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Sample records for airway cell types

  1. Ozone-Induced Type 2 Immunity in Nasal Airways. Development and Lymphoid Cell Dependence in Mice.

    PubMed

    Ong, Chee Bing; Kumagai, Kazuyoshi; Brooks, Phillip T; Brandenberger, Christina; Lewandowski, Ryan P; Jackson-Humbles, Daven N; Nault, Rance; Zacharewski, Timothy R; Wagner, James G; Harkema, Jack R

    2016-03-01

    Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone.

  2. Ozone-Induced Type 2 Immunity in Nasal Airways. Development and Lymphoid Cell Dependence in Mice.

    PubMed

    Ong, Chee Bing; Kumagai, Kazuyoshi; Brooks, Phillip T; Brandenberger, Christina; Lewandowski, Ryan P; Jackson-Humbles, Daven N; Nault, Rance; Zacharewski, Timothy R; Wagner, James G; Harkema, Jack R

    2016-03-01

    Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone. PMID:26203683

  3. Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes

    PubMed Central

    Sun, Xiaoru; Zheng, Minghuan; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Dragos; Chen, Chengshui; Chen, Luonan; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC-specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL) and CD8+ T cells from lungs (T-LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up-regulated and 70% down-regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over-expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types. PMID:24826900

  4. Anti-Inflammatory Effects of Levalbuterol-Induced 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Airway Epithelial Cells

    PubMed Central

    Randall, Matthew J.; Kostin, Shannon F.; Burgess, Edward J.; Hoyt, Laura R.; Ather, Jennifer L.; Lundblad, Lennart K.; Poynter, Matthew E.

    2015-01-01

    production to an extent similar to that of dexamethasone. These results demonstrate that levalbuterol augments expression of 11β-HSD1 in airway epithelial cells, reducing LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production through the conversion of inactive 11-keto corticosteroids into the active 11-hydroxy form in this cell type. PMID:25628603

  5. Vascular endothelial growth factor is a key mediator in the development of T cell priming and its polarization to type 1 and type 17 T helper cells in the airways.

    PubMed

    Kim, You-Sun; Hong, Sung-Wook; Choi, Jun-Pyo; Shin, Tae-Seop; Moon, Hyung-Geun; Choi, Eun-Jung; Jeon, Seong Gyu; Oh, Sun-Young; Gho, Yong Song; Zhu, Zhou; Kim, Yoon-Keun

    2009-10-15

    Chronic inflammatory airway diseases including asthma are characterized by immune dysfunction to inhaled allergens. Our previous studies demonstrated that T cell priming to inhaled allergens requires LPS, which is ubiquitously present in household dust allergens. In this study, we evaluated the role of vascular endothelial growth factor (VEGF) in the development of T cell priming and its polarization to Th1 or Th17 cells when exposed to LPS-contaminated allergens. An asthma mouse model was induced by airway sensitization with LPS-contaminated allergens and then challenged with allergens alone. Therapeutic intervention was performed during allergen sensitization. The present study showed that lung inflammation induced by sensitization with LPS-contaminated allergens was decreased in mice with homozygous disruption of the IL-17 gene; in addition, allergen-specific Th17 immune response was abolished in IL-6 knockout mice. Meanwhile, in vivo production of VEGF was up-regulated by airway exposure of LPS. In addition, airway sensitization of allergen plus recombinant VEGF induced both type 1 and type 17 Th cell (Th1 and Th17) responses. Th1 and Th17 responses induced by airway sensitization with LPS-contaminated allergens were blocked by treatment with a pan-VEGF receptor (VEGFR; VEGFR-1 plus VEGFR-2) inhibitor during sensitization. These effects were accompanied by inhibition of the production of Th1 and Th17 polarizing cytokines, IL-12p70 and IL-6, respectively. These findings indicate that VEGF produced by LPS plays a key role in activation of naive T cells and subsequent polarization to Th1 and Th17 cells.

  6. Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

    PubMed

    Krishna, Venkatramana D; Roach, Erin; Zaidman, Nathan A; Panoskaltsis-Mortari, Angela; Rotschafer, Jessica H; O'Grady, Scott M; Cheeran, Maxim C-J

    2015-01-01

    Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection. PMID:26384331

  7. Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells

    PubMed Central

    Krishna, Venkatramana D.; Roach, Erin; Zaidman, Nathan A.; Panoskaltsis-Mortari, Angela; Rotschafer, Jessica H.; O’Grady, Scott M.; Cheeran, Maxim C-J.

    2015-01-01

    Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection. PMID:26384331

  8. Differential Induction of Type I and Type III Interferons by Swine and Human Origin H1N1 Influenza A Viruses in Porcine Airway Epithelial Cells.

    PubMed

    Krishna, Venkatramana D; Roach, Erin; Zaidman, Nathan A; Panoskaltsis-Mortari, Angela; Rotschafer, Jessica H; O'Grady, Scott M; Cheeran, Maxim C-J

    2015-01-01

    Interferons (IFNs) have been shown to inhibit influenza A virus (IAV) replication and play an essential role in controlling viral infection. Here we studied the kinetics and magnitude of induction of type I and type III IFN transcripts by primary porcine airway epithelial cells (pAECs) in response to swine and human origin IAV. We observed that swine influenza viruses (SIV) replicate more efficiently than the human pandemic influenza A/California/2009 (pH1N1 CA/09) in pAECs. Interestingly, we also found significant difference in kinetics of IFN-β, IFN-λ1 and IFN-λ3 gene expression by these viruses. While there was delay of up to 12 hours post infection (h p.i.) in induction of IFN genes in pAECs infected with swine IAV A/Sw/Illinois/2008 (H1N1 IL/08), human pH1N1 CA/09 rapidly induced IFN-β, IFN-λ1 and IFN-λ3 gene expression as early as 4 h p.i. However, the magnitude of IFN-β and IFN-λ3 induction at 24 h p.i. was not significantly different between the viral strains tested. Additionally, we found that swine H1N1 IL/08 was less sensitive to dsRNA induced antiviral response compared to human pH1N1 CA/09. Our data suggest that the human and swine IAVs differ in their ability to induce and respond to type I and type III interferons in swine cells. Swine origin IAV may have adapted to the pig host by subverting innate antiviral responses to viral infection.

  9. Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation.

    PubMed

    de Vries, M; Heijink, I H; Gras, R; den Boef, L E; Reinders-Luinge, M; Pouwels, S D; Hylkema, M N; van der Toorn, M; Brouwer, U; van Oosterhout, A J M; Nawijn, M C

    2014-08-01

    Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC. PMID:24816488

  10. 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

  11. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  12. 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

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

    PubMed

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

    2012-11-01

    Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl(-)/HCO(3)(-) 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 (I(sc)). 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(+)-nHCO(3)(-) 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 [HCO(3)(-)] as compared with the control lines. Unstimulated equivalent short-circuit current (I(eq)) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both I(eq) and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl(-)/HCO(3)(-) 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

  14. Migration of Airway Smooth Muscle Cells

    PubMed Central

    Gerthoffer, William T.

    2008-01-01

    Migration of smooth muscle cells is a process fundamental to development of hollow organs, including blood vessels and the airways. Migration is also thought to be part of the response to tissue injury. It has also been suggested to contribute to airways remodeling triggered by chronic inflammation. In both nonmuscle and smooth muscle cells numerous external signaling molecules and internal signal transduction pathways contribute to cell migration. The review includes evidence for the functional significance of airway smooth muscle migration, a summary of promigratory and antimigratory agents, and summaries of important signaling pathways mediating migration. Important signaling pathways and effector proteins described include small G proteins, phosphatidylinositol 3-kinases (PI3-K), Rho activated protein kinase (ROCK), p21-activated protein kinases (PAK), Src family tyrosine kinases, and mitogen-activated protein kinases (MAPK). These signaling modules control multiple critical effector proteins including actin nucleating, capping and severing proteins, myosin motors, and proteins that remodel microtubules. Actin filament remodeling, focal contact remodeling and propulsive force of molecular motors are all coordinated to move cells along gradients of chemical cues, matrix adhesiveness, or matrix stiffness. Airway smooth muscle cell migration can be modulated in vitro by drugs commonly used in pulmonary medicine including β-adrenergic agonists and corticosteroids. Future studies of airway smooth muscle cell migration may uncover novel targets for drugs aimed at modifying airway remodeling. PMID:18094091

  15. An uptake of cationized ferritin by alveolar type I cells in airway-instilled goat lung: distribution of anionic sites on the epithelial surface.

    PubMed

    Atwal, O S; Viel, L; Minhas, K J

    1990-07-01

    The present study has investigated ultrastructural localization of anionic sites on the luminal surface of the alveolar epithelium of goat lung by direct airway instillation of cationized ferritin (CF) in the cranial lobe of the right lung through a bronchoscope. The cationic probe decorated preferentially the luminal plasmalemmal vesicles and plasmalemma proper of alveolar type I cell. This indicated the presence of highly charged anionic microdomains at these binding sites. The ligand was internalized in the free plasmalemmal vesicles of alveolar type I cell within 2 min. Heavy decoration of vesicles at 5 min of perfusion indicated that the amount of CF internalization increased with its concentration in the alveoli. It is suggested that exposure of alveolar surface to several gases of ruminal-origin induces changes in the surface charge of luminal plasmalemma of alveolar type I cells. The significance of these anionic plasmalemmal sites is discussed in relation to the adjustment of osmotic pressure gradient across the alveolar-capillary membrane of the ruminant lung.

  16. An uptake of cationized ferritin by alveolar type I cells in airway-instilled goat lung: distribution of anionic sites on the epithelial surface.

    PubMed

    Atwal, O S; Viel, L; Minhas, K J

    1990-07-01

    The present study has investigated ultrastructural localization of anionic sites on the luminal surface of the alveolar epithelium of goat lung by direct airway instillation of cationized ferritin (CF) in the cranial lobe of the right lung through a bronchoscope. The cationic probe decorated preferentially the luminal plasmalemmal vesicles and plasmalemma proper of alveolar type I cell. This indicated the presence of highly charged anionic microdomains at these binding sites. The ligand was internalized in the free plasmalemmal vesicles of alveolar type I cell within 2 min. Heavy decoration of vesicles at 5 min of perfusion indicated that the amount of CF internalization increased with its concentration in the alveoli. It is suggested that exposure of alveolar surface to several gases of ruminal-origin induces changes in the surface charge of luminal plasmalemma of alveolar type I cells. The significance of these anionic plasmalemmal sites is discussed in relation to the adjustment of osmotic pressure gradient across the alveolar-capillary membrane of the ruminant lung. PMID:2390765

  17. 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.

  18. Cell Jamming in the Airway Epithelium.

    PubMed

    Park, Jin-Ah; Fredberg, Jeffrey J

    2016-03-01

    Hallmarks of asthma include chronic airway inflammation, progressive airway remodeling, and airway hyperresponsiveness. The initiation and perpetuation of these processes are attributable at least in part to critical events within the airway epithelium, but the underlying mechanisms remain poorly understood. New evidence now suggests that epithelial cells derived from donors without asthma versus donors with asthma, even in the absence of inflammatory cells or mediators, express modes of collective migration that innately differ not only in the amount of migration but also in the kind of migration. The maturing cell layer tends to undergo a transition from a hypermobile, fluid-like, unjammed phase in which cells readily rearrange, exchange places, and flow, to a quiescent, solid-like, jammed phase in which cells become virtually frozen in place. Moreover, the unjammed phase defines a phenotype that can be perpetuated by the compressive stresses caused by bronchospasm. Importantly, in cells derived from donors with asthma versus donors without asthma, this jamming transition becomes substantially delayed, thus suggesting an immature or dysmature epithelial phenotype in asthma. PMID:27027955

  19. A Multi-Omics Approach Identifies Key Hubs Associated with Cell Type-Specific Responses of Airway Epithelial Cells to Staphylococcal Alpha-Toxin

    PubMed Central

    Richter, Erik; Harms, Manuela; Ventz, Katharina; Gierok, Philipp; Chilukoti, Ravi Kumar; Hildebrandt, Jan-Peter; Mostertz, Jörg; Hochgräfe, Falko

    2015-01-01

    Responsiveness of cells to alpha-toxin (Hla) from Staphylococcus aureus appears to occur in a cell-type dependent manner. Here, we compare two human bronchial epithelial cell lines, i.e. Hla-susceptible 16HBE14o- and Hla-resistant S9 cells, by a quantitative multi-omics strategy for a better understanding of Hla-induced cellular programs. Phosphoproteomics revealed a substantial impact on phosphorylation-dependent signaling in both cell models and highlights alterations in signaling pathways associated with cell-cell and cell-matrix contacts as well as the actin cytoskeleton as key features of early rHla-induced effects. Along comparable changes in down-stream activity of major protein kinases significant differences between both models were found upon rHla-treatment including activation of the epidermal growth factor receptor EGFR and mitogen-activated protein kinases MAPK1/3 signaling in S9 and repression in 16HBE14o- cells. System-wide transcript and protein expression profiling indicate induction of an immediate early response in either model. In addition, EGFR and MAPK1/3-mediated changes in gene expression suggest cellular recovery and survival in S9 cells but cell death in 16HBE14o- cells. Strikingly, inhibition of the EGFR sensitized S9 cells to Hla indicating that the cellular capacity of activation of the EGFR is a major protective determinant against Hla-mediated cytotoxic effects. PMID:25816343

  20. 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

  1. Immunomodulation of airway epithelium cell activation by mesenchymal stromal cells ameliorates house dust mite-induced airway inflammation in mice.

    PubMed

    Duong, Khang M; Arikkatt, Jaisy; Ullah, M Ashik; Lynch, Jason P; Zhang, Vivian; Atkinson, Kerry; Sly, Peter D; Phipps, Simon

    2015-11-01

    Allergic asthma is underpinned by T helper 2 (Th2) inflammation. Redundancy in Th2 cytokine function and production by innate and adaptive immune cells suggests that strategies aimed at immunomodulation may prove more beneficial. Hence, we sought to determine whether administration of mesenchymal stromal cells (MSCs) to house dust mite (HDM) (Dermatophagoides pteronyssinus)-sensitized mice would suppress the development of Th2 inflammation and airway hyperresponsiveness (AHR) after HDM challenge. We report that the intravenous administration of allogeneic donor MSCs 1 hour before allergen challenge significantly attenuated the features of allergic asthma, including tissue eosinophilia, Th2 cytokine (IL-5 and IL-13) levels in bronchoalveolar lavage fluid, and AHR. The number of infiltrating type 2 innate lymphoid cells was not affected by MSC transfer, suggesting that MSCs may modulate the adaptive arm of Th2 immunity. The effect of MSC administration was long lasting; all features of allergic airway disease were significantly suppressed in response to a second round of HDM challenge 4 weeks after MSC administration. Further, we observed that MSCs decreased the release of epithelial cell-derived alarmins IL-1α and high mobility group box-1 in an IL-1 receptor antagonist-dependent manner. This significantly decreased the expression of the pro-Th2 cytokine IL-25 and reduced the number of activated and antigen-acquiring CD11c(+)CD11b(+) dendritic cells in the lung and mediastinal lymph nodes. Our findings suggest that MSC administration can ameliorate allergic airway inflammation by blunting the amplification of epithelial-derived inflammatory cytokines induced by HDM exposure and may offer long-term protection against Th2-mediated allergic airway inflammation and AHR.

  2. 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.

  3. 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

  4. 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.

  5. Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells.

    PubMed

    Murai, Hiroki; Okazaki, Shintaro; Hayashi, Hisako; Kawakita, Akiko; Hosoki, Koa; Yasutomi, Motoko; Sur, Sanjiv; Ohshima, Yusei

    2015-09-01

    Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells.

  6. Airway goblet cells: responsive and adaptable front-line defenders.

    PubMed

    Rogers, D F

    1994-09-01

    Goblet cells are situated in the epithelium of the conducting airways, often with their apical surfaces protruding into the lumen, a location which fits them for a rapid response to inhaled airway insults. Together with the submucosal glands, goblet cells secrete high molecular weight mucus glycoproteins (mucins), which confer upon the airway surface fluid the requisite biochemical and biophysical properties which determine the efficiency of entrapment and transportation of inhaled irritants, particles and micro-organisms. The diversity of glycosylation of airway mucins may be important in facilitating adherence of micro-organisms to mucus prior to mucociliary clearance. Other secretory products, including lipids and "small" glycoproteins, may also be produced by goblet cells. It is possible that goblet cells have the potential to produce markedly more mucus than do the glands. Mucins are tightly packed in the intracellular granules of the goblet cell. The morphology of these granules varies with fixation technique, and release of mucins may be via a combination of merocrine and apocrine secretion. Discharge of mucus is accomplished remarkably rapidly (tens of milliseconds) and vast quantities of mucus are released (size expansions from the granule of many hundredfold). Depending upon species and preparation, goblet cells discharge mucus in response to a wide variety of stimuli, including proteinases, irritant gases, inflammatory mediators, reactive oxygen species, nerve activation and changes in the biophysical environment. Under normal conditions, goblet cell proliferation and differentiation, particularly to ciliated cells, contributes to maintenance of the airway epithelial cell population. In addition to participating in acute airway defence, goblet cells increase in number in response to chronic airway insult, with a resultant increase in output of mucus. The increase in number of cells is via hyperplastic and metaplastic mechanisms. Early triggers for the

  7. Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa1

    PubMed Central

    Hara, Kenichiro; Iijima, Koji; Elias, Martha K.; Seno, Satoshi; Tojima, Ichiro; Kobayashi, Takao; Kephart, Gail M.; Kurabayashi, Masahiko; Kita, Hirohito

    2014-01-01

    While type 2 immune responses to environmental antigens are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. Here we report that IL-33 and thymic stromal lymphopoietin (TSLP) were produced quickly in the lungs of naïve mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and TSLP sensitized naïve animals to an innocuous airway antigen OVA, which resulted in production of type 2 cytokines and IgE antibody and eosinophilic airway inflammation when mice were challenged with the same antigen. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naïve animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa. PMID:24663677

  8. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells

    PubMed Central

    Huang, Sarah X L; Green, Michael D; de Carvalho, Ana Toste; Mumau, Melanie; Chen, Ya-Wen; D’Souza, Sunita L.; Snoeck, Hans-Willem

    2015-01-01

    Lung and airway epithelial cells generated in vitro from human pluripotent stem cells have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of human pluripotent stem cells into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development and takes approximately 50 days. First, definitive endoderm is induced in the presence of high concentrations of Activin A. Subsequently, lung-biased anterior foregut endoderm is specified by sequential inhibition of BMP, TGF-β and Wnt signaling. Anterior foregut endoderm is then ventralized by applying Wnt, BMP, FGF and RA signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, c-AMP and glucocorticoid agonism. This protocol is conducted in defined conditions, does not involve genetic manipulation of the cells, and results in cultures where the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells. PMID:25654758

  9. 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.

