL EXPOSURE OF PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINES ON AIRWAY EPITHELIAL CELL RELEASE OF CHEMOTACTIC MEDIATORS

    EPA Science Inventory

    Asthma is a chronic inflammatory disorder of the airways affecting nearly 15 million individuals nationally. Within the inflamed asthmatic airway there exist complex interactions between many cells and the cytokines they release, in particular mast cells, eosinophils, T-lymphocy...

  4. S-Nitrosoglutathione induces functional DeltaF508-CFTR in airway epithelial cells.

    PubMed

    Andersson, Charlotte; Gaston, Benjamin; Roomans, Godfried M

    2002-09-27

    S-Nitrosoglutathione (GSNO) is an endogenous bronchodilator levels of which are reduced in the airways of cystic fibrosis (CF) patients. GSNO has recently been shown to increase maturation of CFTR in CF cell lines at physiological concentrations. The ability of S-nitrosoglutathione to direct the DeltaF508-CFTR to the plasma membrane and restore the function of the cAMP-dependent chloride transport in cultured human airway epithelial cells has been studied. Immunocytochemistry showed a time- and dose-dependent increase of apically located CFTR after GSNO treatment. Chloride transport studies with the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) showed that GSNO was able to induce a fourfold increase of cAMP-dependent chloride transport. Our data and the fact that endogenous GSNO levels are lower in the airways of CF patients make GSNO an interesting candidate for pharmacological treatment of cystic fibrosis.

  5. Staphylococcus aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line

    PubMed Central

    Gierok, Philipp; Harms, Manuela; Methling, Karen; Hochgräfe, Falko; Lalk, Michael

    2016-01-01

    The Gram positive opportunistic human pathogen Staphylococcus aureus induces a variety of diseases including pneumonia. S. aureus is the second most isolated pathogen in cystic fibrosis patients and accounts for a large proportion of nosocomial pneumonia. Inside the lung, the human airway epithelium is the first line in defence with regard to microbial recognition and clearance as well as regulation of the immune response. The metabolic host response is, however, yet unknown. To address the question of whether the infection alters the metabolome and metabolic activity of airway epithelial cells, we used a metabolomics approach. The nutrition uptake by the human airway epithelial cell line A549 was monitored over time by proton magnetic resonance spectroscopy (1H-NMR) and the intracellular metabolic fingerprints were investigated by gas chromatography and high performance liquid chromatography (GC-MS) and (HPLC-MS). To test the metabolic activity of the host cells, glutamine analogues and labelled precursors were applied after the infection. We found that A549 cells restrict uptake of essential nutrients from the medium after S. aureus infection. Moreover, the infection led to a shutdown of the purine and pyrimidine synthesis in the A549 host cell, whereas other metabolic routes such as the hexosamine biosynthesis pathway remained active. In summary, our data show that the infection with S. aureus negatively affects growth, alters the metabolic composition and specifically impacts the de novo nucleotide biosynthesis in this human airway epithelial cell model. PMID:27834866

  6. Analysis of Notch Signaling-Dependent Gene Expression in Developing Airways Reveals Diversity of Clara Cells

    PubMed Central

    Guha, Arjun; Vasconcelos, Michelle; Zhao, Rui; Gower, Adam C.; Rajagopal, Jayaraj; Cardoso, Wellington V.

    2014-01-01

    Clara cells (CCs) are a morphologically and operationally heterogeneous population of Secretoglobin Scgb1a1-expressing secretory cells that are crucial for airway homeostasis and post-injury repair. Analysis of the extent and origin of CC diversity are limited by knowledge of genes expressed in these cells and their precursors. To identify novel putative markers of CCs and explore the origins of CC diversity, we characterized global changes in gene expression in embryonic lungs in which CCs do not form due to conditional disruption of Notch signaling (RbpjkCNULL). Microarray profiling, Real Time PCR (qRT-PCR), and RNA in situ hybridization (ISH) identified eleven genes downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. ISH revealed that several genes had overlapping but distinct domains of expression of in the normal developing lung (E18.5). Notably, Reg3g, Chad, Gabrp and Lrrc26 were enriched in proximal airways, Hp in the distal airways and Upk3a in clusters of cells surrounding Neuroepithelial Bodies (NEBs). Seven of the eleven genes, including Reg3g, Hp, and Upk3a, were expressed in the adult lung in CCs in a pattern similar to that observed in the developing airways. qRT-PCR-based analysis of gene expression of CCs isolated from different airway regions of B1-EGFP reporter mice corroborated the spatial enrichment in gene expression observed by ISH. Our study identifies candidate markers for CC-precursors and CCs and supports the idea that the diversification of the CC phenotype occurs already during embryonic development. PMID:24586412

  7. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

    PubMed

    Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

    2013-04-11

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

  8. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

    PubMed Central

    2013-01-01

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells. PMID:23577829

  9. Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway.

    PubMed

    Xue, Di; Ma, Yan; Li, Min; Li, Yanan; Luo, Haixia; Liu, Xiaoming; Wang, Yujiong

    2015-01-15

    Mycoplasma ovipneumoniae (M. ovipneumoniae) is a bacterium that specifically infects sheep and goat and causes ovine infectious pleuropneumonia. In an effort to understand the pathogen-host interaction between the M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory response using a primary air-liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (Ovis aries). The ALI culture of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. Exposure of ALI cultures to M. ovipneumoniae led to increased expression of Toll-like receptors (TLRs), and components of the myeloid differentiation factor 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, infection with M. ovipneumoniae failed to induce the expression of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of the MyD88-independent signaling pathway. These results suggest that the MyD88-dependent TLR pathway may play a crucial role in sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating pathogen-host interactions between M. ovipneumoniae and airway epithelial cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. BLUNTING AIRWAYS EOSINOPHILIC INFLAMMATION RESULTS IN A DECREASED AIRWAY NEUTROPHIL RESPONSE TO INHALED LPS IN ATOPIC ASTHMATICS A ROLE FOR CD-14

    EPA Science Inventory

    Recent data demonstrate that atopic inflammation might enhance airway responses to inhaled LPS in individuals with atopic asthma by increasing CD14 expression on airway macrophages. We sought to determine whether blunting airway eosinophilic inflammation decreases CD14 expressio...

  11. BLUNTING AIRWAYS EOSINOPHILIC INFLAMMATION RESULTS IN A DECREASED AIRWAY NEUTROPHIL RESPONSE TO INHALED LPS IN ATOPIC ASTHMATICS A ROLE FOR CD-14

    EPA Science Inventory

    Recent data demonstrate that atopic inflammation might enhance airway responses to inhaled LPS in individuals with atopic asthma by increasing CD14 expression on airway macrophages. We sought to determine whether blunting airway eosinophilic inflammation decreases CD14 expressio...

  12. Distal airway epithelial progenitor cells are radiosensitive to High-LET radiation

    PubMed Central

    McConnell, Alicia M.; Konda, Bindu; Kirsch, David G.; Stripp, Barry R.

    2016-01-01

    Exposure to high-linear energy transfer (LET) radiation occurs in a variety of situations, including charged particle radiotherapy, radiological accidents, and space travel. However, the extent of normal tissue injury in the lungs following high-LET radiation exposure is unknown. Here we show that exposure to high-LET radiation led to a prolonged loss of in vitro colony forming ability by airway epithelial progenitor cells. Furthermore, exposure to high-LET radiation induced clonal expansion of a subset of progenitor cells in the distal airway epithelium. Clonal expansion following high-LET radiation exposure was correlated with elevated progenitor cell apoptosis, persistent γ-H2AX foci, and defects in mitotic progression of distal airway progenitors. We discovered that the effects of high-LET radiation exposure on progenitor cells occur in a p53-dependent manner. These data show that high-LET radiation depletes the distal airway progenitor pool by inducing cell death and loss of progenitor function, leading to clonal expansion. Importantly, high-LET radiation induces greater long-term damage to normal lung tissue than the relative equivalent dose of low-LET γ-rays, which has implications in therapeutic development and risk assessment. PMID:27659946

  13. Matrix metalloproteinase expression and activity in human airway smooth muscle cells

    PubMed Central

    Elshaw, Shona R; Henderson, Neil; Knox, Alan J; Watson, Susan A; Buttle, David J; Johnson, Simon R

    2004-01-01

    Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells.MMPs and TIMPs were examined using quantitative real-time RT–PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity.The most abundant MMPs were pro-MMP-2, pro- MMP-3, active MMP-3 and MT1-MMP. TIMP-1 and TIMP-2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM-conditioned medium to inhibit recombinant MMP-2 in a QF assay. Thrombin increased MMP activity by activation of pro-MMP-2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4.In conclusion, ASM cells express pro-MMP-2, pro and active MMP-3, MMP-9 and MT1-MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP-2 can be activated by thrombin which may contribute to airway remodelling. PMID:15265805

  14. Proteomic Analysis of Primary Human Airway Epithelial Cells Exposed to the Respiratory Toxicant Diacetyl.

    PubMed

    Foster, Matthew W; Gwinn, William M; Kelly, Francine L; Brass, David M; Valente, Ashlee M; Moseley, M Arthur; Thompson, J Will; Morgan, Daniel L; Palmer, Scott M

    2017-02-03

    Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.