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

    PubMed Central

    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

    ABSTRACT 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. PMID:26062017

  11. 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...

  12. Neuropeptides control the dynamic behavior of airway mucosal dendritic cells.

    PubMed

    Voedisch, Sabrina; Rochlitzer, Sabine; Veres, Tibor Z; Spies, Emma; Braun, Armin

    2012-01-01

    The airway mucosal epithelium is permanently exposed to airborne particles. A network of immune cells patrols at this interface to the environment. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated the impact of neuronal signals on key functions of dendritic cells (DC). Using two-photon microscopic time-lapse analysis of living lung sections from CD11c-EYFP transgenic mice we studied the influence of neuropeptides on airway DC motility. Additionally, using a confocal microscopic approach, the phagocytotic capacity of CD11c(+) cells after neuropeptide stimulation was determined. Electrical field stimulation (EFS) leads to an unspecific release of neuropeptides from nerves. After EFS and treatment with the neuropeptides vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP), airway DC in living lung slices showed an altered motility. Furthermore, the EFS-mediated effect could partially be blocked by pre-treatment with the receptor antagonist CGRP(8-37). Additionally, the phagocytotic capacity of bone marrow-derived and whole lung CD11c(+) cells could be inhibited by neuropeptides CGRP, VIP, and Substance P. We then cross-linked these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model altered neuropeptide amounts and DC motility in the airways could be measured. In summary, our data suggest that neuropeptides modulate key features motility and phagocytosis of mouse airway DC. Therefore altered neuropeptide levels in airways during allergic inflammation have impact on regulation of airway immune mechanisms and therefore might contribute to the pathophysiology of asthma.

  13. 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.

  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. Mast cell-derived neurotrophin 4 mediates allergen-induced airway hyperinnervation in early life

    PubMed Central

    Patel, Kruti R.; Aven, Linh; Shao, Fengzhi; Krishnamoorthy, Nandini; Duvall, Melody G.; Levy, Bruce D.; Ai, Xingbin

    2016-01-01

    Asthma often progresses from early episodes of insults. How early life events connect to long-term airway dysfunction remains poorly understood. We demonstrated previously that increased neurotrophin 4 (NT4) levels following early life allergen exposure cause persistent changes in airway smooth muscle (ASM) innervation and airway hyper-reactivity (AHR) in mice. Herein, we identify pulmonary mast cells as a key source of aberrant NT4 expression following early insults. NT4 is selectively expressed by ASM and mast cells in mice, nonhuman primates and humans. We show in mice that mast cell-derived NT4 is dispensable for ASM innervation during development. However, upon insults, mast cells expand in number and degranulate to release NT4 and thus become the major source of NT4 under pathological condition. Adoptive transfer of wild type mast cells, but not NT4−/− mast cells restores ASM hyperinnervation and AHR in KitW-sh/W-sh mice following early life insults. Notably, an infant nonhuman primate model of asthma also exhibits ASM hyperinnervation associated with the expansion and degranulation of mast cells. Together, these findings identify an essential role of mast cells in mediating ASM hyperinnervation following early life insults by producing NT4. This role may be evolutionarily conserved in linking early insults to long-term airway dysfunction. PMID:26860818

  16. Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells.

    PubMed

    Shaykhiev, Renat; Crystal, Ronald G

    2014-12-01

    The airway epithelium is the primary site of the earliest pathologic changes induced by smoking, contributing to the development of chronic obstructive pulmonary disease (COPD). The normal human airway epithelium is composed of several major cell types, including differentiated ciliated and secretory cells, intermediate undifferentiated cells, and basal cells (BC). BC contain the stem/progenitor cell population responsible for maintenance of the normally differentiated airway epithelium. Although inflammatory and immune processes play a significant role in the pathogenesis of COPD, the earliest lesions include hyperplasia of the BC population, suggesting that the disease may start with this cell type. Apart from BC hyperplasia, smoking induces a number of COPD-relevant airway epithelial remodeling phenotypes that are likely initiated in the BC population, including mucous cell hyperplasia, squamous cell metaplasia, epithelial-mesenchymal transition, altered ciliated and nonmucous secretory cell differentiation, and suppression of junctional barrier integrity. Significant progress has been recently made in understanding the biology of human airway BC, including gene expression features, stem/progenitor, and other functions, including interaction with other airway cell types. Accumulating evidence suggests that human airway BC function as both sensors and cellular sources of various cytokines and growth factors relevant to smoking-associated airway injury, as well as the origin of various molecular and histological phenotypes relevant to the pathogenesis of COPD. In the context of these considerations, we suggest that early BC-specific smoking-induced molecular changes are critical to the pathogenesis of COPD, and these represent a candidate target for novel therapeutic approaches to prevent COPD progression in susceptible individuals.

  17. Type II congenital pulmonary airway malformation in an esophageal lung

    PubMed Central

    Martínez-Martínez, Blanca Estela; Furuya, María Elena Yuriko; Martínez-Muñiz, Irma; Vargas, Mario H; Flores-Salgado, Rosalinda

    2013-01-01

    A seven-month-old girl, born prematurely (birth weight 1000 g) from a twin pregnancy, was admitted to hospital due to recurrent pneumonia and atelectasis. She experienced cough and respiratory distress during feeding. The right hemithorax was smaller than the left, with diminished breath sounds and dullness. Chest x-rays revealed decreased lung volume and multiple radiolucent images in the right lung, as well as overdistention of the left lung. An esophagogram revealed three bronchial branches arising from the lower one-third of the esophagus, corresponding to the right lung and ending in a cul-de-sac. A diagnosis of esophageal lung was established. On bronchography, the right lung was absent and the trachea only continued into the left main bronchus. Echocardiography and angiotomography revealed agenesis of the pulmonary artery right branch. The surgical finding was an esophageal right lung, which was removed; the histopathological diagnosis was type II congenital pulmonary airway malformation in an esophageal lung. PMID:23762890

  18. 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...

  19. REGULATION OF CYTOKINE PRODUCTION IN HUMAN ALVEOLAR MACHROPHAGES AND AIRWAY EPITHELIAL CELLS IN RESPONSE TO AMBIENT AIR POLLUTION PARTICLES: FURTHER MECHANISTIC STUDIES

    EPA Science Inventory

    In order to better understand how ambient air particulate matter (PM) affect lung health, the two main airway cell types likely to interact with inhaled particles, alveolar macrophages (AM) and airway epithelial cells have been exposed to particles in vitro and followed for endp...

  20. Platelet membranes induce airway smooth muscle cell proliferation.

    PubMed

    Svensson Holm, Ann-Charlotte B; Bengtsson, Torbjörn; Grenegård, Magnus; Lindström, Eva G

    2011-01-01

    The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localized in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS assay and the results confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GP-ASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants from lysed or filtrated thrombin stimulated platelets induced ASMC proliferation to the same extent as the membrane preparation. We have previously shown that platelet-induced proliferation is dependent on 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.

  1. Upregulation of a disintegrin and metalloproteinase-33 by VEGF in human airway smooth muscle cells: Implications for asthma

    PubMed Central

    Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Zheng, Hong; Zhao, Li-Hong; Kim, Sung-Ho

    2016-01-01

    ABSTRACT Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)–33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma. PMID:27579513

  2. Upregulation of a disintegrin and metalloproteinase-33 by VEGF in human airway smooth muscle cells: Implications for asthma.

    PubMed

    Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Zheng, Hong; Zhao, Li-Hong; Kim, Sung-Ho

    2016-10-17

    Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)-33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma. PMID:27579513

  3. Current Perspectives in Mesenchymal Stromal Cell Therapies for Airway Tissue Defects

    PubMed Central

    Petrella, Francesco; Rizzo, Stefania; Borri, Alessandro; Casiraghi, Monica; Spaggiari, Lorenzo

    2015-01-01

    Lung cancer is the leading cause of cancer death and respiratory diseases are the third cause of death in industrialized countries; for this reason the airways and cardiopulmonary system have been the focus of extensive investigation, in particular of the new emerging branch of regenerative medicine. Mesenchymal stromal cells (MSCs) are a population of undifferentiated multipotent adult cells that naturally reside within the human body, which can differentiate into osteogenic, chondrogenic, and adipogenic lineages when cultured in specific inducing media. MSCs have the ability to migrate and engraft at sites of inflammation and injury in response to cytokines, chemokines, and growth factors at a wound site and they can exert local reparative effects through transdifferentiation and differentiation into specific cell types or via the paracrine secretion of soluble factors with anti-inflammatory and wound-healing activities. Experimental and clinical evidence exists regarding MSCs efficacy in airway defects restoration; although clinical MSCs use, in the daily practice, is not yet completely reached for airway diseases, we can argue that MSCs do not represent any more merely an experimental approach to airway tissue defects restoration but they can be considered as a “salvage” therapeutic tool in very selected patients and diseases. PMID:26167186

  4. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    SciTech Connect

    Doupnik, C.A.; Leikauf, G.D. )

    1990-10-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with (3H)arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. (3H)arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein.

  5. 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

  6. 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

  7. Junctional abnormalities in human airway epithelial cells expressing F508del CFTR

    PubMed Central

    Stauffer, Brandon; Moriarty, Hannah K.; Kim, Agnes H.; McCarty, Nael A.; Koval, Michael

    2015-01-01

    Cystic fibrosis (CF) has a profound impact on airway physiology. Accumulating evidence suggests that intercellular junctions are impaired in CF. We examined changes to CF transmembrane conductance regulator (CFTR) function, tight junctions, and gap junctions in NuLi-1 (CFTRwt/wt) and CuFi-5 (CFTRΔF508/ΔF508) cells. Cells were studied at air-liquid interface (ALI) and compared with primary human bronchial epithelial cells. On the basis of fluorescent lectin binding, the phenotype of the NuLi-1 and CuFi-5 cells at week 8 resembled that of serous, glycoprotein-rich airway cells. After week 7, CuFi-5 cells possessed 130% of the epithelial Na+ channel activity and 17% of the CFTR activity of NuLi-1 cells. In both cell types, expression levels of CFTR were comparable to those in primary airway epithelia. Transepithelial resistance of NuLi-1 and CuFi-5 cells stabilized during maturation in ALI culture, with significantly lower transepithelial resistance for CuFi-5 than NuLi-1 cells. We also found that F508del CFTR negatively affects gap junction function in the airway. NuLi-1 and CuFi-5 cells express the connexins Cx43 and Cx26. While both connexins were properly trafficked by NuLi-1 cells, Cx43 was mistrafficked by CuFi-5 cells. Cx43 trafficking was rescued in CuFi-5 cells treated with 4-phenylbutyric acid (4-PBA), as assessed by intracellular dye transfer. 4-PBA-treated CuFi-5 cells also exhibited an increase in forskolin-induced CFTR-mediated currents. The Cx43 trafficking defect was confirmed using IB3-1 cells and found to be corrected by 4-PBA treatment. These data support the use of NuLi-1 and CuFi-5 cells to examine the effects of F508del CFTR expression on tight junction and gap junction function in the context of serous human airway cells. PMID:26115671

  8. IL1RL1 asthma risk variants regulate airway type 2 inflammation

    PubMed Central

    Gordon, Erin D.; Palandra, Joe; Wesolowska-Andersen, Agata; Ringel, Lando; Rios, Cydney L.; Lachowicz-Scroggins, Marrah E.; Sharp, Louis Z.; Everman, Jamie L.; MacLeod, Hannah J.; Lee, Jae W.; Mason, Robert J.; Matthay, Michael A.; Sheldon, Richard T.; Peters, Michael C.; Nocka, Karl H.; Fahy, John V.; Seibold, Max A.

    2016-01-01

    Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 — rs1420101 and rs11685480 — are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33–driven type 2 inflammation. PMID:27699235

  9. IL1RL1 asthma risk variants regulate airway type 2 inflammation

    PubMed Central

    Gordon, Erin D.; Palandra, Joe; Wesolowska-Andersen, Agata; Ringel, Lando; Rios, Cydney L.; Lachowicz-Scroggins, Marrah E.; Sharp, Louis Z.; Everman, Jamie L.; MacLeod, Hannah J.; Lee, Jae W.; Mason, Robert J.; Matthay, Michael A.; Sheldon, Richard T.; Peters, Michael C.; Nocka, Karl H.; Fahy, John V.; Seibold, Max A.

    2016-01-01

    Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 — rs1420101 and rs11685480 — are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33–driven type 2 inflammation.

  10. Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation.

    PubMed

    Daniels, N J; Hyde, E; Ghosh, S; Seo, K; Price, K M; Hoshino, K; Kaisho, T; Okada, T; Ronchese, F

    2016-01-01

    Allergic airway inflammation is driven by the recognition of inhaled allergen by T helper type 2 (Th2) cells in the airway and lung. Allergen-specific cytotoxic T lymphocytes (CTLs) can strongly reduce airway inflammation, however, the mechanism of their inhibitory activity is not fully defined. We used mouse models to show that allergen-specific CTLs reduced early cytokine production by Th2 cells in lung, and their subsequent accumulation and production of interleukin (IL)-4 and IL-13. In addition, treatment with specific CTLs also increased the proportion of caspase(+) dendritic cells (DCs) in mediastinal lymph node (MLN), and decreased the numbers of CD103(+) and CD11b(+) DCs in the lung. This decrease required expression of the cytotoxic mediator perforin in CTLs and of the appropriate MHC-antigen ligand on DCs, suggesting that direct CTL-DC contact was necessary. Lastly, lung imaging experiments revealed that in airway-challenged mice XCR1-GFP(+) DCs, corresponding to the CD103(+) DC subset, and XCR1-GFP(-) CD11c(+) cells, which include CD11b(+) DCs and alveolar macrophages, both clustered in the areas surrounding the small airways and were closely associated with allergen-specific CTLs. Thus, allergen-specific CTLs reduce allergic airway inflammation by depleting CD103(+) and CD11b(+) DC populations in the lung, and may constitute a mechanism through which allergic immune responses are regulated.

  11. miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production.

    PubMed

    Oglesby, Irene K; Vencken, Sebastian F; Agrawal, Raman; Gaughan, Kevin; Molloy, Kevin; Higgins, Gerard; McNally, Paul; McElvaney, Noel G; Mall, Marcus A; Greene, Catherine M

    2015-11-01

    Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.

  12. 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

  13. Phosphodiesterase 4B is essential for TH2-cell function and development of airway hyperresponsiveness in allergic asthma

    PubMed Central

    Catherine Jin, S.-L.; Goya, Sho; Nakae, Susumu; Wang, Dan; Bruss, Matthew; Hou, Chiaoyin; Umetsu, Dale; Conti, Marco

    2010-01-01

    Background Cyclic AMP (cAMP) signaling modulates functions of inflammatory cells involved in the pathogenesis of asthma, and type 4 cAMP-specific phosphodiesterases (PDE4s) are essential components of this pathway. Induction of the PDE4 isoform PDE4B is necessary for Toll-like receptor signaling in monocytes and macrophages and is associated with T cell receptor/CD3 in T cells; however, its exact physiological function in the development of allergic asthma remains undefined. Objectives We investigated the role of PDE4B in the development of allergen-induced airway hyperresponsiveness (AHR) and TH2-driven inflammatory responses. Methods Wild-type and PDE4B−/− mice were sensitized and challenged with ovalbumin and AHR measured in response to inhaled methacholine. Airway inflammation was characterized by analyzing leukocyte infiltration and cytokine accumulation in the airways. Ovalbumin-stimulated cell proliferation and TH2 cytokine production were determined in cultured bronchial lymph node cells. Results Mice deficient in PDE4B do not develop AHR. This protective effect was associated with a significant decrease in eosinophils recruitment to the lungs and decreased TH2 cytokine levels in the bronchoalveolar lavage fluid. Defects in T-cell replication, TH2 cytokine production, and dendritic cell migration were evident in cells from the airway-draining lymph nodes. Conversely, accumulation of the TH1 cytokine IFN-γ was not affected in PDE4B−/− mice. Ablation of the orthologous PDE4 gene PDE4A has no impact on airway inflammation. Conclusion By relieving a cAMP-negative constraint, PDE4B plays an essential role in TH2-cell activation and dendritic cell recruitment during airway inflammation. These findings provide proof of concept that PDE4 inhibitors with PDE4B selectivity may have efficacy in asthma treatment. PMID:21047676

  14. Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol

    PubMed Central

    Erle, David J.

    2016-01-01

    Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

  15. Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol.

    PubMed

    Khosravi, Ali Reza; Erle, David J

    2016-01-01

    Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

  16. DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

    PubMed Central

    Habibovic, Aida; Hristova, Milena; Heppner, David E.; Danyal, Karamatullah; Ather, Jennifer L.; Janssen-Heininger, Yvonne M.W.; Irvin, Charles G.; Poynter, Matthew E.; Lundblad, Lennart K.; Dixon, Anne E.; Geiszt, Miklos

    2016-01-01

    Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management. PMID:27812543

  17. Transcriptional regulation of cytokine function in airway smooth muscle cells

    PubMed Central

    Clarke, Deborah; Damera, Gautam; Sukkar, Maria B.; Tliba, Omar

    2009-01-01

    The immuno-modulatory properties of airway smooth muscle have become of increasing importance in our understanding of the mechanisms underlying chronic inflammation and structural remodeling of the airway wall in asthma and chronic obstructive pulmonary disease (COPD). ASM cells respond to many cytokines, growth factors and lipid mediators to produce a wide array of immuno-modulatory molecules which may in turn orchestrate and perpetuate the disease process in asthma and COPD. Despite numerous studies of the cellular effects of cytokines on cultured ASM, few have identified intracellular signaling pathways by which cytokines modulate or induce these cellular responses. In this review we provide an overview of the transcriptional mechanisms as well as intracellular signaling pathways regulating cytokine functions in ASM cells. The recent discovery of toll-like receptors in ASM cells represents a significant development in our understanding of the immuno-modulatory capabilities of ASM cells. Thus, we also review emerging evidence of the inflammatory response to toll-like receptor activation in ASM cells. PMID:19393330

  18. Surgical Airway

    PubMed Central

    Patel, Sapna A; Meyer, Tanya K

    2014-01-01

    Close to 3% of all intubation attempts are considered difficult airways, for which a plan for a surgical airway should be considered. Our article provides an overview of the different types of surgical airways. This article provides a comprehensive review of the main types of surgical airways, relevant anatomy, necessary equipment, indications and contraindications, preparation and positioning, technique, complications, and tips for management. It is important to remember that the placement of a surgical airway is a lifesaving procedure and should be considered in any setting when one “cannot intubate, cannot ventilate”. PMID:24741501

  19. Stomatin immunoreactivity in ciliated cells of the human airway epithelium.

    PubMed

    Fricke, Britta; Stewart, Gordon W; Treharne, Kathryn J; Mehta, Anil; Knöpfle, Gisela; Friedrichs, Nicolaus; Müller, Klaus-Michael; von Düring, Monika

    2003-07-01

    Stomatin is a widely distributed 32kD membrane protein of unknown function. In biochemical studies it is associated with cholesterol+sphingomyelin-rich 'rafts' in the cytomembrane. Genetic studies in C. elegans, supported by microscopic studies in mammalian tissue and co-expression studies in oocytes, suggest a functional link with the DEG/ENaC (degenerin/epithelial Na+ channel) superfamily of monovalent ion channels. Since ENaC channels play a prominent role in the physiology of the respiratory epithelium, we have studied the immunolocalization of stomatin in mature and developing human airway epithelium by means of Western blot analysis, immunocytochemistry, and immunoelectron microscopy. Stomatin immunoreactivity (stomatin-IR) was found in the ciliated cells of the conductive airway epithelium in a distinct distribution pattern with the strongest signal along the cilia. Immunogold labelling revealed immunogold particles at the basal bodies, along the cilia, and at the membrane of the microvilli. The presence of stomatin-IR paralleled the stages of ciliogenesis in airway development, and its appearance preceded the elongation of the axoneme and the cilial outgrowth. Due to its presence in the different cellular locations in the ciliated cell, we suggest that stomatin is involved in various cellular functions. From its ultrastructural position, stomatin could be a candidate for a membrane-associated mechanotransducer with a role in the control of ciliary motility. Stomatin as a raft protein might be a microtubule associated protein moving along the outer surface of the microtubules to its terminal site of action in the cilia. Stomatin-IR in microvilli supports the hypothesis of a co-localization with beta- and gamma- ENaC and, in conclusion, their potential functional interaction to control the composition of periciliary mucus electrolytes. PMID:12759749

  20. Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

    SciTech Connect

    Tapparel, Caroline; Sobo, Komla; Constant, Samuel; Huang, Song; Van Belle, Sandra; Kaiser, Laurent

    2013-11-15

    New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent. - Highlights: • A 3D human upper airway epithelia reconstituted in vitro supports HRV-C growth. • HRV-Cs enter and exit preferentially at the apical side of this ALI culture system. • HRV-Cs are acid sensitive. • Temperature sensitivity may be type-dependent for HRV-Cs.

  1. 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.

  2. 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

  3. LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.

    PubMed

    Luz, Simão; Cihil, Kristine M; Brautigan, David L; Amaral, Margarida D; Farinha, Carlos M; Swiatecka-Urban, Agnieszka

    2014-05-23

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.

  4. 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

  5. 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

  6. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

    PubMed

    Gomperts, Brigitte N; Belperio, John A; Rao, P Nagesh; Randell, Scott H; Fishbein, Michael C; Burdick, Marie D; Strieter, Robert M

    2006-02-01

    Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases. PMID:16424223

  7. Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells.

    PubMed

    Foxman, Ellen F; Storer, James A; Fitzgerald, Megan E; Wasik, Bethany R; Hou, Lin; Zhao, Hongyu; Turner, Paul E; Pyle, Anna Marie; Iwasaki, Akiko

    2015-01-20

    Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33-35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I-like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

  8. Cell Surface Human Airway Trypsin-Like Protease Is Lost During Squamous Cell Carcinogenesis.

    PubMed

    Duhaime, Michael J; Page, Khaliph O; Varela, Fausto A; Murray, Andrew S; Silverman, Michael E; Zoratti, Gina L; List, Karin

    2016-07-01

    Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro-cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin-like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non-proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas.

  9. 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...

  10. Variations of chromosomes 2 and 3 gene expression profiles among pulmonary telocytes, pneumocytes, airway cells, mesenchymal stem cells and lymphocytes

    PubMed Central

    Zheng, Minghuan; Sun, Xiaoru; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Sanda M; Chen, Chengshui; Chen, Luonan; Cretoiu, Dragos; Popescu, Laurentiu M; Fang, Hao; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) were identified as a distinct cellular type of the interstitial tissue and defined as cells with extremely long telopodes (Tps). Our previous data demonstrated patterns of mouse TC-specific gene profiles on chromosome 1. The present study focuses on the identification of characters and patterns of TC-specific or TC-dominated gene expression profiles in chromosome 2 and 3, the network of principle genes and potential functional association. We compared gene expression profiles of pulmonary TCs, mesenchymal stem cells, fibroblasts, alveolar type II cells, airway basal cells, proximal airway cells, CD8+T cells from bronchial lymph nodes (T-BL), and CD8+ T cells from lungs (T-LL). We identified that 26 or 80 genes of TCs in chromosome 2 and 13 or 59 genes of TCs up-or down-regulated in chromosome 3, as compared with other cells respectively. Obvious overexpression of Myl9 in chromosome 2 of TCs different from other cells, indicates that biological functions of TCs are mainly associated with tissue/organ injury and ageing, while down-expression of Pltp implies that TCs may be associated with inhibition or reduction of inflammation in the lung. Dominant overexpression of Sh3glb1, Tm4sf1 or Csf1 in chromosome 3 of TCs is mainly associated with tumour promotion in lung cancer, while most down-expression of Pde5 may be involved in the development of pulmonary fibrosis and other acute and chronic interstitial lung disease. PMID:25278030

  11. 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

  12. Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

    PubMed Central

    Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-01-01

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

  13. Mast cell-cholinergic nerve interaction in mouse airways.

    PubMed

    Weigand, Letitia A; Myers, Allen C; Meeker, Sonya; Undem, Bradley J

    2009-07-01

    We addressed the mechanism by which antigen contracts trachea isolated from actively sensitized mice. Trachea were isolated from mice (C57BL/6J) that had been actively sensitized to ovalbumin (OVA). OVA (10 microg ml(-1)) caused histamine release (approximately total tissue content), and smooth muscle contraction that was rapid in onset and short-lived (t(1/2) < 1 min), reaching approximately 25% of the maximum tissue response. OVA contraction was mimicked by 5-HT, and responses to both OVA and 5-HT were sensitive to 10 microm-ketanserin (5-HT(2) receptor antagonist) and strongly inhibited by atropine (1microm). Epithelial denudation had no effect on the OVA-induced contraction. Histological assessment revealed about five mast cells/tracheal section the vast majority of which contained 5-HT. There were virtually no mast cells in the mast cell-deficient (sash -/-) mouse trachea. OVA failed to elicit histamine release or contractile responses in trachea isolated from sensitized mast cell-deficient (sash -/-) mice. Intracellular recordings of the membrane potential of parasympathetic neurons in mouse tracheal ganglia revealed a ketanserin-sensitive 5-HT-induced depolarization and similar depolarization in response to OVA challenge. These data support the hypothesis that antigen-induced contraction of mouse trachea is epithelium-independent, and requires mast cell-derived 5-HT to activate 5-HT(2) receptors on parasympathetic cholinergic neurons. This leads to acetylcholine release from nerve terminals, and airway smooth muscle contraction. PMID:19403609

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

    PubMed

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

    2015-02-01

    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.

  15. Clusterin Modulates Allergic Airway Inflammation by Attenuating CCL20-Mediated Dendritic Cell Recruitment.

    PubMed

    Hong, Gyong Hwa; Kwon, Hyouk-Soo; Moon, Keun-Ai; Park, So Young; Park, Sunjoo; Lee, Kyoung Young; Ha, Eun Hee; Kim, Tae-Bum; Moon, Hee-Bom; Lee, Heung Kyu; Cho, You Sook

    2016-03-01

    Recruitment and activation of dendritic cells (DCs) in the lungs are critical for Th2 responses in asthma, and CCL20 secreted from bronchial epithelial cells (BECs) is known to influence the recruitment of DCs. Because asthma is a disease that is closely associated with oxidative stress, we hypothesized that clusterin, an oxidative stress regulatory molecule, may have a role in the development of allergic airway inflammation. The aim of this study was to examine whether clusterin regulates CCL20 production from the BECs and the subsequent DC recruitment in the lungs. To verify the idea, clusterin knockout (Clu(-/-)), clusterin heterogeneous (Clu(+/-)), and wild-type mice were exposed intranasally to house dust mite (HDM) extract to induce allergic airway inflammation. We found that the total number of immune cells in bronchoalveolar lavage fluid and the lung was increased in Clu(-/-) and Clu(+/-) mice. Of these immune cells, inflammatory DCs (CD11b(+)CD11c(+)) and Ly6C(high) monocyte populations in the lung were significantly increased, which was accompanied by increased levels of various chemokines, including CCL20 in bronchoalveolar lavage fluid, and increased oxidative stress markers in the lung. Moreover, HDM-stimulated human BECs with either up- or downregulated clusterin expression showed that CCL20 secretion was negatively associated with clusterin expression. Interestingly, clusterin also reduced the level of intracellular reactive oxygen species, which is related to induction of CCL20 expression after HDM stimulation. Thus, the antioxidant property of clusterin is suggested to regulate the expression of CCL20 in BECs and the subsequent recruitment of inflammatory DCs in the airway. PMID:26826245

  16. Clusterin Modulates Allergic Airway Inflammation by Attenuating CCL20-Mediated Dendritic Cell Recruitment.

    PubMed

    Hong, Gyong Hwa; Kwon, Hyouk-Soo; Moon, Keun-Ai; Park, So Young; Park, Sunjoo; Lee, Kyoung Young; Ha, Eun Hee; Kim, Tae-Bum; Moon, Hee-Bom; Lee, Heung Kyu; Cho, You Sook

    2016-03-01

    Recruitment and activation of dendritic cells (DCs) in the lungs are critical for Th2 responses in asthma, and CCL20 secreted from bronchial epithelial cells (BECs) is known to influence the recruitment of DCs. Because asthma is a disease that is closely associated with oxidative stress, we hypothesized that clusterin, an oxidative stress regulatory molecule, may have a role in the development of allergic airway inflammation. The aim of this study was to examine whether clusterin regulates CCL20 production from the BECs and the subsequent DC recruitment in the lungs. To verify the idea, clusterin knockout (Clu(-/-)), clusterin heterogeneous (Clu(+/-)), and wild-type mice were exposed intranasally to house dust mite (HDM) extract to induce allergic airway inflammation. We found that the total number of immune cells in bronchoalveolar lavage fluid and the lung was increased in Clu(-/-) and Clu(+/-) mice. Of these immune cells, inflammatory DCs (CD11b(+)CD11c(+)) and Ly6C(high) monocyte populations in the lung were significantly increased, which was accompanied by increased levels of various chemokines, including CCL20 in bronchoalveolar lavage fluid, and increased oxidative stress markers in the lung. Moreover, HDM-stimulated human BECs with either up- or downregulated clusterin expression showed that CCL20 secretion was negatively associated with clusterin expression. Interestingly, clusterin also reduced the level of intracellular reactive oxygen species, which is related to induction of CCL20 expression after HDM stimulation. Thus, the antioxidant property of clusterin is suggested to regulate the expression of CCL20 in BECs and the subsequent recruitment of inflammatory DCs in the airway.

  17. 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

  18. Airway basal cells. The "smoking gun" of chronic obstructive pulmonary disease.

    PubMed

    Crystal, Ronald G

    2014-12-15

    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).

  19. MyD88 in lung resident cells governs airway inflammatory and pulmonary function responses to organic dust treatment.

    PubMed

    Poole, Jill A; Wyatt, Todd A; Romberger, Debra J; Staab, Elizabeth; Simet, Samantha; Reynolds, Stephen J; Sisson, Joseph H; Kielian, Tammy

    2015-01-01

    Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement. PMID:26376975

  20. 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...

  1. Basophils as a primary inducer of the T helper type 2 immunity in ovalbumin-induced allergic airway inflammation

    PubMed Central

    Zhong, Wenwei; Su, Wen; Zhang, Yanjie; Liu, Qi; Wu, Jinhong; Di, Caixia; Zhang, Zili; Xia, Zhenwei

    2014-01-01

    Antigen-induced allergic airway inflammation is mediated by T helper type 2 (Th2) cells and their cytokines, but the mechanism that initiates the Th2 immunity is not fully understood. Recent studies show that basophils play important roles in initiating Th2 immunity in some inflammatory models. Here we explored the role of basophils in ovalbumin (OVA) -induced airway allergic inflammation in BALB/c mice. We found that OVA sensitization and challenge resulted in a significant increase in the amount of basophils in blood and lung, along with the up-regulation of activation marker of CD200R. However, depletion of basophils with MAR-1 or Ba103 antibody attenuated airway inflammation, represented by the significantly decreased amount of the Th2 subset in spleen and draining lymph nodes, interlukin-4 level in lung and OVA-special immunoglobulin E (sIgE) levels in serum. On the other hand, adoptive transfer of basophils from OVA-challenged lung tissue to naive BALB/c mice provoked the Th2 immune response. In addition, pulmonary basophils from OVA-challenged mice were able to uptake DQ-OVA and express MHC class II molecules and CD40 in vivo, as well as to release interleukin-4 following stimulation by IgE–antigen complexes and promote Th2 polarization in vitro. These findings demonstrate that basophils may participate in Th2 immune responses in antigen-induced allergic airway inflammation and that they do so through facilitating antigen presentation and providing interleukin-4. PMID:24383680

  2. 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

  3. Human influenza is more effective than avian influenza at antiviral suppression in airway cells.

    PubMed

    Hsu, Alan Chen-Yu; Barr, Ian; Hansbro, Philip M; Wark, Peter A

    2011-06-01

    Airway epithelial cells are the initial site of infection with influenza viruses. The innate immune responses of airway epithelial cells to infection are important in limiting virus replication and spread. However, relatively little is known about the importance of this innate antiviral response to infection. Avian influenza viruses are a potential source of future pandemics; therefore, it is critical to examine the effectiveness of the host antiviral system to different influenza viruses. We used a human influenza (H3N2) and a low-pathogenic avian influenza (H11N9) to assess and compare the antiviral responses of Calu-3 cells. After infection, H3N2 replicated more effectively than the H11N9 in Calu-3 cells. This was not due to differential expression of sialic acid residues on Calu-3 cells, but was attributed to the interference of host antiviral responses by H3N2. H3N2 induced a delayed antiviral signaling and impaired type I and type III IFN induction compared with the H11N9. The gene encoding for nonstructural (NS) 1 protein was transfected into the bronchial epithelial cells (BECs), and the H3N2 NS1 induced a greater inhibition of antiviral responses compared with the H11N9 NS1. Although the low-pathogenic avian influenza virus was capable of infecting BECs, the human influenza virus replicated more effectively than avian influenza virus in BECs, and this was due to a differential ability of the two NS1 proteins to inhibit antiviral responses. This suggests that the subversion of human antiviral responses may be an important requirement for influenza viruses to adapt to the human host and cause disease.

  4. Pluripotent Allospecific CD8+ Effector T Cells Traffic to Lung in Murine Obliterative Airway Disease

    PubMed Central

    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-γ+ T cells were detected in airway allografts, with significant coexpression of TNF-α and granzyme B. Therefore, using IFN-γ as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-γ 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. PMID:16195540

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

    PubMed

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

    2012-01-01

    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-κB 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.

  6. 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

  7. NADPH oxidase-dependent acid production in airway epithelial cells.

    PubMed

    Schwarzer, Christian; Machen, Terry E; Illek, Beate; Fischer, Horst

    2004-08-27

    The purpose of this study was to determine the role of NADPH oxidase in H(+) secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zinc-blockable plasma membrane H(+) conductance. However, the rate of H(+) secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H(+) secretion was blocked by mucosal ZnCl(2) and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22(phox)) and found Duox1, -2, and p22(phox) to be highly expressed in hTE, as well as the intracellular subunits p40(phox), p47(phox), and p67(phox). In contrast, Calu-3 lacked transcripts for Duox1, p40(phox), and p47(phox). Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na(+)/H(+) exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H(+) production and H(+) secretion, but not in H(+) conduction.

  8. 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

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

    PubMed

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

    2016-01-12

    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

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

    PubMed

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

    2016-01-12

    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.

  11. p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration.