  15. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

    SciTech Connect

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation and migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that Ipt

  16. Azithromycin Treatment Alters Gene Expression in Inflammatory, Lipid Metabolism, and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

    PubMed Central

    Ribeiro, Carla Maria P.; Hurd, Harry; Wu, Yichao; Martino, Mary E. B.; Jones, Lisa; Brighton, Brian; Boucher, Richard C.; O'Neal, Wanda K.

    2009-01-01

    Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help

  17. Role of Orexin in Respiratory and Sleep Homeostasis during Upper Airway Obstruction in Rats

    PubMed Central

    Tarasiuk, Ariel; Levi, Avishag; Berdugo-Boura, Nilly; Yahalom, Ari; Segev, Yael

    2014-01-01

    Study Objectives: Chronic upper airway obstruction (UAO) elicits a cascade of complex endocrine derangements that affect growth, sleep, and energy metabolism. We hypothesized that elevated hypothalamic orexin has a role in maintaining ventilation during UAO, while at the same time altering sleep-wake activity and energy metabolism. Here, we sought to explore the UAO-induced changes in hypothalamic orexin and their role in sleep-wake balance, respiratory activity, and energy metabolism. Interventions: The tracheae of 22-day-old Sprague-Dawley rats were surgically narrowed; UAO and sham-operated control animals were monitored for 7 weeks. We measured food intake, body weight, temperature, locomotion, and sleep-wake activity. Magnetic resonance imaging was used to quantify subcutaneous and visceral fat tissue volumes. In week 7, the rats were sacrificed and levels of hypothalamic orexin, serum leptin, and corticosterone were determined. The effect of dual orexin receptor antagonist (almorexant 300 mg/kg) on sleep and respiration was also explored. Measurements and Results: UAO increased hypothalamic orexin mRNA and protein content by 64% and 65%, respectively. UAO led to 30% chronic sleep loss, excessive active phase sleepiness, decreased body temperature, increased food intake, reduction of abdominal and subcutaneous fat tissue volume, and growth retardation. Administration of almorexant normalized sleep but induced severe breathing difficulties in UAO rats, while it had no effect on sleep or on breathing of control animals. Conclusions: In upper airway obstruction animals, enhanced orexin secretion, while crucially important for respiratory homeostasis maintenance, is also responsible for chronic partial sleep loss, as well as considerable impairment of energy metabolism and growth. Citation: Tarasiuk A, Levi A, Berdugo-Boura N, Yahalom A, Segev Y. Role of orexin in respiratory and sleep homeostasis during upper airway obstruction in rats. SLEEP 2014

  18. Klotho expression is reduced in COPD airway epithelial cells: effects on inflammation and oxidant injury.

    PubMed

    Gao, Wei; Yuan, Cheng; Zhang, Jingying; Li, Lingling; Yu, Like; Wiegman, Coen H; Barnes, Peter J; Adcock, Ian M; Huang, Mao; Yao, Xin

    2015-12-01

    COPD (chronic obstructive pulmonary disease) is associated with sustained inflammation, excessive injury, and accelerated lung aging. Human Klotho (KL) is an anti-aging protein that protects cells against inflammation and damage. In the present study, we quantified KL expression in the lungs of COPD patients and in an ozone-induced mouse model of COPD, and investigated the mechanisms that control KL expression and function in the airways. KL distribution and levels in human and mouse airways were measured by immunohistochemistry and Western blotting. The effect of CSE (cigarette smoke extract) on KL expression was detected in human bronchial epithelial cells. Moreover, the effect of KL on CSE-mediated inflammation and hydrogen peroxide-induced cellular injury/apoptosis was determined using siRNAs. KL expression was decreased in the lungs of smokers and further reduced in patients with COPD. Similarly, 6 weeks of exposure to ozone decreased KL levels in airway epithelial cells. CSE and TNFα (tumour necrosis factor α) decreased KL expression and release from airway epithelial cells, which was associated with enhanced pro-inflammatory cytokine expression. Moreover, KL depletion increased cell sensitivity to cigarette smoke-induced inflammation and oxidative stress-induced cell damage. These effects involved the NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase) and Nrf2 (nuclear factor erythroid 2-related factor 2) pathways. Reduced KL expression in COPD airway epithelial cells was associated with increased oxidative stress, inflammation and apoptosis. These data provide new insights into the mechanisms associated with the accelerated lung aging in COPD development.

  19. Klotho expression is reduced in COPD airway epithelial cells: effects on inflammation and oxidant injury

    PubMed Central

    Gao, Wei; Yuan, Cheng; Zhang, Jingying; Li, Lingling; Yu, Like; Wiegman, Coen H.; Barnes, Peter J.; Adcock, Ian M.; Huang, Mao

    2015-01-01

    COPD (chronic obstructive pulmonary disease) is associated with sustained inflammation, excessive injury, and accelerated lung aging. Human Klotho (KL) is an anti-aging protein that protects cells against inflammation and damage. In the present study, we quantified KL expression in the lungs of COPD patients and in an ozone-induced mouse model of COPD, and investigated the mechanisms that control KL expression and function in the airways. KL distribution and levels in human and mouse airways were measured by immunohistochemistry and Western blotting. The effect of CSE (cigarette smoke extract) on KL expression was detected in human bronchial epithelial cells. Moreover, the effect of KL on CSE-mediated inflammation and hydrogen peroxide-induced cellular injury/apoptosis was determined using siRNAs. KL expression was decreased in the lungs of smokers and further reduced in patients with COPD. Similarly, 6 weeks of exposure to ozone decreased KL levels in airway epithelial cells. CSE and TNFα (tumour necrosis factor α) decreased KL expression and release from airway epithelial cells, which was associated with enhanced pro-inflammatory cytokine expression. Moreover, KL depletion increased cell sensitivity to cigarette smoke-induced inflammation and oxidative stress-induced cell damage. These effects involved the NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase) and Nrf2 (nuclear factor erythroid 2-related factor 2) pathways. Reduced KL expression in COPD airway epithelial cells was associated with increased oxidative stress, inflammation and apoptosis. These data provide new insights into the mechanisms associated with the accelerated lung aging in COPD development. PMID:26201096

  20. T cell-derived IL-17 mediates epithelial changes in the airway and drives pulmonary neutrophilia1

    PubMed Central

    Fogli, Laura K.; Sundrud, Mark S.; Goel, Swati; Bajwa, Sofia; Jensen, Kari; Derudder, Emmanuel; Sun, Amy; Coffre, Maryaline; Uyttenhove, Catherine; van Snick, Jacques; Schmidt-Supprian, Marc; Rao, Anjana; Grunig, Gabriele; Durbin, Joan; Casola, Stefano S.; Rajewsky, Klaus; Koralov, Sergei B.

    2013-01-01

    Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. Here we describe a novel mouse model of spontaneous IL-17-driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We utilized this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases. PMID:23966625

  1. Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice.

    PubMed

    Zhang, Bei; An, Jun; Shimada, Takashi; Liu, Shuang; Maeyama, Kazutaka

    2012-08-01

    Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.

  2. CF airway smooth muscle transcriptome reveals a role for PYK2.

    PubMed

    Cook, Daniel P; Adam, Ryan J; Zarei, Keyan; Deonovic, Benjamin; Stroik, Mallory R; Gansemer, Nicholas D; Meyerholz, David K; Au, Kin Fai; Stoltz, David A

    2017-09-07

    Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction-related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.

  3. Role of neutrophilic inflammation in ozone-induced epithelial alterations in the nasal airways of rats

    NASA Astrophysics Data System (ADS)

    Cho, Hye Youn

    Ozone is a principal oxidant air pollutant in photochemical smog. Epithelial cells lining the centriacinar region of lung and the proximal aspects of nasal passage are primary target sites for ozone-induced injury in laboratory animals. Acute exposure of rats to high ambient concentrations of ozone (e.g., 0.5 ppm) results in neutrophilic inflammation, epithelial hyperplasia and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) lining the proximal nasal airways. The principal purpose of the present study was to investigate the role of pre-metaplastic cellular responses, especially neutrophilic inflammation, in the pathogenesis of ozone-induced MCM in rat NTE. For this purpose, three specific hypotheses-based whole-animal inhalation studies were conducted. Male F344/N rats were exposed in whole-body inhalation chambers to 0 (filtered air) or 0.5 ppm ozone for 1-3 days (8 h/day). Histochemical, immunochemical, molecular and morphometric techniques were used to investigate the ozone-induced cellular and molecular events in the NTE. Two in vitro studies were also conducted to examine the effects of ozone-inducible cytokines (i.e., tumor necrosis factor-alpha; TNF- a, and interleukin-6; IL-6) on mucin gene (rMuc-5AC) expression. Ozone induced a rapid increase of rMuc-5AC mRNA in nasal tissues within hours after the start of exposure. It preceded the appearance of MCM, and persisted with MCM. Ozone-induced neutrophilic inflammation accompanied the mucin gene upregulation, but was resolved when MCM first appeared in the NTE. Antibody-mediated depletion of circulating neutrophils attenuated ozone-induced MCM, although it did not affect the ozone-induced epithelial hyperplasia and mucin mRNA upregulation. In another study, it was found that preexisting neutrophilic rhinitis induced by endotoxin augmented the ozone-induced MCM. However, pre-existing rhinitis did not alter the severity of ozone-induced epithelial hyperplasia and mucin gene upregulation

  4. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers

    PubMed Central

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-01-01

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions. PMID:27886077

  5. Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers.

    PubMed

    Castellani, Stefano; Orlando, Clara; Carbone, Annalucia; Di Gioia, Sante; Conese, Massimo

    2016-11-23

    Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

  6. TLR-2 IS INVOLVED IN AIRWAY EPITHELIAL CELL RESPONE TO AIR POLLUTION PARTICLES

    EPA Science Inventory

    Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of a number of oxidant stress response genes. Components of ambient air PM responsible for stim...

  7. SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES
    Y.M. Kim, A.G. Lenz, R. Silbajoris, I. Jaspers and J.M. Samet. Department of Environmental Sciences and Engineering and Center for Environmental Medicine, University of North Carolina, ...

  8. SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES
    Y.M. Kim, A.G. Lenz, R. Silbajoris, I. Jaspers and J.M. Samet. Department of Environmental Sciences and Engineering and Center for Environmental Medicine, University of North Carolina, ...

  9. CULTURE CONDITIONS AFFECT HUMAN AIRWAY EPITHELIAL CELL RESPONSE TO DIESEL PARTICLE EXPOSURE IN VITRO

    EPA Science Inventory

    Diesel exhaust particles (DEP) are a ubiquitous ambient air contaminant that may contribute to the health effects of particulate matter inhalation. In vitro studies have shown that DEP exposure induces pro-inflammatory proteins in human airway epithelial cells (HAEC) with varying...

  10. TLR-2 IS INVOLVED IN AIRWAY EPITHELIAL CELL RESPONE TO AIR POLLUTION PARTICLES

    EPA Science Inventory

    Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of a number of oxidant stress response genes. Components of ambient air PM responsible for stim...

  11. CULTURE CONDITIONS AFFECT HUMAN AIRWAY EPITHELIAL CELL RESPONSE TO DIESEL PARTICLE EXPOSURE IN VITRO

    EPA Science Inventory

    Diesel exhaust particles (DEP) are a ubiquitous ambient air contaminant that may contribute to the health effects of particulate matter inhalation. In vitro studies have shown that DEP exposure induces pro-inflammatory proteins in human airway epithelial cells (HAEC) with varying...

  12. Beta-adrenergic agonists inhibit corticosteroid-induced apoptosis of airway epithelial cells.

    PubMed

    Tse, Roberta; Marroquin, Bertha A; Dorscheid, Delbert R; White, Steven R

    2003-08-01

    Airway epithelial damage is a feature of persistent asthma. Treatment with inhaled and oral corticosteroids may suppress inflammation and gain clinical control despite continued epithelial damage. We have previously demonstrated that corticosteroids elicit apoptosis of airway epithelial cells in culture. beta-Adrenergic receptor agonists are commonly used in asthma therapy and can inhibit corticosteroid-induced apoptosis of eosinophils. We tested the hypothesis that beta-adrenergic agonists would inhibit corticosteroid-induced airway epithelial cell apoptosis in cultured primary airway epithelial cells and in the cell line 1HAEo-. Albuterol treatment inhibited dexamethasone-induced apoptosis completely but did not inhibit apoptosis induced by Fas receptor activation. The protective effect of albuterol was duplicated by two different analogs of protein kinase A. The protective effect was not associated with increased translocation of the glucocorticoid receptor to the nucleus nor with changes in glucocorticoid receptor-mediated transcriptional activation or repression. We demonstrate that beta-adrenergic agonists can inhibit corticosteroid-induced apoptosis but not apoptosis induced by Fas activation. These data suggest that one potential deleterious effect of corticosteroid therapy in asthma can be prevented by concomitant beta-adrenergic agonist treatment.

  13. SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    SEASONAL EFFECTS OF ULTRAFINE, FINE, AND COARSE PARTICULATE MATTER (PM) ON HUMAN PRIMARY AIRWAY EPITHELIAL CELLS

    Exposure of humans to PM results in increased mortality and morbidity. Recent toxicology studies have shown a number of pathophysiological pulmonary and car...

  14. THE EFFECT OF SIZE FRACTIONED PARTICULATE MATTER ON HUMAN AIRWAY EPITHELIAL CELLS IN VITRO

    EPA Science Inventory

    THE EFFECT OF SIZE FRACTIONATED PARTICULATE MATTER ON HUMAN AIRWAY EPITHELIAL CELLS IN VITRO. LA Dailey1, C Sioutas2, JM Soukup1, S Becker1, RB Devlin1. 1National Health & Environmental Effects Research Laboratory, USEPA, RTP, NC,USA; 2USC, Civil & Environmental Engineering, LA, ...

  15. Neutrophilic airways inflammation in lung cancer: the role of exhaled LTB-4 and IL-8.

    PubMed

    Carpagnano, Giovanna E; Palladino, Grazia P; Lacedonia, Donato; Koutelou, Anna; Orlando, Silvio; Foschino-Barbaro, Maria P

    2011-06-07

    Recent advances in lung cancer biology presuppose its inflammatory origin. In this regard, LTB-4 and IL-8 are recognized to play a crucial role in neutrophil recruitment into airways during lung cancer.Notwithstanding the intriguing hypothesis, the exact role of neutrophilic inflammation in tumour biology remains complex and not completely known.The aim of this study was to give our contribution in this field by investigating LTB-4 and IL-8 in the breath condensate of NSCLC patients and verifying their role in cancer development and progression. We enrolled 50 NSCLC patients and 35 controls. LTB-4 and IL-8 concentrations were measured in the breath condensate and the blood of all the subjects under study using EIA kits. Thirty NSCLC patients and ten controls underwent induced sputum collection and analysis. LTB-4 and IL-8 resulted higher in breath condensate and the blood of NSCLC patients compared to controls. Significantly higher concentrations were found as the cancer stages progressed. A positive correlation was observed between exhaled IL-8 and LTB-4 and the percentage of neutrophils in the induced sputum. The high concentrations of exhaled LTB-4 and IL-8 showed the presence of a neutrophilic inflammation in the airways of NSCLC patients and gave a further support to the inflammatory signalling in lung cancer. These exhaled proteins could represent a suitable non-invasive marker in the diagnosis and monitoring of lung cancer.

  16. A role for M(2) and M(3) muscarinic receptors in the contraction of rat and human small airways.

    PubMed

    Brown, Stephanie M; Koarai, Akira; Sturton, Richard G; Nicholson, Andrew G; Barnes, Peter J; Donnelly, Louise E

    2013-02-28

    Large airway bronchoconstriction acts mainly through cholinergic pathways via muscarinic M3 receptors with some contribution from M2 receptors. By contrast, the mechanisms of small airway contraction are largely unknown. This study used precision cut lung slices to examine the role of muscarinic M2 and M3 receptors in the contractile response of rat and human small airways. In rat small airways, the M3 preferential antagonist, 4-DAMP, inhibited carbachol-mediated contraction (1×10(-6) M) more than that of the M2 selective antagonist, AF-DX116 (pIC50 values: 8.85±0.18 and 6.31±0.19, n=6-8 respectively). Tiotropium, inhibited the contractile response to carbachol with (pIC50: 9.86±0.07, n=6), but could not distinguish between M2 and M3 mediated effects. Similar experiments using human small airways with tiotropium and AF-DX116, gave a pIC50 of 10.35±0.05 and a pKB of 6.37±0.13, n=5 respectively. Therefore, M3 receptors play a key role in muscarinic contraction of small airways in both rats and humans but the effect of M2 receptors cannot be excluded. To investigate the role of M2 receptors, carbachol-induced contraction of small airways was performed in the presence and absence of a β2-agonist in order to elevate intracellular cAMP levels prior to contraction. Isoproterenol-induced relaxation was significantly increased by AF-DX116 (P<0.001) in rat small airways and by AF-DX116 (P<0.01), gallamine (P<0.05) and pertussis toxin (P<0.05) in human small airways. Taken together, these data suggest that cholinergic antagonism of muscarinic receptors in human and rat small airways inhibits airway contraction via direct inhibition of contraction through M3 receptors, and by M2 receptor mediated inhibition of relaxation. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro.

    PubMed

    Fang, Shijing; Crews, Anne L; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong; Adler, Kenneth B

    2013-04-15

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

  18. MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

    PubMed Central

    Fang, Shijing; Crews, Anne L.; Chen, Wei; Park, Joungjoa; Yin, Qi; Ren, Xiu-Rong

    2013-01-01

    Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation. PMID:23377348

  19. Continuous mucociliary transport by primary human airway epithelial cells in vitro

    PubMed Central

    Sears, Patrick R.; Yin, Wei-Ning

    2015-01-01

    Mucociliary clearance (MCC) is an important innate defense mechanism that continuously removes inhaled pathogens and particulates from the airways. Normal MCC is essential for maintaining a healthy respiratory system, and impaired MCC is a feature of many airway diseases, including both genetic (cystic fibrosis, primary ciliary dyskinesia) and acquired (chronic obstructive pulmonary disease, bronchiectasis) disorders. Research into the fundamental processes controlling MCC, therefore, has direct clinical application, but has been limited in part due to the difficulty of studying this complex multicomponent system in vitro. In this study, we have characterized a novel method that allows human airway epithelial cells to differentiate into a mucociliary epithelium that transports mucus in a continuous circular track. The mucociliary transport device allows the measurement and manipulation of all features of mucociliary transport in a controlled in vitro system. In this initial study, the effect of ciliary beat frequency and mucus concentration on the speed of mucociliary transport was investigated. PMID:25979076

  20. Effects of specific prostanoid EP receptor agonists on cell proliferation and intracellular Ca(2+) concentrations in human airway smooth muscle cells.