    PubMed

    Zuo, Wei; Zhang, Ting; Wu, Daniel Zheng'An; Guan, Shou Ping; Liew, Audrey-Ann; Yamamoto, Yusuke; Wang, Xia; Lim, Siew Joo; Vincent, Matthew; Lessard, Mark; Crum, Christopher P; Xian, Wa; McKeon, Frank

    2015-01-29

    Lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis involve the progressive and inexorable destruction of oxygen exchange surfaces and airways, and have emerged as a leading cause of death worldwide. Mitigating therapies, aside from impractical organ transplantation, remain limited and the possibility of regenerative medicine has lacked empirical support. However, it is clinically known that patients who survive sudden, massive loss of lung tissue from necrotizing pneumonia or acute respiratory distress syndrome often recover full pulmonary function within six months. Correspondingly, we recently demonstrated lung regeneration in mice following H1N1 influenza virus infection, and linked distal airway stem cells expressing Trp63 (p63) and keratin 5, called DASC(p63/Krt5), to this process. Here we show that pre-existing, intrinsically committed DASC(p63/Krt5) undergo a proliferative expansion in response to influenza-induced lung damage, and assemble into nascent alveoli at sites of interstitial lung inflammation. We also show that the selective ablation of DASC(p63/Krt5) in vivo prevents this regeneration, leading to pre-fibrotic lesions and deficient oxygen exchange. Finally, we demonstrate that single DASC(p63/Krt5)-derived pedigrees differentiate to type I and type II pneumocytes as well as bronchiolar secretory cells following transplantation to infected lung and also minimize the structural consequences of endogenous stem cell loss on this process. The ability to propagate these cells in culture while maintaining their intrinsic lineage commitment suggests their potential in stem cell-based therapies for acute and chronic lung diseases. PMID:25383540

  12. SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion

    PubMed Central

    Ren, Binhui; Azzegagh, Zoulikha; Jaramillo, Ana M.; Zhu, Yunxiang; Pardo-Saganta, Ana; Bagirzadeh, Rustam; Flores, Jose R.; Han, Wei; Tang, Yong-jun; Tu, Jing; Alanis, Denise M.; Evans, Christopher M.; Guindani, Michele; Roche, Paul A.; Rajagopal, Jayaraj; Chen, Jichao; Davis, C. William; Tuvim, Michael J.; Dickey, Burton F.

    2015-01-01

    Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated. PMID:26182382

  13. Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle.

    PubMed

    Tate, R J; Tolan, D; Pyne, S

    1999-07-01

    Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.

  14. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  15. RACK1, a PKC Targeting Protein, Is Exclusively Localized to Basal Airway Epithelial Cells

    PubMed Central

    Slager, Rebecca E.; DeVasure, Jane M.; Pavlik, Jaqueline A.; Sisson, Joseph H.; Wyatt, Todd A.

    2008-01-01

    The novel isoform of protein kinase C (PKC), PKCɛ, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCɛ signaling in the airways and the potential role of the PKCɛ-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, β-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCɛ signaling in the basal airway cell may involve RACK1; however, PKCɛ regulation in ciliated cells uses RACK1-independent pathways. (J Histochem Cytochem 56:7–14, 2008) PMID:17875659

  16. Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro.

    PubMed

    Tapparel, Caroline; Sobo, Komla; Constant, Samuel; Huang, Song; Van Belle, Sandra; Kaiser, Laurent

    2013-11-01

    New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent.

  17. 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. PMID:25204635

  18. HIF-1 expression is associated with CCL2 chemokine expression in airway inflammatory cells: implications in allergic airway inflammation

    PubMed Central

    2012-01-01

    Background The pathogenesis of allergic airway inflammation in asthmatic patients is complex and characterized by cellular infiltrates and activity of many cytokines and chemokines. Both the transcription factor hypoxia inducible factor-1 (HIF-1) and chemokine CCL2 have been shown to play pivotal roles in allergic airway inflammation. The interrelationship between these two factors is not known. We hypothesized that the expression of HIF-1 and CCL2 may be correlated and that the expression of CCL2 may be under the regulation of HIF-1. Several lines of evidence are presented to support this hypothesis. Methods The effects of treating wild-type OVA (ovalbumin)-sensitized/challenged mice with ethyl-3,4-dihydroxybenzoate (EDHB), which upregulate HIF, on CCL2 expression, were determined. Mice conditionally knocked out for HIF-1β was examined for their ability to mount an allergic inflammatory response and CCL2 expression in the lung after intratracheal exposure to ovalbumin. The association of HIF-1α and CCL2 levels was also measured in endobronchial biopsies and bronchial fluid of asthma patients after challenge. Results We show that both HIF-1α and CCL2 were upregulated during an OVA (ovalbumin)-induced allergic response in mice. The levels of HIF-1α and CCL2 were significantly increased following treatment with a pharmacological agent which upregulates HIF-1α, ethyl-3,4-dihydroxybenzoate (EDHB). In contrast, the expression levels of HIF-1α and CCL2 were decreased in the lungs of mice that have been conditionally knocked out for ARNT (HIF-1β) following sensitization with OVA when compared to levels in wild type mice. In asthma patients, the levels of HIF-1α and CCL2 increased after challenge with the allergen. Conclusions These data suggest that CCL2 expression is regulated, in part, by HIF-1 in the lung. These findings also demonstrate that both CCL2 and HIF-1 are implicated in the pathogenesis of allergic airway inflammation. PMID:22823210

  19. 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

  20. Cells and Culture Systems Used to Model the Small Airway Epithelium.

    PubMed

    Bhowmick, Rudra; Gappa-Fahlenkamp, Heather

    2016-06-01

    The pulmonary epithelium is divided into upper, lower, and alveolar (or small) airway epithelia and acts as the mechanical and immunological barrier between the external environment and the underlying submucosa. Of these, the small airway epithelium is the principal area of gas exchange and has high immunological activity, making it a major area of cell biology, immunology, and pharmaceutical research. As animal models do not faithfully represent the human pulmonary system and ex vivo human lung samples have reliability and availability issues, cell lines, and primary cells are widely used as small airway epithelial models. In vitro, these cells are mostly cultured as monolayers (2-dimensional cultures), either media submerged or at air-liquid interface. However, these 2-dimensional cultures lack a three dimension-a scaffolding extracellular matrix, which establishes the intercellular network in the in vivo airway epithelium. Therefore, 3-dimensional cell culture is currently a major area of development, where cells are cultured in a matrix or are cultured in a manner that they develop ECM-like scaffolds between them, thus mimicking the in vivo phenotype more faithfully. This review focuses on the commonly used small airway epithelial cells, their 2-dimensional and 3-dimensional culture techniques, and their comparative phenotype when cultured under these systems. PMID:27071933

  1. The deubiquitinating enzyme USP10 regulates the endocytic recycling of CFTR in airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Barnaby, Roxanna L; Stanton, Bruce A

    2010-01-01

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cyclic AMP-regulated chloride channel that plays an important role in regulating the volume of the lung airway surface liquid, and thereby mucociliary clearance and elimination of pathogens from the lung. In epithelial cells, cell surface CFTR abundance is determined in part by regulating both CFTR endocytosis from the apical plasma membrane and recycling back to the plasma membrane. We recently reported, using an activity-based chemical screen to identify active deubiquitinating enzymes (DUBs) in human airway epithelial cells, that Ubiquitin Specific Protease-10 (USP10) is located and active in the early endosomal compartment and regulates the deubiquitination of CFTR and thereby promotes its endocytic recycling. siRNA-mediated knockdown of USP10 increased the multi-ubiquitination and lysosomal degradation of CFTR and decreased the endocytic recycling and the half-life of CFTR in the apical membrane, as well as CFTR-mediated chloride secretion. Overexpression of wild-type USP10 reduced CFTR multi-ubiquitination and degradation, while overexpression of a dominant-negative USP10 promoted increased multi-ubiquitination and lysosomal degradation of CFTR. In the current study, we show localization and activity of USP10 in the early endosomal compartment of primary bronchial epithelial cells, as well as an interaction between CFTR and USP10 in this compartment. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes, thereby enhancing the endocytic recycling and cell surface expression of CFTR.

  2. 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.

  3. 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.

  4. 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

  5. CD8+IL-17+ T Cells Mediate Neutrophilic Airway Obliteration in T-bet–Deficient Mouse Lung Allograft Recipients

    PubMed Central

    Dodd-o, Jeffrey M.; Coon, Tiffany A.; Miller, Hannah L.; Ganguly, Sudipto; Popescu, Iulia; O'Donnell, Christopher P.; Cardenes, Nayra; Levine, Melanie; Rojas, Mauricio; Weathington, Nathaniel M.; Zhao, Jing; Zhao, Yutong; McDyer, John F.

    2015-01-01

    Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet−/− recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet−/− recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8+ T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ–dominant responses in WT mice. CD4+ T cells produced IL-17 but not IFN-γ responses in T-bet−/− recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8+IFN-γ+ responses in both T-bet−/− and WT mice but had no attenuating effect on lung rejection pathology in T-bet−/− recipients or on the development of obliterative airway inflammation that occurred only in T-bet−/− recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade–resistant rejection pathology and airway inflammation in T-bet−/− recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet−/− allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet–deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade–resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8+IL-17+ T cells. Our data support T-bet–deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation

  6. Human Airway Epithelial Cell Responses to Single Walled Carbon Nanotube Exposure: Nanorope-Residual Body Formation

    SciTech Connect

    Panessa-Warren, Barbara J.; Warren, John B.; Kisslinger, Kim; Crosson, Kenya; Maye, Mathew M.

    2012-11-01

    This investigation examines the 'first contact responses' of in vitro human epithelial airway cells exposed to unrefined single walled carbon nanotubes (SWCNTs) [containing metal catalyst, carbon black, amorphous carbon, graphitic shells, and SWCNTs], and refined acid/peroxide cleaned and cut SWCNTs at low and high dose exposures (0.16 ug/L and 1.60 ug/L) for 2, 3 and 3.5 hours. FTIR, X-ray compositional analysis, morphological TEM analysis and UV-Vis were used to physicochemically characterize the SWCNTs in this study. Following SWCNT exposure to human lung NCI-H292 epithelial monolayers, the airway cells were prepared for light microscopy vital staining, or fixed in glutaraldehyde for SEM/TEM imaging to determine SWCNT binding, uptake, intracellular processing and organellar/SWCNT fate within the exposure period. At 2 hr exposures to both unrefined Carbolex, and refined SWCNTs (at both high and low doses), there were no increases in lung cell necrosis compared to controls. However high dose, 3 hr exposures to unrefined Carbolex material produced severe cell damage (apical and basal plasma membrane holes, decreased mitochondria, numerous intracellular vesicles containing nanomaterial and membrane fragments) and increased cell necrosis. The refined SWCNTs exposed for 3 hr at low dose produced no increase in cell death, although high dose exposure produced significant cell death. By TEM, Acid/peroxide cleaned SWCNT 3 hr exposures at high and low doses, revealed SWCNTs attachment to cell surface mucin, and SWCNT uptake into the cells during membrane recycling. Membranes and SWCNTs were seen within cytoplasmic lamellar body-type vesicles, where vesicular contents were bio-degraded, eventually forming long SWCNT-nanoropes, which were subsequently released into the cytoplasm as clusters of attached nanoropes, as the vesicle membranes fragmented. These Nanorope-Residual Bodies did not cause damage to the surrounding organelles or cytoplasm, and seemed very stabile in the

  7. 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.

  8. Apoptotic Cells Activate NKT Cells through T Cell Ig-Like Mucin-Like–1 Resulting in Airway Hyperreactivity

    PubMed Central

    Lee, Hyun-Hee; Meyer, Everett H.; Goya, Sho; Pichavant, Muriel; Kim, Hye Young; Bu, Xia; Umetsu, Sarah E.; Jones, Jennifer C.; Savage, Paul B.; Iwakura, Yoichiro; Casasnovas, Jose M.; Kaplan, Gerardo; Freeman, Gordon J.; DeKruyff, Rosemarie H.; Umetsu, Dale T.

    2011-01-01

    T cell Ig-like mucin-like–1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1–dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma. PMID:20889552

  9. 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.

  10. Selective regulation of MAP kinases and Chemokine expression after ligation of ICAM-1 on human airway epithelial cells

    PubMed Central

    Krunkosky, Thomas M; Jarrett, Carla L

    2006-01-01

    Background Intercellular adhesion molecule 1 (ICAM-1) is an immunoglobulin-like cell adhesion molecule expressed on the surface of multiple cell types, including airway epithelial cells. It has been documented that cross-linking ICAM-1 on the surface of leukocytes results in changes in cellular function through outside-inside signaling; however, the effect of cross-linking ICAM-1 on the surface of airway epithelial cells is currently unknown. The objective of this study was to investigate whether or not cross-linking ICAM-1 on the surface of airway epithelial cells phosphorylated MAP kinases or stimulated chemokine expression and secretion. Methods The human lung adenocarcinoma (A549) cells and primary cultures of normal human bronchial epithelial (NHBE) cells were used in these studies. To increase ICAM-1 surface expression, cultures were stimulated with TNFα to enhance ICAM-1 surface expression. Following ICAM-1 upregulation, ICAM-1 was ligated with a murine anti-human ICAM-1 antibody and subsequently cross-linked with a secondary antibody (anti-mouse IgG(ab')2) in the presence or absence of the MAP kinase inhibitors. Following treatments, cultures were assessed for MAPK activation and chemokine gene expression and secretion. Control cultures were treated with murine IgG1 antibody or murine IgG1 antibody and anti-mouse IgG(ab')2 to illustrate specificity. Data were analyzed for significance using a one-way analysis of variance (ANOVA) with Bonferroni post-test correction for multiple comparisons, and relative gene expression was analyzed using the 2-ΔΔCT method. Results ICAM-1 cross-linking selectively phosphorylated both ERK and JNK MAP kinases as detected by western blot analysis. In addition, cross-linking resulted in differential regulation of chemokine expression. Specifically, IL-8 mRNA and protein secretion was not altered by ICAM-1 cross-linking, in contrast, RANTES mRNA and protein secretion was induced in both epithelial cultures. These events were

  11. Streptomycin treatment alters the intestinal microbiome, pulmonary T cell profile and airway hyperresponsiveness in a cystic fibrosis mouse model

    PubMed Central

    Bazett, Mark; Bergeron, Marie-Eve; Haston, Christina K.

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator deficient mouse models develop phenotypes of relevance to clinical cystic fibrosis (CF) including airway hyperresponsiveness, small intestinal bacterial overgrowth and an altered intestinal microbiome. As dysbiosis of the intestinal microbiota has been recognized as an important contributor to many systemic diseases, herein we investigated whether altering the intestinal microbiome of BALB/c Cftrtm1UNC mice and wild-type littermates, through treatment with the antibiotic streptomycin, affects the CF lung, intestinal and bone disease. We demonstrate that streptomycin treatment reduced the intestinal bacterial overgrowth in Cftrtm1UNC mice and altered the intestinal microbiome similarly in Cftrtm1UNC and wild-type mice, principally by affecting Lactobacillus levels. Airway hyperresponsiveness of Cftrtm1UNC mice was ameliorated with streptomycin, and correlated with Lactobacillus abundance in the intestine. Additionally, streptomycin treated Cftrtm1UNC and wild-type mice displayed an increased percentage of pulmonary and mesenteric lymph node Th17, CD8 + IL-17+ and CD8 + IFNγ+ lymphocytes, while the CF-specific increase in respiratory IL-17 producing γδ T cells was decreased in streptomycin treated Cftrtm1UNC mice. Bone disease and intestinal phenotypes were not affected by streptomycin treatment. The airway hyperresponsiveness and lymphocyte profile of BALB/c Cftrtm1UNC mice were affected by streptomycin treatment, revealing a potential intestinal microbiome influence on lung response in BALB/c Cftrtm1UNC mice. PMID:26754178

  12. Airway epithelial cell-derived insulin-like growth factor-1 triggers skewed CD8(+) T cell polarization.

    PubMed

    Zou, Jian-Yong; Huang, Shao-hong; Li, Yun; Chen, Hui-guo; Rong, Jian; Ye, Sheng

    2014-10-01

    Skewed CD8(+) T cell responses are important in airway inflammation. This study investigates the role of the airway epithelial cell-derived insulin-like growth factor 1 (IGF1) in contributing to CD8(+) T cell polarization. Expression of IGF1 in the airway epithelial cell line, RPMI2650 cells, was assessed by quantitative real time RT-PCR and Western blotting. The role of IGF1 in regulating CD8(+) T cell activation was observed by coculture of mite allergen-primed RPMI2650 cells and naïve CD8(+) T cells. CD8(+) T cell polarization was assessed by the carboxyfluorescein succinimidyl ester-dilution assay and the determination of cytotoxic cytokine levels in the culture medium. Exposure to mite allergen, Der p1, increased the expression of IGF1 by RPMI2650 cells. The epithelial cell-derived IGF1 prevented the activation-induced cell death by inducing the p53 gene hypermethylation. Mite allergen-primed RPMI2650 cells induced an antigen-specific CD8(+) T cell polarization. We conclude that mite allergens induce airway epithelial cell line, RPMI2650 cells, to produce IGF1; the latter contributes to antigen-specific CD8(+) T cell polarization. PMID:24844927

  13. 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

  14. 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.

  15. Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells

    PubMed Central

    Shkumatov, Artem; Thompson, Michael; Choi, Kyoung M.; Sicard, Delphine; Baek, Kwanghyun; Kim, Dong Hyun; Tschumperlin, Daniel J.; Prakash, Y. S.

    2015-01-01

    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. PMID:25724668

  16. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells

    PubMed Central

    Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A.

    2015-01-01

    Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development. PMID:25758640

  17. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

    PubMed

    Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

    2015-07-15

    Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development. PMID:25758640

  18. 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...

  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. A crucial role of sialidase Neu1 in hyaluronan receptor function of CD44 in T helper type 2-mediated airway inflammation of murine acute asthmatic model.