    PubMed

    Mori, Akemi; Ito, Satoru; Morioka, Masataka; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2011-05-20

    Increased airway smooth muscle mass due to cell proliferation contributes to airway hyper-responsiveness and remodeling in patients with asthma. Prostaglandin E2 (PGE2) inhibits proliferation of airway smooth muscle cells, but the role of prostanoid EP receptor subtypes in mechanisms involved has not been fully elucidated yet. We investigated the effects of specific prostanoid EP receptor agonists on cell proliferation and intracellular Ca(2+) concentrations ([Ca(2+)]i) in human airway smooth muscle cells. Cell numbers were assessed by mitochondria-dependent reduction of 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate to formazan (WST-1 assay). RT-PCR data showed that human airway smooth muscle cells express EP2, EP3, and EP4 but not EP1 receptor mRNA. PGE2 (1nM-1μM) inhibited cell proliferation induced by 5% fetal bovine serum (FBS) in a concentration-dependent manner. (16S)-9-deoxy-9β-chloro-15-deoxy-16-hydroxy-17, 17-trimethylene-19, 20-didehydro PGE2 sodium salt (ONO-AE1-259-01; EP2 receptor agonist) and 16-(3-methoxymethyl)phenyl-ω-tetranor-3,7-dithia PGE2 (ONO-AE1-329; EP4 receptor agonist) inhibited the 5% FBS-induced cell proliferation. ONO-AE1-259-01 and ONO-AE1-329 also significantly increased the cytosolic cAMP levels. In contrast, 11,15-O-dimethyl PGE2 (ONO-AE-248; EP3 receptor agonist) elicited an oscillatory increase in [Ca(2+)]i but did not affect the cell growth or cAMP levels. [(17S)-2,5-ethano-6-oxo-17,20-dimethyl PGE1] (ONO-DI-004; EP1 receptor agonist) did not affect cell growth, cAMP levels, or [Ca(2+)]i. In conclusion, PGE2 inhibits FBS-induced cell proliferation mostly via EP2 and EP4 receptor activation and subsequent cAMP elevation. The EP3 receptor agonist causes an increase in [Ca(2+)]i without affecting cell growth. There is no functional expression of the EP1 receptor. Research on prostanoid EP receptors may lead to novel therapeutic strategies for treatment of asthma. Copyright © 2011 Elsevier B

  1. Loss of GFI1 impairs pulmonary neuroendorine cell proliferation, but the neuroendocrine phenotype has limited impact on post-naphthalene airway repair

    PubMed Central

    Linnoila, R Ilona; Jensen-Taubman, Sandra; Kazanjian, Avedis; Grimes, H Leighton

    2010-01-01

    Naphthalene exposure kills lung airway epithelial (Clara) cells, but is rapidly followed by Clara cell reconstitution coincident with proliferation of pulmonary neuroendocrine cells (PNEC). Although a role for mature PNEC in the reconstitution process has been excluded, the reconstituting progenitor cells have been suggested to enter a transient neuroendocrine (NE) differentiation phase before differentiating to Clara cells. Furthermore, these progenitors were suggested to be the target population for transformation to a NE tumor; small cell lung cancer (SCLC). Although the NE phenotype is central to SCLC oncogenesis, the relevance of NE differentiation to post naphthalene reconstitution remains to be determined. The Growth factor independent-1 (Gfi1) transcription factor is expressed in SCLC and is required for the NE differentiation of PNEC. Gfi1−/− mice display a 70% reduction in airway cells that express NE markers, and cells that stain for NE markers show weak expression of some markers. Therefore, to determine the relevance of the NE phenotype to post-naphthalene reconstitution, we examined post-naphthalene reconstitution in Gfi1−/− mice. Our analyses indicate that the post-naphthalene regeneration process includes both airway epithelial proliferation and apoptosis. Gfi1 deletion lowered both airway epithelial proliferation and apoptosis; however, the post-naphthalene rate of increase in growth and apoptosis was not significantly different between Gfi1−/− mice and wild-type littermates. Moreover, the timing and extent of CC10 + cell regeneration was unaffected by Gfi1 deletion. These data suggest that neither Gfi1 nor the NE phenotype play a dominant role in the regeneration process. However, the few Gfi1−/− cells capable of NE differentiation show a significant reduction in post-naphthalene proliferation. The modest proliferation seen in Gfi1−/− NE cells is consistent with the previously proposed role for Gfi1 in controlling

  2. Airway basal cells of healthy smokers express an embryonic stem cell signature relevant to lung cancer.

    PubMed

    Shaykhiev, Renat; Wang, Rui; Zwick, Rachel K; Hackett, Neil R; Leung, Roland; Moore, Malcolm A S; Sima, Camelia S; Chao, Ion Wa; Downey, Robert J; Strulovici-Barel, Yael; Salit, Jacqueline; Crystal, Ronald G

    2013-09-01

    Activation of the human embryonic stem cell (hESC) signature genes has been observed in various epithelial cancers. In this study, we found that the hESC signature is selectively induced in the airway basal stem/progenitor cell population of healthy smokers (BC-S), with a pattern similar to that activated in all major types of human lung cancer. We further identified a subset of 6 BC-S hESC genes, whose coherent overexpression in lung adenocarcinoma (AdCa) was associated with reduced lung function, poorer differentiation grade, more advanced tumor stage, remarkably shorter survival, and higher frequency of TP53 mutations. BC-S shared with hESC and a considerable subset of lung carcinomas a common TP53 inactivation molecular pattern which strongly correlated with the BC-S hESC gene expression. These data provide transcriptome-based evidence that smoking-induced reprogramming of airway BC toward the hESC-like phenotype might represent a common early molecular event in the development of aggressive lung carcinomas in humans.

  3. Triggers of airway inflammation.

    PubMed

    Kerrebijn, K F

    1986-01-01

    Most asthmatics have hyperresponsive airways. This makes them more sensitive than non-asthmatics to bronchoconstricting environmental exposures which, in their turn, may enhance responsiveness. Airway inflammation is considered to be a key determinant of airway hyperresponsiveness: the fact that chronic airway inflammation in cystic fibrosis does not lead to airway hyperresponsiveness of any importance indicates, however, that the role of airway inflammation is complex and incompletely elucidated. The main inducers of airway inflammation are viral infections, antigens, occupational stimuli and pollutants. Although exercise, airway cooling and hyper- or hypotonic aerosols are potent stimuli of bronchoconstriction, it is questionable if airway inflammation is involved in their mode of action. Each of the above-mentioned stimuli is discussed, with emphasis laid on the relation of symptoms to mechanisms.

  4. Intratracheal therapy with autologous bone marrow-derived mononuclear cells reduces airway inflammation in horses with recurrent airway obstruction.

    PubMed

    Barussi, Fernanda C M; Bastos, Fernanda Z; Leite, Lidiane M B; Fragoso, Felipe Y I; Senegaglia, Alexandra C; Brofman, Paulo R S; Nishiyama, Anita; Pimpão, Cláudia T; Michelotto, Pedro V

    2016-10-01

    This research evaluated the effects of bone marrow-derived mononuclear cells (BMMCs) on the inflammatory process in the equine recurrent airway obstruction (RAO). Eight horses in RAO clinical score were divided into cell therapy group (Gcel) treated with a single intratracheal dose of BMMCs, and dexamethasone group (Gdex) treated with 21days of oral dexamethasone. The horses were clinically revaluated on days 7 and 21, together with cytological evaluation of the BALF, and detection of inflammatory markers (interleukins [IL]-10, -4, and -17, and interferon γ and α). There were decreases in respiratory effort and clinical score on days 7 and 21(p<0.05) for both groups. The percentage of neutrophils decreased and macrophages increased on days 7 and 21 (p<0.005) in both groups. IL-10 levels increased in the Gcel group on day 21 compared to days 0 and 7 (p<0.05), but this was not observed in the Gdex group. The quantification of IL-4, IL-17, IFN-γ, and IFN-α did not change between evaluations in both groups. These preliminary results suggest that BMMCs may ameliorate the inflammatory response of RAO. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells.

    PubMed

    Sharma, Pawan; Panebra, Alfredo; Pera, Tonio; Tiegs, Brian C; Hershfeld, Alena; Kenyon, Lawrence C; Deshpande, Deepak A

    2016-02-15

    Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K(+) channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as

  6. Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells

    PubMed Central

    Sharma, Pawan; Panebra, Alfredo; Pera, Tonio; Tiegs, Brian C.; Hershfeld, Alena; Kenyon, Lawrence C.

    2015-01-01

    Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K+ channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as

  7. Pycnogenol Ameliorates Asthmatic Airway Inflammation and Inhibits the Function of Goblet Cells.

    PubMed

    Liu, Zhaoe; Han, Bo; Chen, Xing; Wu, Qiaoling; Wang, Lijun; Li, Gang

    2016-11-01

    Pycnogenol(®) (PYC) is utilized in the treatment of various diseases ranging from chronic inflammation to circulatory diseases, but its efficacy and functional mechanism in pediatric asthma continue to remain obscure. Therefore, the purpose of this study was to investigate the effectiveness and molecular mechanism of PYC on regulation of asthmatic airway inflammation. We found that PYC with tail intravenous injection of 50 mg/kg or intragastric administration of 100 mg/kg all reduced ovalbumin (OVA)-induced airway injury. Pharmacokinetics of PYC was evaluated by high-performance liquid chromatography assay, indicating that PYC was quickly absorbed into the blood after intragastric administration, and PYC metabolism was later improved gradually with increase of time after PYC administration. PYC has a higher bioavailability of 71.96%, and it was more easily absorbed by the body. PYC inhibited the number of total inflammatory cells and levels of interleukin (IL)-4, IL-5, IL-9, and IL-13 in bronchoalveolar lavage fluid of OVA-induced mice. PYC inhibited IL-13 secretion from the Th2 cells, thereby causing a reduction in expression of the signaling molecules in JAK/STAT6 pathway in airway epithelial cells. STAT6 silence suppressed IL-13-increased acetylcholine level. STAT6 overexpression promoted expression of goblet cell metaplasia-associated molecules (FOXA3, SPDEF, and Muc5ac). PYC suppressed OVA-induced expression of FOXA3, SPDEF, and Muc5ac in lung. Our findings indicate that PYC has a higher bioavailability and it prevents emergence of OVA-induced airway injury and airway inflammation in mice by inhibiting IL-13/JAK/STAT6 pathway and blocking release of acetylcholine to reduce goblet cell metaplasia.

  8. Chronic exposure to ozone causes tolerance to airway hyperresponsiveness in guinea pigs: lack of SOD role.

    PubMed

    Vargas, M H; Romero, L; Sommer, B; Zamudio, P; Gustin, P; Montaño, L M

    1998-05-01

    Tolerance to respiratory effects of O3 has been demonstrated for anatomic and functional changes, but information about tolerance to O3-induced airway hyperresponsiveness (AHR) is scarce. In guinea pigs exposed to air or O3 (0.3 parts/million, 4 h/day, for 1, 3, 6, 12, 24, or 48 days, studied 16-18 h later), pulmonary insufflation pressure changes induced by intravenous substance P (SP, 0.032-3.2 micro ug/kg) were measured, then the animals were subjected to bronchoalveolar lavage (BAL). Bronchial rings with or without phosphoramidon were also evaluated 3 h after air or a single O3 exposure. O3 caused in vivo AHR (increased sensitivity) to SP after 1, 3, 6, 12, and 24 days of exposure compared with control. However, after 48 days of exposure, O3 no longer caused AHR. Total cell, macrophage, neutrophil, and eosinophil counts in BAL were increased in most O3-exposed groups. When data from all animals were pooled, we found a highly significant correlation between degree of airway responsiveness and total cells (r = 0.55), macrophages (r = 0.54), neutrophils (r = 0.47), and eosinophils (r = 0.53), suggesting that airway inflammation is involved in development of AHR to SP. Superoxide dismutase (SOD) levels in BAL fluids were increased (P < 0.05) after 1, 3, 6, and 12 days of O3 exposure and returned to basal levels after 24 and 48 days of exposure. O3 failed to induce hyperresponsiveness to SP in bronchial rings, and phosphoramidon increased responses to SP in air- and O3-exposed groups, suggesting that neutral endopeptidase inactivation was not involved in O3-induced AHR to SP in vivo. We conclude that chronic exposure to 0. 3 ppm O3, a concentration found in highly polluted cities, resulted in tolerance to AHR to SP in guinea pigs by an SOD-independent mechanism.

  9. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

    PubMed

    Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C

    2015-12-01

    Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

  10. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone

    PubMed Central

    Pearson, Helen; Britt, Rodney D.; Pabelick, Christine M.; Prakash, Y.S.; Amrani, Yassine; Pandya, Hitesh C.

    2016-01-01

    Background Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Methods Cultured fetal human ASM cells stimulated with TNF-α (0–20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. Results CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Conclusion Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases. PMID:26331770

  11. Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells.

    PubMed

    Olsen, Colin E; Liguori, Andrew E; Zong, Yue; Lantz, R Clark; Burgess, Jefferey L; Boitano, Scott

    2008-08-01

    As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

  12. The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases

    PubMed Central

    Rokicki, Marek; Wojtacha, Jacek; Dżeljijli, Agata

    2016-01-01

    The report presents the cellular structure of the respiratory system as well as the history of club cells (Clara cells), their ultrastructure, and location in the airways and human organs. The authors discuss the biochemical structure of proteins secreted by these cells and their importance for the integrity and regeneration of the airway epithelium. Their role as progenitor cells for the airway epithelium and their involvement in the biotransformation of toxic xenobiotics introduced into the lungs during breathing is emphasized. This is followed by a discussion of the clinical aspects associated with club cells, demonstrating that tracking the serum concentration of club cell-secreted proteins is helpful in the diagnosis of a number of lung tissue diseases. Finally, suggestions are provided regarding the possible use of proteins secreted by club cells in the treatment of serious respiratory conditions. PMID:27212975

  13. ω-3 Polyunsaturated fatty acids accelerate airway repair by activating FFA4 in club cells.

    PubMed

    Lee, Kyoung-Pil; Park, Soo-Jin; Kang, Saeromi; Koh, Jung-Min; Sato, Koichi; Chung, Hae-Young; Okajima, Fumikazu; Im, Dong-Soon

    2017-06-01

    A G protein-coupled receptor (GPCR) named free fatty acid receptor 4 (FFA4, also known as GPR120) was found to act as a GPCR for ω-3 polyunsaturated fatty acids. Its expression has been reported in lung epithelial club cells. We investigated whether supplementation of the ω-3 fatty acids benefits lung health. Omacor (7.75 mg/kg), clinically prescribed preparation of ω-3 fatty acids, and FFA4-knockout mice were utilized in a naphthalene-induced mouse model of acute airway injury (1 injection of 30 mg/kg ip). Naphthalene injection induced complete destruction of bronchiolar epithelial cells within a day. Appearance of bronchiolar epithelial cells was observed after 21 days in control mice. It was found, however, that supplementation of Omacor accelerated the recovery. The appearance of bronchiolar epithelial cells was observed between 7 and 14 days after naphthalene injury in Omacor-treated mice. In isolated club cells, ω-3 fatty acids were found to stimulate cell proliferation and migration but to inhibit cell differentiation. With the use of pharmacological tools and FFA4-knockout mice, FFA4 was found to be responsible for ω-3 fatty acids-induced proliferation in vitro in club cells. Furthermore, accelerated recovery from naphthalene-induced airway injury in Omacor-treated mice was not observed in FFA4-knockout mice in vivo. Present findings indicate that ω-3 fatty acids-induced proliferation of bronchiole epithelial cells through FFA4 is responsible for Omacor-induced accelerated recovery from airway injury. Therefore, intermittent administration of Omacor needs to be tested for acute airway injury because ω-3 fatty acids stimulate proliferation but inhibit differentiation of club cells. Copyright © 2017 the American Physiological Society.

  14. SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

    PubMed Central

    Ahmad, Shama; Nichols, David P.; Strand, Matthew; Rancourt, Raymond C.; Randell, Scott H.; White, Carl W.; Ahmad, Aftab

    2011-01-01

    Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target. PMID:22096575

  15. Near Equilibrium Calculus of Stem Cells in Application to the Airway Epithelium Lineage

    PubMed Central

    Sun, Zheng; Plikus, Maksim V.; Komarova, Natalia L.