    PubMed

    Katoh, S; Maeda, S; Fukuoka, H; Wada, T; Moriya, S; Mori, A; Yamaguchi, K; Senda, S; Miyagi, T

    2010-08-01

    CD44 is a highly glycosylated cell adhesion molecule that is involved in lymphocyte infiltration of inflamed tissues. We have demonstrated previously that sialic acid residues of CD44 negatively regulates its receptor function and CD44 plays an important role in the accumulation of T helper type 2 (Th2) cells in the airway of a murine model of acute asthma. Here we evaluated the role of sialidase in the hyaluronic acid (HA) receptor function of CD44 expressed on CD4+ T cells, as well as in the development of a mite antigen-induced murine model of acute asthma. Splenic CD4+ T cell binding of HA was examined with flow cytometry. Expression of sialidases (Neu1, Neu2, Neu3 and Neu4) in spleen cells was evaluated by quantitative real-time reverse transcription-polymerase chain reaction. Airway inflammation and airway hyperresponsiveness (AHR) were evaluated in the asthmatic Neu1-deficient mouse strain SM/J model. Splenic CD4+ T cells from asthmatic model mice displayed increased HA receptor activity of CD44 after culture with the antigen, along with characteristic parallel induction of sialidase (Neu1) expression. This induction of HA binding was suppressed significantly by a sialidase inhibitor and was not observed in SM/J mice. Th2 cytokine concentration and absolute number of Th2 cells in the bronchoalveolar lavage fluid, and AHR were decreased in SM/J mice. In conclusion, HA receptor activity of CD44 and acute asthmatic reactions, including Th2-mediated airway inflammation and AHR, are dependent upon Neu1 enzymatic activity. Our observation suggests that Neu1 may be a target molecule for the treatment of asthma.

  1. Pulmonary dendritic cell distribution and prevalence in guinea pig airways: effect of ovalbumin sensitization and challenge.

    PubMed

    Lawrence, T E; Millecchia, L L; Frazer, D G; Fedan, J S

    1997-08-01

    We characterized the localization and prevalence of dendritic cells (DC) in guinea pig airways before and after s.c. sensitization and aerosol challenge with ovalbumin (OVA). DC, eosinophils, macrophages, T cells and B cells in lung and trachea were identified and quantified in frozen sections using monoclonal antibodies and computer-assisted image analysis. Airway reactivity of conscious animals to inhaled methacholine was examined. In unsensitized animals, DC were localized primarily within the lamina propria of the trachea and bronchi, in the submucosa of the trachea and in the adventitia of the bronchi. In contrast to reported studies on rats, few DC were noted in the epithelium. After OVA challenge, sensitized animals demonstrated an early obstructive response and a late-phase response that was well developed by 18 hr. Challenge with OVA increased DC prevalence in the lamina propria and submucosa of the trachea and in the lamina propria and adventitia of the bronchi. There was widespread eosinophilia throughout the airways, but no changes in B cells or T cells were evident. Macrophages were increased in the epithelium of both OVA-treated and saline-treated animals. At 18 hr after challenge, sensitized guinea pigs but not saline-treated controls were hyperreactive to inhaled methacholine. Except for macrophages, none of these effects were observed after saline treatment. Our findings indicate that inflammation in the airways of OVA-sensitized guinea pigs involves infiltration of DC, which is seen at the time animals are hyperreactive to inhaled methacholine. PMID:9262368

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

    PubMed

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

    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

  3. 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

  4. 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

  5. Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response

    PubMed Central

    Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Groshong, Steve D.; Ito, Yoko; Travanty, Emily A.; Leete, Jennifer; Holmes, Kathryn V.; Mason, Robert J.

    2009-01-01

    The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6–8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host. PMID:19741068

  6. 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

  7. 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.

  8. 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. PMID:26201096

  9. Cystic fibrosis transmembrane conductance regulator trafficking modulates the barrier function of airway epithelial cell monolayers.

    PubMed

    LeSimple, Pierre; Liao, Jie; Robert, Renaud; Gruenert, Dieter C; Hanrahan, John W

    2010-04-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane glycoprotein which functions as an anion channel and influences diverse cellular processes. We studied its role in the development of epithelial tightness by expressing wild-type (WT-CFTR) or mutant (Delta F508-CFTR) CFTR in human airway epithelial cell monolayers cultured at the air-liquid interface. Green fluorescent protein (GFP)-tagged WT or Delta F508 constructs were expressed in the CF bronchial cell line CFBE41o(-) using adenoviruses, and the results were compared with those obtained using CFBE41o(-) lines stably complemented with wild-type or mutant CFTR. As predicted, GFP-Delta WT-CFTR reached the apical membrane whereas GFP-F508-CFTR was only detected intracellularly. Although CFTR expression would be expected to reduce transepithelial resistance (TER), expressing GFP-CFTR significantly increased the TER of CFBE41o(-) monolayers whilst GFP-Delta F508-CFTR had no effect. Similar results were obtained with cell lines stably overexpressing Delta F508-CFTR or WT-CFTR. Preincubating Delta F508-CFTR monolayers at 29 degrees C reduced mannitol permeability and restored TER, and the effect on TER was reversible during temperature oscillations. Expression of GFP-Delta F508-CFTR or GFP-WT-CFTR in a cell line already containing endogenous WT-CFTR (Calu-3) did not alter TER. The CFTR- and temperature-dependence of TER were not affected by the CFTR inhibitor CFTR(inh)172 or low-chloride medium; therefore the effect of CFTR on barrier function was unrelated to its ion channel activity. Modulation of TER was blunted but not eliminated by genistein, implying the involvement of tyrosine phosphorylation and other mechanisms. Modulation of CFTR trafficking was correlated with an increase in tight junction depth. The results suggest that CFTR trafficking is required for the normal organisation and function of tight junctions. A reduction in barrier function caused by endoplasmic reticulum

  10. TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

    PubMed

    Suzuki, Shingo; Sargent, R Geoffrey; Illek, Beate; Fischer, Horst; Esmaeili-Shandiz, Alaleh; Yezzi, Michael J; Lee, Albert; Yang, Yanu; Kim, Soya; Renz, Peter; Qi, Zhongxia; Yu, Jingwei; Muench, Marcus O; Beyer, Ashley I; Guimarães, Alessander O; Ye, Lin; Chang, Judy; Fine, Eli J; Cradick, Thomas J; Bao, Gang; Rahdar, Meghdad; Porteus, Matthew H; Shuto, Tsuyoshi; Kai, Hirofumi; Kan, Yuet W; Gruenert, Dieter C

    2016-01-01

    Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways. PMID:26730810

  11. 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...

  12. 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...

  13. 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...

  14. Trehalose-Mediated Autophagy Impairs the Anti-Viral Function of Human Primary Airway Epithelial Cells

    PubMed Central

    Wu, Qun; Jiang, Di; Huang, Chunjian; van Dyk, Linda F.; Li, Liwu; Chu, Hong Wei

    2015-01-01

    Human rhinovirus (HRV) is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5) effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I) and IFN-β promoter stimulator 1 (IPS-1), two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations. PMID:25879848

  15. 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, ...

  16. The loss of Hoxa5 function promotes Notch-dependent goblet cell metaplasia in lung airways

    PubMed Central

    Boucherat, Olivier; Chakir, Jamila; Jeannotte, Lucie

    2012-01-01

    Summary Hox genes encode transcription factors controlling complex developmental processes in various organs. Little is known, however, about how HOX proteins control cell fate. Herein, we demonstrate that the goblet cell metaplasia observed in lung airways from Hoxa5−/− mice originates from the transdifferentiation of Clara cells. Reduced CC10 expression in Hoxa5−/− embryos indicates that altered cell specification occurs prior to birth. The loss of Hoxa5 function does not preclude airway repair after naphthalene exposure, but the regenerated epithelium presents goblet cell metaplasia and less CC10-positive cells, demonstrating the essential role of Hoxa5 for correct differentiation. Goblet cell metaplasia in Hoxa5−/− mice is a FOXA2-independent process. However, it is associated with increased Notch signaling activity. Consistent with these findings, expression levels of activated NOTCH1 and the effector gene HEY2 are enhanced in patients with chronic obstructive pulmonary disease. In vivo administration of a γ-secretase inhibitor attenuates goblet cell metaplasia in Hoxa5−/− mice, highlighting the contribution of Notch signaling to the phenotype and suggesting a potential therapeutic strategy to inhibit goblet cell differentiation and mucus overproduction in airway diseases. In summary, the loss of Hoxa5 function in lung mesenchyme impacts on epithelial cell fate by modulating Notch signaling. PMID:23213461

  17. 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

  18. Airway Epithelial Cells are the Site of Expression of a Mammalian Antimicrobial Peptide Gene

    NASA Astrophysics Data System (ADS)

    Diamond, Gill; Jones, Douglas E.; Bevins, Charles L.

    1993-05-01

    We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5'exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.

  19. 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.

  20. 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

  1. Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells.

    PubMed

    Berger, John P; Simet, Samantha M; DeVasure, Jane M; Boten, Jessica A; Sweeter, Jenea M; Kharbanda, Kusum K; Sisson, Joseph H; Wyatt, Todd A

    2014-08-01

    Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3-7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3 to 7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.

  2. T-cell-intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy.

    PubMed

    Perez-Lloret, Jimena; Okoye, Isobel S; Guidi, Riccardo; Kannan, Yashaswini; Coomes, Stephanie M; Czieso, Stephanie; Mengus, Gabrielle; Davidson, Irwin; Wilson, Mark S

    2016-02-01

    There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ(+)CD4(+) T-helper 2 (TH2) cells orchestrate the type-2-driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081-E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4(gfp+)αβ(+)CD4(+) TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell-intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24(-/-) T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1-regulated signaling. Following this prediction, we found that Trim24(-/-) T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β-mediated activation in vitro and in vivo, and fail to respond to IL-1β-exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell-intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity. PMID:26787865

  3. Lung airway-surveilling CXCR3hi memory CD8+ T cells are critical for protection against Influenza A virus

    PubMed Central

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

    2013-01-01

    Summary 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 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. PMID:24238342

  4. 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.

  5. 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

  6. 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.

  7. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis

    PubMed Central

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β−NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  8. Transcription Factor Runx3 Is Induced by Influenza A Virus and Double-Strand RNA and Mediates Airway Epithelial Cell Apoptosis.

    PubMed

    Gan, Huachen; Hao, Qin; Idell, Steven; Tang, Hua

    2015-01-01

    Influenza A virus (IAV) targets airway epithelial cells and exploits the host cell machinery to replicate, causing respiratory illness in annual epidemics and pandemics of variable severity. The high rate of antigenic drift (viral mutation) and the putative antigenic shift (reassortant strains) have raised the need to find the host cell inducible factors modulating IAV replication and its pathogenesis to develop more effective antiviral treatment. In this study, we found for the first time that transcription factor Runx3, a developmental regulator and tumor suppressor, was induced by IAV H1N1 and H3N2, viral RNA, a synthetic analog of viral double-stranded RNA (dsRNA) polyinosinic-polycytidylic acid, and type-II interferon-γ (IFNγ) in human airway epithelial cells. Whereas Runx3 was essentially not induced by type-I IFNα and type-III IFNλ, we show that Runx3 induction by IAV infection and viral RNA is mediated through the innate immune receptor MDA5 and the IκB kinase-β-NF-κB pathway. Moreover, we provide substantial evidence indicating that Runx3 plays a crucial role in airway epithelial cell apoptosis induced by IAV infection and dsRNA through the activation of extrinsic and intrinsic apoptosis pathways. Thus, we have identified Runx3 as an inducible and important transcription factor modulating IAV-induced host epithelial cell apoptosis. PMID:26643317

  9. 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

  10. Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

    PubMed

    Favia, Maria; Mancini, Maria T; Bezzerri, Valentino; Guerra, Lorenzo; Laselva, Onofrio; Abbattiscianni, Anna C; Debellis, Lucantonio; Reshkin, Stephan J; Gambari, Roberto; Cabrini, Giulio; Casavola, Valeria

    2014-07-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

  11. Gene Transfer by Guanidinium-Cholesterol Cationic Lipids into Airway Epithelial Cells in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Oudrhiri, Noufissa; Vigneron, Jean-Pierre; Peuchmaur, Michel; Leclerc, Tony; Lehn, Jean-Marie; Lehn, Pierre

    1997-03-01

    Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis (guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

  12. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses.

    PubMed

    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

  13. Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats

    SciTech Connect

    Desouza, Ivani A. . E-mail: ivanidesouza@fcm.unicamp.br; Franco-Penteado, Carla F.; Camargo, Enilton A.; Lima, Carmen S.P.; Teixeira, Simone A.; Muscara, Marcelo N.; De Nucci, Gilberto; Antunes, Edson

    2006-11-15

    Staphylocococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5-250 ng/trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB{sub 4} and PGE{sub 2}) and cytokines (TNF-{alpha}, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 {mu}g/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE{sub 2}, LTB{sub 4}, TNF-{alpha}, IL-6) and anti-inflammatory mediators (IL-10)

  14. Phenotypic traits of the hypothalamic PVN cells innervating airway-related vagal preganglionic neurons.

    PubMed

    Kc, Prabha; Karibi-Ikiriko, Abere; Rust, Cheryl F; Jayam-Trouth, Annapurni; Haxhiu, Musa A

    2006-12-01

    The paraventricular nucleus of the hypothalamus (PVN) integrates multiple inputs via projections from arginine vasopressin (AVP)- and oxytocin (OXT)-containing neurons to the brain stem and spinal cord as well as regulates respiratory and cardiovascular stress-related responses, which also affect airway function. In the present study, we used immunocytochemistry and the retrograde transneuronal tracer, Bartha strain of pseudorabies virus expressing green fluorescent protein (PRV-GFP), to localize AVP- and OXT-producing neurons that project to airway-related vagal preganglionic neurons (AVPNs) innervating intrapulmonary airways. PRV-GFP was microinjected into the upper right lung lobe, and after 4 days survival, hypothalamic tissue sections were processed for co-expression of PRV-GFP and AVP or PRV-GFP and OXT. In addition, in a separate group of five rats, Phaseolus vulgaris leucoagglutinin (PHAL), an anterograde tracer, was injected unilaterally into the PVN and cholera toxin beta subunit was microinjected into the tracheal wall. Analysis of five successfully infected animals showed that 14% of PRV-GFP labeled neurons express AVP traits and 18% of transneuronally-labeled neurons contain OXT. Furthermore, the identified AVPNs innervating extrathoracic trachea receive axon terminals of the PVN neurons. The results indicate that AVP- and OXT-producing PVN cells, via direct projections to the AVPNs, could modulate cholinergic outflow to the airways, as a part of overall changes in response to stress.

  15. Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells.

    PubMed

    Rubenstein, Ronald C; Lockwood, Shannon R; Lide, Ellen; Bauer, Rebecca; Suaud, Laurence; Grumbach, Yael

    2011-01-01

    The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o⁻ model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o⁻ parental and ΔF508-CFTR-overexpressing cells after treatment with 1 μM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o⁻ cells that stably express wild-type (wt) CFTR. CFBE41o⁻ wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o⁻ wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may

  16. Upper airways sensory irritation responses of mice exposed to mainstream smoke from four cigarette types.

    PubMed

    Williams, Chandra D; Potts, Ryan J; Steichen, Thomas J; Doolittle, David J; Ayres, Paul H

    2010-01-01

    Relative sensory irritation responses for Swiss-Webster mice exposed nose-only to mainstream tobacco smoke were evaluated for several cigarette types using a smoking regimen consisting of a 35-ml puff, 2 s in duration, taken once per minute. The degree of sensory irritation for each cigarette type was evaluated as the smoke concentration inducing a 50% reduction in breathing frequency. The smoke concentration inducing 50% respiratory depression is called the RD(50) value. Study findings suggest that mainstream tobacco smoke from the Eclipse cigarette, which primarily heats rather than burns tobacco, yielded an RD(50) that was significantly higher (approximately twofold) than a tobacco-burning leading ultralight or the 2R4F or 1R5F reference cigarettes. This is indicative of reduced upper airways irritation by Eclipse that may be due to its distinct design. Study findings suggest that the irritating nature of mainstream tobacco smoke from different cigarette types can be evaluated effectively in terms of smoke concentration using the relative sensory irritation assessment. These findings constitute the first report about use of the RD(50) sensory irritation response during comparative evaluations of mainstream tobacco smoke. PMID:19555219

  17. Upper airways sensory irritation responses of mice exposed to mainstream smoke from four cigarette types.

    PubMed

    Williams, Chandra D; Potts, Ryan J; Steichen, Thomas J; Doolittle, David J; Ayres, Paul H

    2010-01-01

    Relative sensory irritation responses for Swiss-Webster mice exposed nose-only to mainstream tobacco smoke were evaluated for several cigarette types using a smoking regimen consisting of a 35-ml puff, 2 s in duration, taken once per minute. The degree of sensory irritation for each cigarette type was evaluated as the smoke concentration inducing a 50% reduction in breathing frequency. The smoke concentration inducing 50% respiratory depression is called the RD(50) value. Study findings suggest that mainstream tobacco smoke from the Eclipse cigarette, which primarily heats rather than burns tobacco, yielded an RD(50) that was significantly higher (approximately twofold) than a tobacco-burning leading ultralight or the 2R4F or 1R5F reference cigarettes. This is indicative of reduced upper airways irritation by Eclipse that may be due to its distinct design. Study findings suggest that the irritating nature of mainstream tobacco smoke from different cigarette types can be evaluated effectively in terms of smoke concentration using the relative sensory irritation assessment. These findings constitute the first report about use of the RD(50) sensory irritation response during comparative evaluations of mainstream tobacco smoke.