    2016-01-01

    Homeostatic maintenance of tissues is orchestrated by well tuned networks of cellular signaling. Such networks regulate, in a stochastic manner, fates of all cells within the respective lineages. Processes such as symmetric and asymmetric divisions, differentiation, de-differentiation, and death have to be controlled in a dynamic fashion, such that the cell population is maintained at a stable equilibrium, has a sufficiently low level of stochastic variation, and is capable of responding efficiently to external damage. Cellular lineages in real tissues may consist of a number of different cell types, connected by hierarchical relationships, albeit not necessarily linear, and engaged in a number of different processes. Here we develop a general mathematical methodology for near equilibrium studies of arbitrarily complex hierarchical cell populations, under regulation by a control network. This methodology allows us to (1) determine stability properties of the network, (2) calculate the stochastic variance, and (3) predict how different control mechanisms affect stability and robustness of the system. We demonstrate the versatility of this tool by using the example of the airway epithelium lineage. Recent research shows that airway epithelium stem cells divide mostly asymmetrically, while the so-called secretory cells divide predominantly symmetrically. It further provides quantitative data on the recovery dynamics of the airway epithelium, which can include secretory cell de-differentiation. Using our new methodology, we demonstrate that while a number of regulatory networks can be compatible with the observed recovery behavior, the observed division patterns of cells are the most optimal from the viewpoint of homeostatic lineage stability and minimizing the variation of the cell population size. This not only explains the observed yet poorly understood features of airway tissue architecture, but also helps to deduce the information on the still largely hypothetical

  16. Near Equilibrium Calculus of Stem Cells in Application to the Airway Epithelium Lineage.

    PubMed

    Sun, Zheng; Plikus, Maksim V; Komarova, Natalia L

    2016-07-01

    Homeostatic maintenance of tissues is orchestrated by well tuned networks of cellular signaling. Such networks regulate, in a stochastic manner, fates of all cells within the respective lineages. Processes such as symmetric and asymmetric divisions, differentiation, de-differentiation, and death have to be controlled in a dynamic fashion, such that the cell population is maintained at a stable equilibrium, has a sufficiently low level of stochastic variation, and is capable of responding efficiently to external damage. Cellular lineages in real tissues may consist of a number of different cell types, connected by hierarchical relationships, albeit not necessarily linear, and engaged in a number of different processes. Here we develop a general mathematical methodology for near equilibrium studies of arbitrarily complex hierarchical cell populations, under regulation by a control network. This methodology allows us to (1) determine stability properties of the network, (2) calculate the stochastic variance, and (3) predict how different control mechanisms affect stability and robustness of the system. We demonstrate the versatility of this tool by using the example of the airway epithelium lineage. Recent research shows that airway epithelium stem cells divide mostly asymmetrically, while the so-called secretory cells divide predominantly symmetrically. It further provides quantitative data on the recovery dynamics of the airway epithelium, which can include secretory cell de-differentiation. Using our new methodology, we demonstrate that while a number of regulatory networks can be compatible with the observed recovery behavior, the observed division patterns of cells are the most optimal from the viewpoint of homeostatic lineage stability and minimizing the variation of the cell population size. This not only explains the observed yet poorly understood features of airway tissue architecture, but also helps to deduce the information on the still largely hypothetical

  17. Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression

    PubMed Central

    Hays, L E; Zodrow, D M; Yates, J E; Deffebach, M E; Jacoby, D B; Olson, S B; Pankow, J F; Bagby, G C

    2008-01-01

    Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis. PMID:18475298

  18. The potential role of natural agents in treatment of airway inflammation.

    PubMed

    Sharafkhaneh, Amir; Velamuri, Suryakanta; Badmaev, Vladimir; Lan, Charlie; Hanania, Nicola

    2007-12-01

    Obstructive airway diseases including asthma, chronic obstructive pulmonary disease and cystic fibrosis present with dyspnea and variety of other symptoms. Physiologically, they are characterized by maximal expiratory flow limitation and pathologically, by inflammation of the airways and the lung parenchyma. Inflammation plays a major role in the gradual worsening of the lung function resulting in worsening symptoms. For many years, scientists focused their efforts in identifying various pathways involved in the chronic inflammation present in these diseases. Further, studies are underway to identify various molecular targets in these pathways for the purpose of developing novel therapeutic agents. Natural agents have been used for thousands of years in various cultures for the treatment of several medical conditions and have mostly proven to be safe. Recent in vivo and in vitro studies show potential anti-inflammatory role for some of the existing natural agents. This review provides an overview of the literature related to the anti-inflammatory effects of some of the natural agents which have potential value in the treatment of inflammatory lung diseases.

  19. Pseudomonas aeruginosa Airway Infection Recruits and Modulates Neutrophilic Myeloid-Derived Suppressor Cells

    PubMed Central

    Öz, Hasan H.; Zhou, Benyuan; Voss, Pina; Carevic, Melanie; Schroth, Carolin; Frey, Nina; Rieber, Nikolaus; Hector, Andreas; Hartl, Dominik

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes infections mainly in patients with cystic fibrosis (CF) lung disease. Despite innate and adaptive immune responses upon infection, P. aeruginosa is capable of efficiently escaping host defenses, but the underlying immune mechanisms remain poorly understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that are functionally characterized by their potential to suppress T- and natural killer (NK)-cell responses. Here we demonstrate, using an airway in vivo infection model, that P. aeruginosa recruits and activates neutrophilic MDSCs, which functionally suppress T-cell responses. We further show that the CF gene defect (CF transmembrane conductance regulator, CFTR) modulates the functionality, but not the recruitment or generation of neutrophilic MDSCs. Collectively, we define a mechanism by which P. aeruginosa airway infection undermines host immunity by modulating neutrophilic MDSCs in vivo. PMID:27965936

  20. The role of airway stent placement in the management of tracheobronchial stenosis caused by inoperable advanced lung cancer.

    PubMed

    Furukawa, Kinya; Ishida, Junzo; Yamaguchi, Gaku; Usuda, Jitsuo; Tsutsui, Hidemitsu; Saito, Makoto; Konaka, Chimori; Kato, Harubumi

    2010-04-01

    To our knowledge there is no in-depth report on the benefits of airway stenting, which focuses specifically on patients with inoperable advanced lung cancer causing severe central airway obstruction. We evaluated the role of airway stenting as one aspect of the multidisciplinary management of advanced lung cancer. We performed airway stenting in 40 lung cancer patients, placing a total of 58 stents. Stenting was done as a final modality in 22 patients with terminal-stage lung cancer (group A). The other 18 patients received additional therapy after stenting (group B), 12 (66.7%) of whom were treatment-naïve on admission. The performance status (PS) and Hugh-Jones classification (H-J) scores improved in both groups after stenting: from 3.56 to 2.48 (P = 0.001) and 4.29 to 3.20 (P = 0.004) in group A, and from 3.15 to 1.25 (P < 0.001) and 4.10 to 2.10 (P < 0.001) in group B, respectively. The median survival time and 1-year survival rate after stenting were 1.6 months and 5.1%, respectively, in group A, and 5.6 months and 25.0%, respectively, in group B. Airway stenting followed by adjuvant therapy may improve the survival of treatment-naïve patients with severe symptomatic airway obstruction caused by advanced lung cancer.

  1. In Vivo Imaging of Tracheal Epithelial Cells in Mice during Airway Regeneration

    PubMed Central

    Kim, Jun Ki; Vinarsky, Vladimir; Wain, John; Zhao, Rui; Jung, Keehoon; Choi, Jinwoo; Lam, Adam; Pardo-Saganta, Ana; Breton, Sylvie; Rajagopal, Jayaraj

    2012-01-01

    Many human lung diseases, such as asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans, and cystic fibrosis, are characterized by changes in the cellular composition and architecture of the airway epithelium. Intravital fluorescence microscopy has emerged as a powerful approach in mechanistic studies of diseases, but it has been difficult to apply this tool for in vivo respiratory cell biology in animals in a minimally invasive manner. Here, we describe a novel miniature side-view confocal probe capable of visualizing the epithelium in the mouse trachea in vivo at a single-cell resolution. We performed serial real-time endotracheal fluorescence microscopy in live transgenic reporter mice to view the three major cell types of the large airways, namely, basal cells, Clara cells, and ciliated cells. As a proof-of-concept demonstration, we monitored the regeneration of Clara cells over 18 days after a sulfur dioxide injury. Our results show that in vivo tracheal microscopy offers a new approach in the study of altered, regenerating, or metaplastic airways in animal models of lung diseases. PMID:22984086

  2. Effects of the cationic protein poly-L-arginine on airway epithelial cells in vitro.

    PubMed Central

    Shahana, Shahida; Kampf, Caroline; Roomans, Godfried M

    2002-01-01

    BACKGROUND: Allergic asthma is associated with an increased number of eosinophils in the airway wall. Eosinophils secrete cationic proteins, particularly major basic protein (MBP). AIM: To investigate the effect of synthetic cationic polypeptides such as poly-L-arginine, which can mimic the effect of MBP, on airway epithelial cells. METHODS: Cultured airway epithelial cells were exposed to poly-L-arginine, and effects were determined by light and electron microscopy. RESULTS: Poly-L-arginine induced apoptosis and necrosis. Transmission electron microscopy showed mitochondrial damage and changes in the nucleus. The tight junctions were damaged, as evidenced by penetration of lanthanum. Scanning electron microscopy showed a damaged cell membrane with many pores. Microanalysis showed a significant decrease in the cellular content of magnesium, phosphorus, sodium, potassium and chlorine, and an increase in calcium. Plakoglobin immunoreactivity in the cell membrane was decreased, indicating a decrease in the number of desmosomes CONCLUSIONS: The results point to poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contacts and generalized cell damage. PMID:12137242

  3. In vivo imaging of tracheal epithelial cells in mice during airway regeneration.

    PubMed

    Kim, Jun Ki; Vinarsky, Vladimir; Wain, John; Zhao, Rui; Jung, Keehoon; Choi, Jinwoo; Lam, Adam; Pardo-Saganta, Ana; Breton, Sylvie; Rajagopal, Jayaraj; Yun, Seok Hyun

    2012-12-01

    Many human lung diseases, such as asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans, and cystic fibrosis, are characterized by changes in the cellular composition and architecture of the airway epithelium. Intravital fluorescence microscopy has emerged as a powerful approach in mechanistic studies of diseases, but it has been difficult to apply this tool for in vivo respiratory cell biology in animals in a minimally invasive manner. Here, we describe a novel miniature side-view confocal probe capable of visualizing the epithelium in the mouse trachea in vivo at a single-cell resolution. We performed serial real-time endotracheal fluorescence microscopy in live transgenic reporter mice to view the three major cell types of the large airways, namely, basal cells, Clara cells, and ciliated cells. As a proof-of-concept demonstration, we monitored the regeneration of Clara cells over 18 days after a sulfur dioxide injury. Our results show that in vivo tracheal microscopy offers a new approach in the study of altered, regenerating, or metaplastic airways in animal models of lung diseases.