  18. In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

    PubMed

    Broadbent, Lindsay; Villenave, Remi; Guo-Parke, Hong; Douglas, Isobel; Shields, Michael D; Power, Ultan F

    2016-01-01

    The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis. PMID:27464691

  19. ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO UTAH VALLEY PARTICULATE MATTER

    EPA Science Inventory

    Exposure to ambient particulate matter (PM) in the Utah Valley (UV) has previously been associated with a variety of adverse health effects. To investigate intracellular signaling mechanisms for pulmonary responses to UV PM inhalation, human primary airway epithelial cells (NHBE)...

  20. Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

    PubMed

    Statt, Sarah; Ruan, Jhen-Wei; Hung, Li-Yin; Chang, Ching-Yun; Huang, Chih-Ting; Lim, Jae Hyang; Li, Jian-Dong; Wu, Reen; Kao, Cheng-Yuan

    2015-11-01

    Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

  1. Ineffective correction of PPARγ signaling in cystic fibrosis airway epithelial cells undergoing repair.

    PubMed

    Bou Saab, J; Bacchetta, M; Chanson, M

    2016-09-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) represents a potential target to treat airway mucus hypersecretion in cystic fibrosis (CF). We aimed to determine if PPARγ is altered in CF human airway epithelial cells (HAECs), if PPARγ contributes to mucin expression and HAEC differentiation, and if PPARγ ligand therapy corrects the CF phenotype. To this end, well-differentiated CF and NCF HAEC primary cultures were wounded to monitor the expression of key genes involved in PPARγ activation and mucus homeostasis, and to evaluate the effect of a PPARγ agonist, at different times of repair. Hydroxyprostaglandin dehydrogenase (HPGD) converts prostaglandin E2 to 15-keto PGE2 (15kPGE2), an endogenous PPARγ ligand. Interestingly, PPARγ and HPGD expression dramatically decreased in CF HAECs. These changes were accompanied by an increase in the expression of MUC5B. The correlation between PPARγ and MUC5B was confirmed in an airway epithelial cell line after CFTR knock-down. Exposure of HAECs to 15kPGE2 did not correct the CF phenotype but revealed a defect in the process of basal cell (BC) differentiation. The HPGD/PPARγ axis is deregulated in primary HAEC cultures from CF patients, which may impact the maturation of BCs to differentiated luminal cells. Importantly, PPARγ therapy was inefficient in correcting the CF defect. PMID:27484450

  2. Reduced expression of Tis7/IFRD1 protein in murine and human cystic fibrosis airway epithelial cell models homozygous for the F508del-CFTR mutation.

    PubMed

    Blanchard, Elise; Marie, Solenne; Riffault, Laure; Bonora, Monique; Tabary, Olivier; Clement, Annick; Jacquot, Jacky

    2011-08-01

    12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o(-) cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o(-) cells compared to normal bronchial epithelial cells 16HBE14o(-). Surprisingly, messenger RNA level of IFRD1 in CFBE41o(-) cells was found elevated. Treating CFBE41o(-) cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.

  3. 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.

  4. T cell derived IL-10 is dispensable for tolerance induction in a murine model of allergic airway inflammation.

    PubMed

    Kunz, Stefanie; Dolch, Anja; Surianarayanan, Sangeetha; Dorn, Britta; Bewersdorff, Mayte; Alessandrini, Francesca; Behrendt, Rayk; Karp, Christopher L; Muller, Werner; Martin, Stefan F; Roers, Axel; Jakob, Thilo

    2016-08-01

    Regulatory mechanisms initiated by allergen-specific immunotherapy are mainly attributed to T cell derived IL-10. However, it has not been shown that T cell derived IL-10 is required for successful tolerance induction (TI). Here, we analyze cellular sources and the functional relevance of cell type specific IL-10 during TI in a murine model of allergic airway inflammation. While TI was effective in IL-10 competent mice, neutralizing IL-10 prior to tolerogenic treatment completely abrogated the beneficial effects. Cellular sources of IL-10 during TI were identified by using transcriptional reporter mice as T cells, B cells, and to a lesser extent DCs. Interestingly, TI was still effective in mice with T cell, B cell, B and T cell, or DC-specific IL-10 deficiency. In contrast, TI was not possible in mice lacking IL-10 in all hematopoetic cells, while it was effective in bone marrow (BM) chimera that lacked IL-10 only in nonhematopoetic cells. Taken together, allergen-specific tolerance depends on IL-10 from hematopoetic sources. The beneficial effects of allergen-specific immunotherapy cannot solely be attributed to IL-10 from T cells, B cells, or even DCs, suggesting a high degree of cellular redundancy in IL-10-mediated tolerance. PMID:27287239

  5. Management of the difficult airway.

    PubMed

    Strauss, Robert A; Noordhoek, Roseanna

    2010-03-01

    The oral and maxillofacial surgeon frequently encounters and manages difficult airways. Knowledge of and calm progression by practitioner and staff through different means to ventilate and manage a difficult airway are crucial. Practitioners should become comfortable with different types of alternative or rescue airways in order to intervene quickly in case of emergent or unanticipated airway compromise.

  6. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2010-03-01

    Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients. PMID:19947928

  7. Ex Vivo and In Vivo Lentivirus-Mediated Transduction of Airway Epithelial Progenitor Cells.

    PubMed

    Leoni, Giulia; Wasowicz, Marguerite Y; Chan, Mario; Meng, Cuixiang; Farley, Raymond; Brody, Steven L; Inoue, Makoto; Hasegawa, Mamoru; Alton, Eric W F W; Griesenbach, Uta

    2015-01-01

    A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration. PMID:26471068

  8. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2010-01-27

    Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.

  9. HB-EGF-Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse.

    PubMed

    Wang, Qing; Li, Hequan; Yao, Yinan; Lu, Guohua; Wang, Yuehong; Xia, Dajing; Zhou, Jianying

    2016-03-01

    The airway smooth muscle (ASM) cells' proliferation, migration, and their progenitor's migration are currently regarded as causative factors for ASM remodeling in asthma. Heparin-binding epidermal growth factor (HB-EGF), a potent mitogen and chemotactic factor, could promote ASM cell proliferation through MAPK pathways. In this study, we obtained primary ASM cells and their progenitors from C57BL/6 mice and went on to explore the role of HB-EGF in these cells migration and the underlying mechanisms. We found that recombinant HB-EGF (rHB-EGF) intratracheal instillation accelerated ASM layer thickening in an OVA-induced asthmatic mouse. Modified Boyden chamber assay revealed that rHB-EGF facilitate ASM cell migration in a dose-dependent manner and ASM cells from asthmatic mice had a greater migration ability than that from normal counterparts. rHB-EGF could stimulate the phosphorylation of ERK1/2 and p38 in ASM cells but further migration assay showed that only epidermal growth factor receptor inhibitor (AG1478) or p38 inhibitor (SB203580), but not ERK1/2 inhibitor (PD98059), could inhibit rHB-EGF-mediated ASM cells migration. Actin cytoskeleton experiments exhibited that rHB-EGF could cause actin stress fibers disassembly and focal adhesions formation of ASM cells through the activation of p38. Finally, airway instillation of rHB-EGF promoted the recruitment of bone marrow-derived smooth muscle progenitor cells, which were transferred via caudal vein, migrating into the airway from the circulation. These observations demonstrated that ASM remodeling in asthma might have resulted from HB-EGF-mediated ASM cells and their progenitor cells migration, via p38 MAPK-dependent actin cytoskeleton remodeling.

  10. Rhinoviral infection and asthma: the detection and management of rhinoviruses by airway epithelial cells.

    PubMed

    Parker, L C; Stokes, C A; Sabroe, I

    2014-01-01

    Human rhinoviruses (HRV) have been linked to the development of childhood asthma and recurrent acute asthma exacerbations throughout life, and contribute considerably to the healthcare and economic burden of this disease. However, the ability of HRV infections to trigger exacerbations, and the link between allergic status and HRV responsiveness, remains incompletely understood. Whilst the receptors on human airway cells that detect and are utilized by most HRV group A and B, but not C serotypes are known, how endosomal pattern recognition receptors (PRRs) detect HRV replication products that are generated within the cytoplasm remains somewhat of an enigma. In this article, we explore a role for autophagy, a cellular homeostatic process that allows the cell to encapsulate its own cytosolic constituents, as the crucial mechanism controlling this process and regulating the innate immune response of airway epithelial cells to viral infection. We will also briefly describe some of the recent insights into the immune responses of the airway to HRV, focusing on neutrophilic inflammation that is a potentially unwanted feature of the acute response to viral infection, and the roles of IL-1 and Pellinos in the regulation of responses to HRV.

  11. [Effect of heparin on airway goblet cell secretion in sensitized guinea pigs].

    PubMed

    Nakata, J; Tamaoki, J; Takeyama, K; Takeda, Y; Yamawaki, I; Kondo, M; Nagai, A

    1998-10-01

    Heparin and related proteoglycans are released from mast cells and possess anti-inflammatory and anti-complement activities. To elucidate whether heparin affects goblet cell secretion in asthmatic airways and, if so, what the mechanism of action is, we studied guinea pigs sensitized with ovalbumin (OVA) by determining the mucus score (MS) of tracheal goblet cells stained with Alcian blue and PAS. Inhalation of OVA caused a rapid decrease in MS in a dose-dependent manner, with the maximal decrease being from 545 +/- 26 to 192 +/- 35 (p < 0.001), indicating an increase in goblet cell mucus discharge. This effect was selectively inhibited by the histamine H2 receptor blockade with cimetidine. Prior inhalation of heparin inhibited OVA-induced goblet cell secretion in a dose-dependent fashion, but had no effect on histamine-induced goblet cell secretion. The OVA-induced histamine release from the tracheal tissue was likewise inhibited by heparin. These results suggest that allergic challenge stimulates airway goblet cell secretion mainly through the release of histamine and the concomitant activation of histamine H2 receptors on goblet cells, and that heparin protects against this effect by inhibiting the histamine release from mast cells.

  12. Airborne lipid antigens mobilize resident intravascular NKT cells to induce allergic airway inflammation.

    PubMed

    Scanlon, Seth T; Thomas, Seddon Y; Ferreira, Caroline M; Bai, Li; Krausz, Thomas; Savage, Paul B; Bendelac, Albert

    2011-09-26

    Airborne exposure to microbial cell wall lipids such as lipopolysaccharide triggers innate immune responses that regulate susceptibility to allergic airway inflammation. α-Glycosylceramides represent another widespread class of microbial lipids that directly stimulate innate-like, IL-4- and IL-13-producing, CD1d-restricted NKT cells. In this study, we demonstrate that NKT cells constitutively accumulate and reside in the microvasculature of the mouse lung. After a single airborne exposure to lipid antigen, they promptly extravasate to orchestrate the formation of peribronchiolar and interstitial lymphohistiocytic granulomas containing numerous eosinophils. Concomitant airborne exposure to ovalbumin (OVA) induces the priming of OVA-specific Th2 cells and IgE antibodies by the same dendritic cell coexpressing CD1d and MHC class II. Although NKT cell activation remains confined to the lipid-exposed lung and draining lymph nodes, Th2 cells recirculate and seed the lung of a parabiotic partner, conferring susceptibility to OVA challenge months after the initial exposure, in a manner independent of NKT cells and CD1d. Thus, transient recruitment and activation of lung-resident intravascular NKT cells can trigger long-term susceptibility to allergic airway inflammation.

  13. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation.

  14. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation. PMID:26276826

  15. T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis

    PubMed Central

    1994-01-01

    Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma. PMID:7520473

  16. Pseudomonas aeruginosa pyocyanin modulates mucin glycosylation with sialyl-Lewisx to increase binding to airway epithelial cells

    PubMed Central

    Choi, Woosuk; Choe, Shawn; Miao, Jinfeng; Xu, Ying; Powell, Rebecca; Lin, Jingjun; Kuang, Zhizhou; Gaskins, H Rex; Lau, Gee W.

    2015-01-01

    Cystic fibrosis (CF) patients battle life-long pulmonary infections with the respiratory pathogen Pseudomonas aeruginosa (PA). An overabundance of mucus in CF airways provides a favorable niche for PA growth. When compared to that of non-CF individuals, mucus of CF airways is enriched in sialyl-Lewisx, a preferred binding receptor for PA. Notably, the levels of sialyl-Lewisx directly correlate with infection severity in CF patients. However, the mechanism by which PA causes increased sialylation remains uncharacterized. In this study, we examined the ability of PA virulence factors to modulate sialyl-Lewisx modification in airway mucins. We found pyocyanin (PCN) to be a potent inducer of sialyl-Lewisx in both mouse airways and in primary and immortalized CF and non-CF human airway epithelial cells. PCN increased the expression of C2/4GnT and ST3Gal-IV, two of the glycosyltransferases responsible for the stepwise biosynthesis of sialyl-Lewisx, through a TNF-α-mediated phosphoinositol-specific phospholipase C (PI-PLC) dependent pathway. Furthermore, PA bound more efficiently to airway epithelial cells pre-exposed to PCN through a flagellar cap-dependent manner. Importantly, antibodies against sialyl-Lewisx and anti-TNF-α attenuated PA binding. These results indicate that PCN secretes PCN to induce a favorable environment for chronic colonization of CF lungs by increasing the glycosylation of airway mucins with sialyl-Lewisx. PMID:26555707

  17. Allergen-induced airway responses.

    PubMed

    Gauvreau, Gail M; El-Gammal, Amani I; O'Byrne, Paul M

    2015-09-01

    Environmental allergens are an important cause of asthma and can contribute to loss of asthma control and exacerbations. Allergen inhalation challenge has been a useful clinical model to examine the mechanisms of allergen-induced airway responses and inflammation. Allergen bronchoconstrictor responses are the early response, which reaches a maximum within 30 min and resolves by 1-3 h, and late responses, when bronchoconstriction recurs after 3-4 h and reaches a maximum over 6-12 h. Late responses are followed by an increase in airway hyperresponsiveness. These responses occur when IgE on mast cells is cross-linked by an allergen, causing degranulation and the release of histamine, neutral proteases and chemotactic factors, and the production of newly formed mediators, such as cysteinyl leukotrienes and prostaglandin D2. Allergen-induced airway inflammation consists of an increase in airway eosinophils, basophils and, less consistently, neutrophils. These responses are mediated by the trafficking and activation of myeloid dendritic cells into the airways, probably as a result of the release of epithelial cell-derived thymic stromal lymphopoietin, and the release of pro-inflammatory cytokines from type 2 helper T-cells. Allergen inhalation challenge has also been a widely used model to study potential new therapies for asthma and has an excellent negative predictive value for this purpose. PMID:26206871

  18. Broncho-Vaxom Attenuates Allergic Airway Inflammation by Restoring GSK3β-Related T Regulatory Cell Insufficiency

    PubMed Central

    Zhong, Hua; Yu, Dehong; Zeng, Xianping; Deng, Mengxia; Sun, Yueqi; Wen, Weiping; Li, Huabin

    2014-01-01

    Background Oral administration of bacterial extracts (eg, Broncho-Vaxom (BV)) has been proposed to attenuate asthma through modulating Treg cells. However, the underlying mechanism has not been fully characterized. This study sought to assess the effects of oral administration of BV on GSK-3β expression and Treg cells in ovalbumin (OVA)-induced asthmatic mice models. Method Asthmatic mice models were established with OVA challenge and treated with oral administration of BV. Next, infiltration of inflammatory cells including eosinophil and neutrophils, mucous metaplasia, levels of Th1/Th2/Treg-typed cytokines and expression of GSK3β and Foxp3 were examined in asthmatic mice models by histological analysis, Bio-Plex and western blot, respectively. Moreover, the frequencies of Treg cells were evaluated in cultured splenocytes by flow cytometry in the presence of BV or GSK3β siRNA interference. Results We found significant decrease of infiltrated inflammatory cells in bronchoalveolar lavage fluid (BALF) in asthmatic mice models after oral administration of BV. Oral administration of BV was shown to significantly suppress mucus metaplasia, Th2-typed cytokine levels and GSK3β expression while increasing Foxp3 production in asthmatic mice models. Moreover, BV significantly enhanced GSK3β-related expansion of Treg cells in cultured spleen cells in vitro. Conclusion Our findings provide evidence that oral administration of BV is capable of attenuating airway inflammation in asthmatic mice models, which may be associated with GSK3β-related expansion of Treg cells. PMID:24667347

  19. Atopy and Inhaled Corticosteroid Use Associate with Fewer IL-17+ Cells in Asthmatic Airways

    PubMed Central

    Fattahi, Fatemeh; Brandsma, Corry-Anke; Lodewijk, Monique; Reinders-Luinge, Marjan; Postma, Dirkje S.; Timens, Wim; Hylkema, Machteld N.; ten Hacken, Nick H. T.

    2016-01-01

    Background Interleukin (IL)-17 plays a critical role in numerous immune and inflammatory responses and was recently suggested to contribute to the pathogenesis of nonatopic (non-eosinophil/neutrophil-dominant) asthma. We aimed to compare expression of IL-17 in bronchial airways between atopic and nonatopic asthmatics, with/without inhaled corticosteroid (ICS) use and to identify its major cellular source. Methods Bronchial biopsies from 114 patients with mild-to-moderate asthma were investigated: 33 nonatopic, 63 non-corticosteroid users, 90 nonsmokers. IL-17 expression was correlated with atopy and inflammatory cell counts (EPX, NP57, CD3, CD4, CD8, CD20, CD68), taking ICS use and smoking into account. Multiple linear regression analyses were used to determine the independent factors as well as the most relevant inflammatory cells contributing to IL-17 expression. Double immunostainings were performed to confirm the major cellular source of IL-17. Results In non-ICS users, nonatopic asthmatics had more IL-17+ cells in the airway wall than atopic asthmatics. In both atopic and nonatopic asthmatics, ICS use was associated with lower numbers of IL-17+ cells, independent of smoking. The number of IL-17+ cells was associated with the number of neutrophils (B: 0.26, 95% CI: 0.17–0.35) and eosinophils (B: 0.18, 95% CI: 0.07–0.29). The majority of IL-17+ cells were neutrophils, as confirmed by double immunostaining. Conclusions We show for the first time that atopy and ICS use are associated with lower numbers of IL-17+ cells in asthmatic airways. Importantly, IL-17+ cells were mostly neutrophils which conflicts with the paradigm that lymphocytes (Th17) are the main source of IL-17. PMID:27552197

  20. Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma.