  4. Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

    PubMed Central

    Perez-Zoghbi, Jose F.; Bai, Yan

    2010-01-01

    Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca2+ signaling, and Ca2+ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca2+]i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca2+ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca2+ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca2+ oscillations. NOC-5 inhibited the increase of [Ca2+]i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP3) in airway SMCs. The effect of NO on the Ca2+ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca2+ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca2+-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca2+ via the IP3 receptor to decrease the frequency of agonist-induced Ca2+ oscillations. PMID:20176853

  5. γ-Secretase Inhibitor Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation

    PubMed Central

    Zhang, Weixi; Zhang, Xueya; Sheng, Anqun; Weng, Cuiye; Zhu, Tingting; Zhao, Wei; Li, Changchong

    2015-01-01

    T helper 17 (Th17) cells play an important role in the pathogenesis of allergic asthma. Th17 cell differentiation requires Notch signaling. γ-Secretase inhibitor (GSI) blocks Notch signaling; thus, it may be considered as a potential treatment for allergic asthma. The aim of this study was to evaluate the effect of GSI on Th17 cell differentiation in a mouse model of allergic asthma. OVA was used to induce mouse asthma model in the presence and absence of GSI. GSI ameliorated the development of OVA-induced asthma, including suppressing airway inflammation responses and reducing the severity of clinical signs. GSI also significantly suppressed Th17-cell responses in spleen and reduced IL-17 levels in serum. These findings suggest that GSI directly regulates Th17 responses through a Notch signaling-dependent pathway in mouse model of allergic asthma, supporting the notion that GSI is a potential therapeutic agent for the treatment of allergic asthma. PMID:26339131

  6. The pharmacology of bitter taste receptors and their role in human airways.

    PubMed

    Devillier, Philippe; Naline, Emmanuel; Grassin-Delyle, Stanislas

    2015-11-01

    The receptors involved in bitter taste perception (bitter taste receptors--T2Rs) constitute a family of G-protein-coupled receptors, of which around 29 subtypes have been identified in humans. T2R expression was initially thought to be confined to the oral cavity but has recently been described in a range of other tissues (such as the heart, gut, nasal cavity and lungs) and cell types (chemosensory, smooth muscle, endothelial, epithelial and inflammatory cells). Although it is still not clear whether endogenous T2R agonists exist, the T2R receptors recognize many natural and synthetic compounds, such as the acyl-homoserine lactones produced by bacteria, caffeine, chloroquine, and erythromycin. In the upper airways, T2Rs are involved in neurogenic inflammation and bacterial clearance. Their known effects in the lungs are exerted at three different levels. Firstly, T2R agonists increase the beating frequency of cilia on epithelial cells. Secondly, the T2Rs induce bronchial smooth muscle cells to relax. Thirdly, the T2R receptors expressed on immune cells (such as macrophages and mast cells) modulate production of pro-inflammatory mediators. Furthermore, T2R agonists are effective in inhibiting lung inflammation or smooth muscle contraction in ex vivo and asthma animal models, and are known to be involved in bacterial killing in the nasal cavity and enhancing lung function in humans. This review focuses on the pharmacology and physiological functions of T2R receptors in the upper and lower airways. It presents recently acquired knowledge suggesting that T2Rs may become valuable drug targets in the treatment of diseases such as asthma and chronic rhinosinusitis. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Functional activity of L-carnitine transporters in human airway epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2016-02-01

    Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects.

  8. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    NASA Astrophysics Data System (ADS)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-09-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO2 NPs (size range 4-33 nm), two preparations of CeO2 NPs (9-36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15-240 μg/cm2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm2, in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO2 and CeO2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured nanomaterials.

  9. Arsenic Alters ATP-Dependent Ca2+ Signaling in Human Airway Epithelial Cell Wound Response

    PubMed Central

    Sherwood, Cara L.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2011-01-01

    Arsenic is a natural metalloid toxicant that is associated with occupational inhalation injury and contaminates drinking water worldwide. Both inhalation of arsenic and consumption of arsenic-tainted water are correlated with malignant and nonmalignant lung diseases. Despite strong links between arsenic and respiratory illness, underlying cell responses to arsenic remain unclear. We hypothesized that arsenic may elicit some of its detrimental effects on the airway through limitation of innate immune function and, specifically, through alteration of paracrine ATP (purinergic) Ca2+ signaling in the airway epithelium. We examined the effects of acute (24 h) exposure with environmentally relevant levels of arsenic (i.e., < 4μM as Na-arsenite) on wound-induced Ca2+ signaling pathways in human bronchial epithelial cell line (16HBE14o-). We found that arsenic reduces purinergic Ca2+ signaling in a dose-dependent manner and results in a reshaping of the Ca2+ signaling response to localized wounds. We next examined arsenic effects on two purinergic receptor types: the metabotropic P2Y and ionotropic P2X receptors. Arsenic inhibited both P2Y- and P2X-mediated Ca2+ signaling responses to ATP. Both inhaled and ingested arsenic can rapidly reach the airway epithelium where purinergic signaling is essential in innate immune functions (e.g., ciliary beat, salt and water transport, bactericide production, and wound repair). Arsenic-induced compromise of such airway defense mechanisms may be an underlying contributor to chronic lung disease. PMID:21357385

  10. Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice

    SciTech Connect

    Sun, Daqing; Wang, Jing; Yang, Niandi; Ma, Haixin

    2016-08-12

    Matrine has been demonstrated to attenuate allergic airway inflammation. Elevated suppressor of cytokine signaling 3 (SOCS3) was correlated with the severity of asthma. The aim of this study was to investigate the effect of matrine on SOCS3 expression in airway inflammation. In this study, we found that matrine significantly inhibited OVA-induced AHR, inflammatory cell infiltration, goblet cell differentiation, and mucous production in a dose-dependent manner in mice. Matrine also abrogated the level of interleukin (IL)-4 and IL-13, but enhanced interferon (IFN)-γ expression, both in BALF and in lung homogenates. Furthermore, matrine impeded TNF-α-induced the expression of IL-6 and adhesion molecules in airway epithelial cells (BEAS-2B and MLE-12). Additionally, we found that matrine inhibited SOCS3 expression, both in asthmatic mice and TNF-α-stimulated epithelial cells via suppression of the NF-κB signaling pathway by using pcDNA3.1-SOCS3 plasmid, SOCS3 siRNA, or nuclear factor kappa-B (NF-κB) inhibitor PDTC. Conclusions: Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice. - Highlights: • Matrine attenuates asthmatic symptoms and regulates Th1/Th2 balance in vivo. • Matrine suppresses inflammation responses in vitro. • Matrine decreases SOCS3 expression both in vivo and in vitro. • Matrine inhibits SOCS3 expression by suppressing NF-κB signaling.

  11. Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells

    PubMed Central

    Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina A; O'Neal, Wanda; Gabriel, Sherif; Abdullah, Lubna; Davis, C William; Boucher, Richard C; Lazarowski, Eduardo R

    2007-01-01

    The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca2+-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca2+-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca2+-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y2 receptor-stimulated increase of cytosolic Ca2+ concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL. PMID:17656429

  12. Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells.

    PubMed

    Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina A; O'Neal, Wanda; Gabriel, Sherif; Abdullah, Lubna; Davis, C William; Boucher, Richard C; Lazarowski, Eduardo R

    2007-10-01

    The efficiency of the mucociliary clearance (MCC) process that removes noxious materials from airway surfaces depends on the balance between mucin secretion, airway surface liquid (ASL) volume, and ciliary beating. Effective mucin dispersion into ASL requires salt and water secretion onto the mucosal surface, but how mucin secretion rate is coordinated with ion and, ultimately, water transport rates is poorly understood. Several components of MCC, including electrolyte and water transport, are regulated by nucleotides in the ASL interacting with purinergic receptors. Using polarized monolayers of airway epithelial Calu-3 cells, we investigated whether mucin secretion was accompanied by nucleotide release. Electron microscopic analyses of Calu-3 cells identified subapical granules that resembled goblet cell mucin granules. Real-time confocal microscopic analyses revealed that subapical granules, labelled with FM 1-43 or quinacrine, were competent for Ca(2+)-regulated exocytosis. Granules containing MUC5AC were apically secreted via Ca(2+)-regulated exocytosis as demonstrated by combined immunolocalization and slot blot analyses. In addition, Calu-3 cells exhibited Ca(2+)-regulated apical release of ATP and UDP-glucose, a substrate of glycosylation reactions within the secretory pathway. Neither mucin secretion nor ATP release from Calu-3 cells were affected by activation or inhibition of the cystic fibrosis transmembrane conductance regulator. In SPOC1 cells, an airway goblet cell model, purinergic P2Y(2) receptor-stimulated increase of cytosolic Ca(2+) concentration resulted in secretion of both mucins and nucleotides. Our data suggest that nucleotide release is a mechanism by which mucin-secreting goblet cells produce paracrine signals for mucin hydration within the ASL.