    PubMed

    Pei, Qing-Mei; Jiang, Ping; Yang, Min; Qian, Xue-Jiao; Liu, Jiang-Bo; Kim, Sung-Ho

    2016-10-01

    Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. PMID:27587274

  1. Following damage, the majority of bone marrow-derived airway cells express an epithelial marker

    PubMed Central

    MacPherson, Heather; Keir, Pamela A; Edwards, Carol J; Webb, Sheila; Dorin, Julia R

    2006-01-01

    Background Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells. Methods Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations. Results The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0 – 1.6% with whole marrow and 0.6 – 1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any

  2. Unjamming and cell shape in the asthmatic airway epithelium

    PubMed Central

    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; 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.; Lisa Manning, M.; Butler, James P.; Drazen, Jeffrey M.; Fredberg, Jeffrey J.

    2015-01-01

    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. PMID:26237129

  3. Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

    PubMed Central

    Nita-Lazar, Mihai; Banerjee, Aditi; Feng, Chiguang; Amin, Mohammed N.; Frieman, Matthew B.; Chen, Wilbur H.; Cross, Alan S.; Wang, Lai-Xi; Vasta, Gerardo R.

    2015-01-01

    The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence motif, with critical regulatory roles in development and immune homeostasis. In this study, we examined the possibility that galectins expressed in the airway epithelial cells might play a significant role in viral or pneumococcal adhesion to airway epithelial cells. Our results in a mouse model for influenza and pneumococcal infection revealed that the murine lung expresses a diverse galectin repertoire, from which selected galectins, including galectin 1 (Gal1) and galectin 3 (Gal3), are released to the bronchoalveolar space. Further, the results showed that influenza and subsequent S. pneumoniae infections significantly alter the glycosylation patterns of the airway epithelial surface and modulate galectin expression. In vitro studies on the human airway epithelial cell line A549 were consistent with the observations made in the mouse model, and further revealed that both Gal1 and Gal3 bind strongly to IAV and S. pneumoniae, and that exposure of the cells to viral neuraminidase or

  4. Nitrite Modulates Bacterial Antibiotic Susceptibility and Biofilm Formation in Association with Airway Epithelial Cells

    PubMed Central

    Zemke, Anna C; Shiva, Sruti; Burn, Jane L.; Moskowitz, Samuel M.; Pilewski, Joseph M.; Gladwin, Mark T.; Bomberger, Jennifer M.

    2014-01-01

    Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, while the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects in other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain ‘poisons’, can be safely nebulized at high concentration in humans. PMID:25229185

  5. RANTES expression induced by Toll-like receptor 4 ligand in rat airway smooth muscle cells.

    PubMed

    Okayasu, Kaori; Tamaoka, Meiyo; Takayama, Satoshi; Miyazaki, Yasunari; Sumi, Yuki; Inase, Naohiko; Yoshizawa, Yasuyuki

    2010-01-01

    Airway smooth muscle cells (ASMCs) have been reported to express Toll-like receptors (TLRs) and take part in the pathogenesis of asthma exacerbation. Though TLRs were found to activate epidermal growth factor receptor (EGFR) in airway epithelial cells, little is known about the association of TLR ligands with EGFR signaling pathways in ASMCs. Using primary cultured ASMCs from Brown Norway rats, TLR4, eotaxin, and RANTES mRNA were examined by real-time quantitative RT-PCR after stimulation with the TLR4 ligand, lipopolysaccharides (LPS). The concentration of RANTES protein in culture supernatant was measured by ELISA. The effect of EGFR signaling inhibitors on RANTES expression was examined as well. Phosphorylation of EGFR after stimulation was examined by Western Blotting. Rat ASMCs expressed TLR4 and eotaxin, and LPS upregulated RANTES production. The EGFR tyrosine kinase inhibitor AG1478, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the matrix metalloproteinase (MMP) inhibitor GM6001 inhibited RANTES expression induced by LPS. LPS phosphorylated EGFR. TLR4 activation can induce RANTES expression via EGFR transactivation and PI3K/Akt pathway in rat ASMCs. MMP-induced EGFR proligand cleavage and ligand binding to EGFR seem to be involved in this pathway. These findings may be critical in the pathogenesis of asthma exacerbation by airway infection. PMID:23896774

  6. 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

  7. 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

  8. Hyperoxia promotes polarization of the immune response in ovalbumin-induced airway inflammation, leading to a TH17 cell phenotype

    PubMed Central

    Nagato, Akinori C; Bezerra, Frank S; Talvani, André; Aarestrup, Beatriz J; Aarestrup, Fernando M

    2015-01-01

    Previous studies have demonstrated that hyperoxia-induced stress and oxidative damage to the lungs of mice lead to an increase in IL-6, TNF-α, and TGF-β expression. Together, IL-6 and TGF-β have been known to direct T cell differentiation toward the TH17 phenotype. In the current study, we tested the hypothesis that hyperoxia promotes the polarization of T cells to the TH17 cell phenotype in response to ovalbumin-induced acute airway inflammation. Airway inflammation was induced in female BALB/c mice by intraperitoneal sensitization and intranasal introduction of ovalbumin, followed by challenge methacholine. After the methacholine challenge, animals were exposed to hyperoxic conditions in an inhalation chamber for 24 h. The controls were subjected to normoxia or aluminum hydroxide dissolved in phosphate buffered saline. After 24 h of hyperoxia, the number of macrophages and lymphocytes decreased in animals with ovalbumin-induced airway inflammation, whereas the number of neutrophils increased after ovalbumin-induced airway inflammation. The results showed that expression of Nrf2, iNOS, T-bet and IL-17 increased after 24 of hyperoxia in both alveolar macrophages and in lung epithelial cells, compared with both animals that remained in room air, and animals with ovalbumin-induced airway inflammation. Hyperoxia alone without the induction of airway inflammation lead to increased levels of TNF-α and CCL5, whereas hyperoxia after inflammation lead to decreased CCL2 levels. Histological evidence of extravasation of inflammatory cells into the perivascular and peribronchial regions of the lungs was observed after pulmonary inflammation and hyperoxia. Hyperoxia promotes polarization of the immune response toward the TH17 phenotype, resulting in tissue damage associated with oxidative stress, and the migration of neutrophils to the lung and airways. Elucidating the effect of hyperoxia on ovalbumin-induced acute airway inflammation is relevant to preventing or

  9. Synthesis and evaluation of airway targeted PLGA nanoparticles for drug delivery in obstructive lung diseases.

    PubMed

    Vij, Neeraj

    2012-01-01

    Chronic airway inflammation is a hallmark of chronic obstructive airway diseases, including asthma, COPD (chronic obstructive pulmonary disease), and CF (cystic fibrosis). It is also a major challenge in delivery and therapeutic efficacy of nano-based delivery systems in these chronic airway conditions as nanoparticle (NP) need to bypass airways defense mechanisms as we recently discussed. NPs which are capable of overcoming airways defense mechanisms should allow targeted drug delivery to disease cells. Over the last decade there has been increasing interest in development of targeted NPs for cancer but relatively little effort on designing novel systems for treating chronic inflammatory and obstructive airway conditions. Here we describe methods for preparing drug loaded multifunctional nanoparticles for targeted delivery to specific cell types in airways. The formulations and methods for selective drug delivery, discussed here are currently under preclinical development in our laboratory for treating chronic airway conditions such as COPD, CF, and asthma.

  10. Airway Surface Mycosis in Chronic Th2-Associated Airway Disease

    PubMed Central

    Porter, Paul; Lim, Dae Jun; Maskatia, Zahida Khan; Mak, Garbo; Tsai, Chu-Lin; Citardi, Martin J; Fakhri, Samer; Shaw, Joanne L.; Fothergil, Annette; Kheradmand, Farrah; Corry, David B; Luong, Amber

    2014-01-01

    Background Environmental fungi have been linked to T helper type 2 (Th2) cell-related airway inflammation and the Th2-associated chronic airway diseases asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and allergic fungal rhinosinusitis (AFRS), but whether these organisms participate directly or indirectly in disease pathology remains unknown. Objective To determine the frequency of fungus isolation and fungus-specific immunity in Th2-associated and non-associated airway disease patients. Methods Sinus lavage fluid and blood were collected from sinus surgery patients (n=118) including CRS patients with and without nasal polyps and AFRS and non-CRS/non-asthmatic control patients. Asthma status was deteremined from medical history. Sinus lavage fluids were cultured and directly examined for evidence of viable fungi. Peripheral blood mononuclear cells were restimulated with fungal antigens in an enzyme linked immunocell spot (ELISpot) assay to determine total memory fungus-specific IL-4-secreting cells. These data were compared to fungus-specific IgE levels measured from plasma by ELISA. Results Filamentous fungi were significantly more commonly cultured from Th2-associated airway disease subjects (asthma, CRSwNP, or AFRS: n=68) compared to non-Th2-associated control patients (n=31); 74% vs 16% respectively, p<0.001. Both fungus-specific IL-4 ELISpot (n=48) and specific IgE (n=70) data correlated with Th2-associated diseases (sensitivity 73% and specificity 100% vs. 50% and 77%, respectively). Conclusions The frequent isolation of fungi growing directly within the airways accompanied by specific immunity to these organisms only in patients with Th2-associated chronic airway diseases suggests that fungi participate directly in the pathogenesis of these conditions. Efforts to eradicate airway fungi from the airways should be considered in selected patients. Clinical Implications Airway fungi may contribute to the expression of sinusitis with nasal polyps and

  11. Intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells

    SciTech Connect

    Verhaeghe, Catherine; Tabruyn, Sebastien P.; Oury, Cecile; Bours, Vincent . E-mail: vbours@ulg.ac.be; Griffioen, Arjan W.

    2007-05-11

    Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications.

  12. Regulation of TB vaccine-induced airway luminal T cells by respiratory exposure to endotoxin.

    PubMed

    Chen, Xuerong; Xiu, Fangming; Horvath, Carly N; Damjanovic, Daniela; Thanthrige-Don, Niroshan; Jeyanathan, Mangalakumari; Xing, Zhou

    2012-01-01

    Tuberculosis (TB) vaccine-induced airway luminal T cells (ALT) have recently been shown to be critical to host defense against pulmonary TB. However, the mechanisms that maintain memory ALT remain poorly understood. In particular, whether respiratory mucosal exposure to environmental agents such as endotoxin may regulate the size of vaccine-induced ALT population is still unclear. Using a murine model of respiratory genetic TB vaccination and respiratory LPS exposure, we have addressed this issue in the current study. We have found that single or repeated LPS exposure increases the number of antigen-specific ALT which are capable of robust secondary responses to pulmonary mycobacterial challenge. To investigate the potential mechanisms by which LPS exposure modulates the ALT population, we have examined the role of ALT proliferation and peripheral T cell recruitment. We have found that LPS exposure-increased ALT is not dependent on increased ALT proliferation as respiratory LPS exposure does not significantly increase the rate of proliferation of ALT. But rather, we find it to be dependent upon the recruitment of peripheral T cells into the airway lumen as blockade of peripheral T cell supplies markedly reduces the initially increased ALT. Thus, our data suggest that environmental exposure to airborne agents such as endotoxin has a profound modulatory effect on TB vaccine-elicited T cells within the respiratory tract. Our study provides a new, M.tb antigen-independent mechanism by which the respiratory mucosal anti-TB memory T cells may be maintained.

  13. 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

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

    PubMed

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

    2016-09-08

    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 Ca(2+) is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca(2+) elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca(2+) elevations in AECs through the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca(2+) 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.

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

    PubMed

    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 Ca(2+) is an important signaling event in this process, yet the fundamental mechanism by which allergens induce Ca(2+) elevations in AECs remains poorly understood. Here we find that extracts from dust mite and cockroach induce sustained Ca(2+) elevations in AECs through the activation of Ca(2+) release-activated Ca(2+) (CRAC) channels encoded by Orai1 and STIM1. CRAC channel activation occurs, at least in part, through allergen mediated stimulation of PAR2 receptors. The ensuing Ca(2+) 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

  16. Proteinases release /sup 35/S-labeled macromolecules from cultured airway epithelial cells

    SciTech Connect

    Varsano, S.; Borson, D.B.; Gold, M.; Forsberg, S.; Basbaum, C.B.; Nadel, J.A.

    1986-03-05

    To determine whether proteinases release radiolabeled macromolecules from airway cells devoid of secretory granules, they studied canine cultured tracheal epithelial cells grown to confluency. At this time the cells are bound by tight junctions, maintain anion transport, have a well developed glycocalyx, but contain no secretory granules. They labeled the cells with /sup 35/SO/sub 4/ (50..mu..ci/ml/24h) then changed the medium every 20 min and measured nondialyzable /sup 35/S released into the medium. Two h later, the rate of spontaneous release of /sup 35/S-labeled-macromolecules was 5700 +/- 1600 CPM/20 min (mean +/- SD). At this time trypsin, thermolysin, pseudomonas elastase and alkaline proteinase, each released /sup 35/S-labeled-macromolecules, whereas aspergillus acid proteinase did not. In more detailed studies, trypsin released /sup 35/S in a concentration dependent fashion, with a threshold below 10 units/ml and a response to 1000 units/ml of 1092 +/- 173% (mean +/- SD; n=5 cultures) above pre-trypsin baseline. Sepharose CL4B chromatography of the radiolabeled materials released by trypsin showed a void volume fraction (MW greater than or equal to 10/sup 6/), and a second, included fraction (MW 2-3 x 10/sup 5/). These results indicate that cultured airway epithelial cells synthesize macromolecules and release them into the medium, and that proteinases increase the rate of macromolecule release markedly.

  17. Airway Inflammation and Hypersensitivity Induced by Chronic Smoking

    PubMed Central

    Kou, Yu Ru; Kwong, Kevin; Lee, Lu-Yuan

    2011-01-01

    Airway hypersensitivity, characterized by enhanced excitability of airway sensory nerves, is a prominent pathophysiological feature in patients with airway inflammatory diseases. Although the underlying pathogenic mechanism is not fully understood, chronic airway inflammation is believed to be primarily responsible. Cigarette smoking is known to cause chronic airway inflammation, accompanied by airway hyperresponsiveness. Experimental evidence indicates that enhanced excitability of vagal bronchopulmonary sensory nerves and increased tachykinin synthesis in these nerves resulting from chronic inflammation are important contributing factors to the airway hyperresponsiveness. Multiple inflammatory mediators released from various types of structural and inflammatory cells are involved in the smoking-induced airway inflammation, which is mainly regulated by redox-sensitive signaling pathways and transcription factors. Furthermore, recent studies have reported potent sensitizing and stimulatory effects of these inflammatory mediators such as prostanoids and reactive oxygen species on these sensory nerves. In summary, these studies using cigarette smoking as an experimental approach have identified certain potentially important cell signaling pathways and underlying mechanisms of the airway hypersensitivity induced by chronic airway inflammation. PMID:21397052

  18. The intubation scoop (i-scoop) - a new type of laryngoscope for difficult and normal airways.

    PubMed

    Raymondos, K; Seidel, T; Sander, B; Gerdes, A; Goetz, F; Helmstädter, V; Panning, B; Dieck, T

    2014-09-01

    The i-scoop is an intubation device with a curved guiding bar with laterally located lenses at its tip, rather than a blade. Twenty-five anaesthesiologists intubated a manikin that simulated first a normal and then a difficult airway. All participants were able to intubate the difficult airway with a good view of the glottis using the i-scoop. None was able to intubate using seven other laryngoscopes (Macintosh laryngoscope, GlideScope(®) GVL and AVL, McGrath(®) (Series 5/MAC), C-MAC(®) , A.P. Advance(™) ). Intubation was successful only with the Airtraq(®) (n = 10), the Airway Scope (n = 5), the C-MAC D-Blade (n = 2), the A.P. Advance DAB (n = 1) and the GlideScope DL Trainer (n = 1) (p < 0.001, success rate of i-scoop vs all 12 laryngoscopes combined). In contrast to all other videolaryngoscopes, intubation of the normal airway with the i-scoop was achieved even faster than with the Macintosh laryngoscope (p < 0.02). The i-scoop outperformed all other laryngoscopes in both difficult and normal airways, and therefore has potential as an easier and safer alternative to present devices.

  19. Chromium(VI) stimulates Fyn to initiate innate immune gene induction in human airway epithelial cells

    PubMed Central

    Nemec, Antonia A.; Zubritsky, Lindsey M.; Barchowsky, Aaron

    2009-01-01

    Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes and we hypothesized this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation. PMID:19994902

  20. Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

    PubMed

    Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

    2013-12-01

    Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells.

  1. Rapamycin attenuates airway hyperreactivity, goblet cells, and IgE in experimental allergic asthma.

    PubMed

    Mushaben, Elizabeth M; Kramer, Elizabeth L; Brandt, Eric B; Khurana Hershey, Gurjit K; Le Cras, Timothy D

    2011-12-01

    The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease. PMID:22021618

  2. Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

    PubMed Central

    Berntsen, P.; Park, C. Y.; Rothen-Rutishauser, B.; Tsuda, A.; Sager, T. M.; Molina, R. M.; Donaghey, T. C.; Alencar, A. M.; Kasahara, D. I.; Ericsson, T.; Millet, E. J.; Swenson, J.; Tschumperlin, D. J.; Butler, J. P.; Brain, J. D.; Fredberg, J. J.; Gehr, P.; Zhou, E. H.