  13. Careers in Airway Science.

    ERIC Educational Resources Information Center

    Federal Aviation Administration (DOT), Washington, DC.

    The Federal Aviation Administration (FAA) has initiated the Airway Science curriculum as a method of preparing the next generation of aviation technicians and managers. This document: (1) discusses the FAA's role in the Airway Science program; (2) describes some of the career fields that FAA offers to Airway Science graduates (air traffic control…

  14. The effect of ozone on inflammatory cell infiltration and airway hyperresponsiveness in the guinea pig lung

    SciTech Connect

    Schultheis, A.J.H.

    1993-01-01

    Inflammatory cells may contribute to the development of exaggerated bronchoconstrictor responses since a persistent link has been noted between pulmonary inflammation and airway hyperresponsiveness. In these studies guinea pigs were exposed to 2.0 ppm ozone for 4 hours, then immediately sacrificed or allowed to breathe filtered air for up to 14 days. Following ozone exposure there was an immediate massive neutrophil infiltration into the lung. Neutrophils in lung digest dropped to control values within 3-12 hours post-ozone but remained elevated in BAL fluid for 3 days. There was probable eosinophil degranulation within the first 24 hours post-ozone. Guinea pigs were hyperresponsive to vigal stimulation through 3 days post-ozone. Although they were also hyperresponsive to ACh, responses to MCh were unchanged. Neuronal M[sub 2] receptors were dysfunctional through 3 days post-ozone. There was resolution of inflammation, airway responsiveness, and neuronal M[sub 2] receptor function by 14 days post-exposure. This investigation has (1) confirmed an immediate lung inflammation following acute ozone exposure; (2) established that cells in BAL give a distorted reflection of inflammatory events in lung digest; (3) demonstrated that ozone-induced hyperresponsiveness is at least partially due to efferent cholinergic mechanisms without functional changes of muscarinic receptors on airway smooth muscle; (4) shown that ACh may not be an appropriate agent to test ozone-induced airway hyperresponsiveness; and (5) demonstrated that inhibitory neuronal M[sub 2] receptors are dysfunctional following ozone exposure. There was close linkage between these events, suggesting that they may be causally related. This investigation proposes a specific mechanism, dysfunction of neuronal M[sub 2] receptors, by which inflammatory cells could cause airway hyperresponsiveness following acute ozone exposure.

  15. Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

    PubMed

    Shkumatov, Artem; Thompson, Michael; Choi, Kyoung M; Sicard, Delphine; Baek, Kwanghyun; Kim, Dong Hyun; Tschumperlin, Daniel J; Prakash, Y S; Kong, Hyunjoon

    2015-06-01

    Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-β1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs. Copyright © 2015 the American Physiological Society.

  16. Antigen-specific Treg regulate Th17-mediated lung neutrophilic inflammation, B cell recruitment and polymeric IgA and IgM levels in the airways

    PubMed Central

    Jaffar, Zeina; Ferrini, Maria E.; Girtsman, Teri A.; Roberts, Kevan

    2010-01-01

    Summary Th17 cells play key roles in mediating autoimmunity, inflammation and mucosal host defense against pathogens. To determine whether naturally occurring Treg (nTreg) limit Th17-mediated pulmonary inflammation, OVA-specific CD4+ Th17 cells and expanded CD4+CD25+Foxp3+ nTreg were cotransferred into BALB/c mice that were then exposed to OVA aerosols. Th17 cells, when transferred alone, accumulated in the lungs and posterior mediastinal LN and evoked a pronounced airway hyperreactivity (AHR) and neutrophilic inflammation, characterized by B cell recruitment and elevated IgA and IgM levels. Cotransfer of antigen-specific nTreg markedly reduced the Th17-induced pulmonary inflammation and associated neutrophilia, B cell influx and polymeric Ig levels in the airways, but did not inhibit AHR. Moreover, the regulation appeared restricted to the site of mucosal inflammation, since transfer of nTreg did not affect the Th17 response developing in the lung draining LN, as evidenced by unaltered levels of IL-17 production and low numbers of Foxp3+ Treg. Our findings suggest a crucial role for Th17 cells in mediating airway B cell influx and IgA response and demonstrate that antigen-specific nTreg suppress Th17-mediated lung inflammation. These results provide new insights into how Th17 responses are limited and may facilitate development of novel approaches for controlling Th17-induced inflammation. PMID:19830731

  17. Deletion of airway cilia results in noninflammatory bronchiectasis and hyperreactive airways

    PubMed Central

    Gilley, Sandra K.; Stenbit, Antine E.; Pasek, Raymond C.; Sas, Kelli M.; Steele, Stacy L.; Amria, May; Bunni, Marlene A.; Estell, Kimberly P.; Schwiebert, Lisa M.; Flume, Patrick; Gooz, Monika; Haycraft, Courtney J.; Yoder, Bradley K.; Miller, Caroline; Pavlik, Jacqueline A.; Turner, Grant A.; Sisson, Joseph H.

    2013-01-01

    The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88− mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88− mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function. PMID:24213915

  18. "Epithelial Cell TRPV1-Mediated Airway Sensitivity as a Mechanism for Respiratory Symptoms Associated with Gulf War Illness?

    DTIC Science & Technology

    2010-06-01

    TITLE: “Epithelial Cell TRPV1 -Mediated Airway Sensitivity as a Mechanism for Respiratory Symptoms Associated with Gulf War Illness” PRINCIPAL...66,),&$7,212) E7(/(3+21(180%(5 ,QFOXGHDUHDFRGH 01-06-2010 Annual Report 1 JUN 2009 - 31 MAY 2010 Epithelial Cell TRPV1 -Mediated Airway...express functional TRPV1 . More recently we found that these cells also express another important irritant receptor, namely TRPA1. Activation of

  19. Effects of micropatterned curvature on the motility and mechanical properties of airway smooth muscle cells.

    PubMed

    Xu, Jimin; Chen, Cheng; Jiang, Xuemei; Xu, Rong; Tambe, Dhananjay; Zhang, Xiaojuan; Liu, Lina; Lan, Bo; Cai, Kaiyong; Deng, Linhong

    2011-12-02

    Geometric features such as size and shape of the microenvironment are known to alter cell behaviors such as growth, differentiation, apoptosis, and migration. Little is known, however, about the effect of curvature on cell behaviors despite that many cells reside in curved space of tubular organs such as the bronchial airways. To address this question, we fabricated micropatterned strips that mimic airway walls with varying curvature. Then, we cultured airway smooth muscle cells (ASMCs) on these strips and investigated the cells' motility and mechanical properties using time-lapse imaging microscopy and optical magnetic twisting cytometry (OMTC). We found that both motility and mechanical properties of the ASMCs were influenced by the curvature. In particular, when the curvature increased from 0 to 1/150 μm(-1), the velocity of cell migration first decreased (0-1/750 μm(-1)), and then increased (1/750-1/150 μm(-1)). In contrast, the cell stiffness increased and then decreased. Thus, at the intermediate curvature (1/750 μm(-1)) the ASMCs were the least motile, but most stiff. The contractility instead decreased consistently as the curvature increased. The level of F-actin, and vinculin expression within the ASMCs appeared to correlate with the contractility and motility, respectively, in relation to the curvature. These results may provide valuable insights to understanding the heterogeneity of airway constrictions in asthma as well as the developing and functioning of other tubular organs and tissue engineering. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Expression and regulation of immune-modulatory enzyme indoleamine 2,3-dioxygenase (IDO) by human airway epithelial cells and its effect on T cell activation

    PubMed Central

    Sewell, Herb F.; Knox, Alan; Ghaemmaghami, Amir M.

    2016-01-01

    Indoleamine 2,3-dioxygenase (IDO) catalyzes the degradation of tryptophan, which plays a critical role in immune suppression through regulating the production of a series of metabolites that are generally referred to as kynurenines. It has become increasingly clear that epithelial cells (ECs) play an active role in maintaining lung homeostasis by modulating the function of immune cells via producing cytokines, chemokines, and anti-microbial mediators. In this study we assessed the regulation of IDO activity and expression in human primary ECs and EC lines under steady state conditions and in response to bacterial and allergenic stimuli. We also investigated the potential immune modulatory functions of IDO expression in human airway ECs. Our data clearly show that airway ECs produce IDO, which is down-regulated in response to allergens and TLR ligands while up-regulated in response to IFN-γ. Using gene silencing, we further demonstrate that IDO plays a key role in the EC-mediated suppression of antigen-specific and polyclonal proliferation of T cells. Interestingly, our data also show that ECs lose their inhibitory effect on T cell activation in response to different TLR agonists mimicking bacterial or viral infections. In conclusion, our work provides an understanding of how IDO is regulated in ECs as well as demonstrates that “resting” ECs can suppress T cell activation in an IDO dependent manner. These data provide new insight into how ECs, through the production of IDO, can influence downstream innate and adaptive responses as part of their function in maintaining immune homeostasis in the airways. PMID:27613847