    2010-01-01

    The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40–100 nm and less than 44 μm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 μm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 μM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases. PMID:20356875

  3. Rapamycin attenuates airway hyperreactivity, goblet cells, and IgE in experimental allergic asthma.

    PubMed

    Mushaben, Elizabeth M; Kramer, Elizabeth L; Brandt, Eric B; Khurana Hershey, Gurjit K; Le Cras, Timothy D

    2011-12-01

    The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues, promotes cell growth/differentiation, and regulates immune responses. Although inhibition of mTOR with rapamycin has potent immunosuppressive activity, mixed effects have been reported in OVA-induced models of allergic asthma. We investigated the impact of two rapamycin treatment protocols on the major characteristics of allergic asthma induced by the clinically relevant allergen, house dust mite (HDM). In protocol 1, BALB/c mice were exposed to 10 intranasal HDM doses over a period of 24 d and treated with rapamycin simultaneously during the sensitization/exposure period. In protocol 2, rapamycin was administered after the mice had been sensitized to HDM (i.p. injection) and prior to initiation of two intranasal HDM challenges over 4 d. Airway hyperreactivity (AHR), IgE, inflammatory cells, cytokines, leukotrienes, goblet cells, and activated T cells were assessed. In protocol 1, rapamycin blocked HDM-induced increases in AHR, inflammatory cell counts, and IgE, as well as attenuated goblet cell metaplasia. In protocol 2, rapamycin blocked increases in AHR, IgE, and T cell activation and reduced goblet cell metaplasia, but it had no effect on inflammatory cell counts. Increases in IL-13 and leukotrienes were also blocked by rapamycin, although increases in IL-4 were unaffected. These data demonstrated that rapamycin can inhibit cardinal features of allergic asthma, including increases in AHR, IgE, and goblet cells, most likely as a result of its ability to reduce the production of two key mediators of asthma: IL-13 and leukotrienes. These findings highlight the importance of the mTOR pathway in allergic airway disease.

  4. Do Airway Epithelium Air–Liquid Cultures Represent the In Vivo Airway Epithelium Transcriptome?

    PubMed Central

    Dvorak, Anna; Tilley, Ann E.; Shaykhiev, Renat; Wang, Rui; Crystal, Ronald G.

    2011-01-01

    Human airway epithelial cells cultured in vitro at the air–liquid interface (ALI) form a pseudostratified epithelium that forms tight junctions and cilia, and produces mucin. These cells are widely used in models of differentiation, injury, and repair. To assess how closely the transcriptome of ALI epithelium matches that of in vivo airway epithelial cells, we used microarrays to compare the transcriptome of human large airway epithelial cells cultured at the ALI with the transcriptome of large airway epithelium obtained via bronchoscopy and brushing. Gene expression profiling showed that global gene expression correlated well between ALI cells and brushed cells, but with some differences. Gene expression patterns mirrored differences in proportions of cell types (ALIs have higher percentages of basal cells, whereas brushed cells have higher percentages of ciliated cells), that is, ALI cells expressed higher levels of basal cell–related genes, and brushed cells expressed higher levels of cilia-related genes. Pathway analysis showed that ALI cells had increased expression of cell cycle and proliferation genes, whereas brushed cells had increased expression of cytoskeletal organization and humoral immune response genes. Overall, ALI cells provide a good representation of the in vivo airway epithelial transcriptome, but for some biologic questions, the differences between in vitro and in vivo environments need to be considered. PMID:20525805

  5. Aggregates of mutant CFTR fragments in airway epithelial cells of CF lungs: new pathologic observations.

    PubMed

    Du, Kai; Karp, Philip H; Ackerley, Cameron; Zabner, Joseph; Keshavjee, Shaf; Cutz, Ernest; Yeger, Herman

    2015-03-01

    Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl(-) channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary "conformational protein-associated" diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.

  6. T-cell–intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy

    PubMed Central

    Perez-Lloret, Jimena; Okoye, Isobel S.; Guidi, Riccardo; Kannan, Yashaswini; Coomes, Stephanie M.; Czieso, Stephanie; Mengus, Gabrielle; Davidson, Irwin; Wilson, Mark S.

    2016-01-01

    There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ+CD4+ T-helper 2 (TH2) cells orchestrate the type-2–driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081–E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4gfp+αβ+CD4+ TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell–intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24−/− T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1–regulated signaling. Following this prediction, we found that Trim24−/− T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β–mediated activation in vitro and in vivo, and fail to respond to IL-1β–exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell–intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity. PMID:26787865

  7. Variation in the Type, Rate, and Selection of Patients for Out-of-hospital Airway Procedures Among Injured Children and Adults

    PubMed Central

    Newgard, Craig D.; Koprowicz, Kent; Wang, Henry; Monnig, Aaron; Kerby, Jeffrey D.; Sears, Gena K.; Davis, Daniel P.; Bulger, Eileen; Stephens, Shannon W.; Daya, Mohamud R.

    2014-01-01

    Objectives The objective was to compare the type, rate, and selection of injured patients for out-of-hospital airway procedures among emergency medical services (EMS) agencies in 10 sites across North America. Methods The authors analyzed a consecutive patient, prospective cohort registry of injured adults and children with an out-of-hospital advanced airway attempt, collected from December 1, 2005, through February 28, 2007, by 181 EMS agencies in 10 sites across the United States and Canada. Advanced airway procedures were defined as orotracheal intubation, nasotracheal intubation, supraglottic airway, or cricothyrotomy. Airway procedure rates were calculated based on age-specific population values for the 10 sites and the number of injured patients with field physiologic abnormality (systolic blood pressure of ≤90 mm Hg, respiratory rate of <10 or >29 breaths / min, Glasgow Coma Scale [GCS] score of ≤12). Descriptive measures were used to compare patients between sites. Results A total 1,738 patients had at least one advanced airway attempt and were included in the analysis. There was wide variation between sites in the types of airway procedures performed, including orotracheal intubation (63% to 99%), supraglottic airways (0 to 27%), nasotracheal intubation (0 to 21%), and cricothyrotomy (0 to 2%). Use of rapid sequence intubation (RSI) varied from 0% to 65%. The population-adjusted rates of field airway intervention (by site) ranged from 1.2 to 22.8 per 100,000 adults and 0.2 to 4.0 per 100,000 children. Among trauma patients with physiologic abnormality, some sites performed airway procedures in almost 50% of patients, while other sites used these procedures in fewer than 10%. There was also large variation in demographic characteristics, physiologic measures, mechanism of injury, mode of transport, field cardiopulmonary resuscitation, and unadjusted mortality among airway patients. Conclusions Among 10 sites across North America, there was wide

  8. Mucosal-associated invariant T cells in autoimmunity, immune-mediated diseases and airways disease.

    PubMed

    Hinks, Timothy S C

    2016-05-01

    Mucosal-associated invariant T (MAIT) cells are a novel class of innate-like T cells, expressing a semi-invariant T-cell receptor (TCR) and able to recognize small molecules presented on the non-polymorphic MHC-related protein 1. Their intrinsic effector-memory phenotype, enabling secretion of pro-inflammatory cytokines, and their relative abundance in humans imply a significant potential to contribute to autoimmune processes. However, as MAIT cells were unknown until recently and specific immunological tools were unavailable, little is known of their roles in disease. Here I review observations from clinical studies and animal models of autoimmune and immune-mediated diseases including the roles of MAIT cells in systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and airways diseases. MAIT cell deficiencies are frequently observed in peripheral blood, and at sites of disease such as the airways in asthma. However, MAIT cells have a specific sensitivity to suppression by therapeutic corticosteroids that may confound many of these observations, as may the tendency of the surface marker CD161 to activation-induced down-regulation. Nonetheless, the dependence on bacteria for the development of MAIT cells suggests a potentially important protective role linking the influences of early life microbial exposures and subsequent development of autoimmunity. Conversely, MAIT cells could contribute to chronic inflammation either through TCR-independent activation, or potentially by TCR recognition of as yet undiscovered ligands. Future research will be greatly facilitated by the immunological tools that are now available, including murine genetic models and human and murine specific tetramers.

  9. Mucosal-associated invariant T cells in autoimmunity, immune-mediated diseases and airways disease.

    PubMed

    Hinks, Timothy S C

    2016-05-01

    Mucosal-associated invariant T (MAIT) cells are a novel class of innate-like T cells, expressing a semi-invariant T-cell receptor (TCR) and able to recognize small molecules presented on the non-polymorphic MHC-related protein 1. Their intrinsic effector-memory phenotype, enabling secretion of pro-inflammatory cytokines, and their relative abundance in humans imply a significant potential to contribute to autoimmune processes. However, as MAIT cells were unknown until recently and specific immunological tools were unavailable, little is known of their roles in disease. Here I review observations from clinical studies and animal models of autoimmune and immune-mediated diseases including the roles of MAIT cells in systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and airways diseases. MAIT cell deficiencies are frequently observed in peripheral blood, and at sites of disease such as the airways in asthma. However, MAIT cells have a specific sensitivity to suppression by therapeutic corticosteroids that may confound many of these observations, as may the tendency of the surface marker CD161 to activation-induced down-regulation. Nonetheless, the dependence on bacteria for the development of MAIT cells suggests a potentially important protective role linking the influences of early life microbial exposures and subsequent development of autoimmunity. Conversely, MAIT cells could contribute to chronic inflammation either through TCR-independent activation, or potentially by TCR recognition of as yet undiscovered ligands. Future research will be greatly facilitated by the immunological tools that are now available, including murine genetic models and human and murine specific tetramers. PMID:26778581

  10. Reversal of established CD4+ type 2 T helper-mediated allergic airway inflammation and eosinophilia by therapeutic treatment with DNA vaccines limits progression towards chronic inflammation and remodelling

    PubMed Central

    Jarman, Elizabeth R; Lamb, Jonathan R

    2004-01-01

    Immunostimulatory DNA-based vaccines can prevent the induction of CD4+ type 2 T helper (Th2) cell-mediated airway inflammation in experimental models, when administered before or at the time of allergen exposure. Here we demonstrate their efficacy in limiting the progression of an established response to chronic pulmonary inflammation and airway remodelling on subsequent allergen challenge. Mice exhibiting Th2-mediated airway inflammation induced following sensitization and challenge with group 1 allergen derived from Dermatophagoides pteronyssinus group species (Der p 1), a major allergen of house dust mite, were treated with pDNA vaccines. Their airways were rechallenged and the extent of inflammation assessed. In plasma DNA (pDNA)-vaccinated mice, infiltration of inflammatory cells, goblet cell hyperplasia and mucus production were reduced and subepithelial fibrosis attenuated. The reduction in eosinophil numbers correlated with a fall in levels of the profibrotic mediator transforming growth factor (TGF)-β1 in bronchoalveolar lavage (BAL) and lung tissue. In addition to lung epithelial cells and resident alveolar macrophages, infiltrating eosinophils, the principle inflammatory cells recruited following allergen exposure, were a major source of TGF-β1. Protection, conferred irrespective of the specificity of the pDNA construct, did not correlate with a sustained increase in systemic interferon (IFN)-γ production but in a reduction in levels of the Th2 pro-inflammatory cytokines. Notably, there was a reduction in levels of interleukin (IL)-5 and IL-13 produced by systemic Der p 1 reactive CD4+ Th2 cells on in vitro stimulation as well as in IL-4 and IL-5 levels in BAL fluid. These data suggest that suppression of CD4+ Th2-mediated inflammation and eosinophilia were sufficient to attenuate progression towards airway remodelling. Immunostimulatory DNA may therefore have a therapeutic application in treatment of established allergic asthma in patients. PMID

  11. Types of Stem Cells

    MedlinePlus

    ... PDF) Download an introduction to stem cells and stem cell research. Stem Cell Glossary Stem cell terms to know. ... stem cells blog from the International Society for Stem Cell Research. Learn About Stem Cells From Lab to You ...

  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. IL-17A mediates a selective gene expression profile in asthmatic human airway smooth muscle cells.

    PubMed

    Dragon, Stéphane; Hirst, Stuart J; Lee, Tak H; Gounni, Abdelilah S

    2014-06-01

    Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1β or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal-regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells.

  14. 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.

  15. Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells.

    PubMed

    Zhao, Rui; Fallon, Timothy R; Saladi, Srinivas Vinod; Pardo-Saganta, Ana; Villoria, Jorge; Mou, Hongmei; Vinarsky, Vladimir; Gonzalez-Celeiro, Meryem; Nunna, Naveen; Hariri, Lida P; Camargo, Fernando; Ellisen, Leif W; Rajagopal, Jayaraj

    2014-07-28

    Our understanding of how stem cells are regulated to maintain appropriate tissue size and architecture is incomplete. We show that Yap (Yes-associated protein 1) is required for the actual maintenance of an adult mammalian stem cell. Without Yap, adult airway basal stem cells are lost through their unrestrained differentiation, resulting in the simplification of a pseudostratified epithelium into a columnar one. Conversely, Yap overexpression increases stem cell self-renewal and blocks terminal differentiation, resulting in epithelial hyperplasia and stratification. Yap overexpression in differentiated secretory cells causes them to partially reprogram and adopt a stem cell-like identity. In contrast, Yap knockdown prevents the dedifferentiation of secretory cells into stem cells. We then show that Yap functionally interacts with p63, the cardinal transcription factor associated with myriad epithelial basal stem cells. In aggregate, we show that Yap regulates all of the cardinal behaviors of airway epithelial stem cells and determines epithelial architecture.

  16. The Role of Lysophosphatidic Acid on Airway Epithelial Cell Denudation in a Murine Heterotopic Tracheal Transplant Model

    PubMed Central

    Tando, Yukiko; Ota, Chiharu; Yamada, Mitsuhiro; Kamata, Satoshi; Yamaya, Mutsuo; Kano, Kuniyuki; Okudaira, Shinichi; Aoki, Junken; Kubo, Hiroshi

    2015-01-01

    Background Chronic rejection is the major leading cause of morbidity and mortality after lung transplantation. Obliterative bronchiolitis (OB), a fibroproliferative disorder of the small airways, is the main manifestation of chronic lung allograft rejection. However, there is currently no treatment for the disease. We hypothesized that lysophosphatidic acid (LPA) participates in the progression of OB. The aim of this study was to reveal the involvement of LPA on the lesion of OB. Methods Ki16198, an antagonist specifically for LPA1 and LPA3, was daily administered into the heterotopic tracheal transplant model mice at the day of transplantation. At days 10 and 28, the allografts were isolated and evaluated histologically. The messenger RNA levels of LPAR in microdissected mouse airway regions were assessed to reveal localization of lysophosphatidic acid receptors. The human airway epithelial cell was used to evaluate the mechanism of LPA-induced suppression of cell adhesion to the extracellular matrix (ECM). Results The administration of Ki16198 attenuated airway epithelial cell loss in the allograft at day 10. Messenger RNAs of LPA1 and LPA3 were detected in the airway epithelial cells of the mice. Lysophosphatidic acid inhibited the attachment of human airway epithelial cells to the ECM and induced cell detachment from the ECM, which was mediated by LPA1 and Rho-kinase pathway. However, Ki16198 did not prevent obliteration of allograft at day 28. Conclusions The LPA signaling is involved in the status of epithelial cells by distinct contribution in 2 different phases of the OB lesion. This finding suggests a role of LPA in the pathogenesis of OB. PMID:27500235

  17. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

    PubMed Central

    Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee

    2015-01-01

    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases. PMID:26599511

  18. Spatial and temporal traction response in human airway smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Tolic-Norrelykke, Iva Marija; Butler, James P.; Chen, Jianxin; Wang, Ning

    2002-01-01

    Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

  19. Lipidome and Transcriptome Profiling of Pneumolysin Intoxication Identifies Networks Involved in Statin-Conferred Protection of Airway Epithelial Cells

    PubMed Central

    Statt, Sarah; Ruan, Jhen-Wei; Huang, Chih-Ting; Wu, Reen; Kao, Cheng-Yuan

    2015-01-01

    Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin. PMID:26023727

  20. 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.

  1. Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

    PubMed Central

    Kalbe, Benjamin; Knobloch, Jürgen; Schulz, Viola M.; Wecker, Christine; Schlimm, Marian; Scholz, Paul; Jansen, Fabian; Stoelben, Erich; Philippou, Stathis; Hecker, Erich; Lübbert, Hermann; Koch, Andrea; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca2+ increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases. PMID:27540365

  2. Inhibitory action of NoxA1 on dual oxidase activity in airway cells.

    PubMed

    Pacquelet, Sandrine; Lehmann, Mandy; Luxen, Sylvia; Regazzoni, Karine; Frausto, Monika; Noack, Deborah; Knaus, Ulla G

    2008-09-01

    Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.

  3. Establishment and transformation of telomerase-immortalized human small airway epithelial cells by heavy ions

    NASA Astrophysics Data System (ADS)

    Zhao, Y. L.; Piao, C. Q.; Hei, T. K.

    Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of 1 GeV/nucleon of 56Fe ions accelerated at the Brookhaven National Laboratory. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

  4. Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

    PubMed Central

    2012-01-01

    Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells. PMID:22694362

  5. Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

    PubMed Central

    Spiess, Page C.; Morin, Dexter; Williams, Chase R.; Buckpitt, Alan R.

    2010-01-01

    Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, β-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA. PMID:19843705

  6. Precursor B Cells Increase in the Lung during Airway Allergic Inflammation: A Role for B Cell-Activating Factor

    PubMed Central

    Malmhäll, Carina; Rådinger, Madeleine; Ramos-Ramirez, Patricia; Lu, You; Deák, Tünde; Semitekolou, Maria; Gaga, Mina; Sjöstrand, Margareta; Lötvall, Jan; Bossios, Apostolos

    2016-01-01

    Background B cells, key cells in allergic inflammation, differentiate in the bone marrow and their precursors include pro-B, pre-B and immature B cells. Eosinophil progenitor cells increase in the lung after allergen exposure. However, the existence and possible role of B cell precursors in the lung during allergic inflammation remains elusive. Methods A BALB/c mouse model of allergic airway inflammation was utilized to perform phenotypic and quantification analyses of pro-B and pre-B cells in the lung by flow cytometry. B cell maturation factors IL-7 and B cell-activating factor (BAFF) and their receptors (CD127 and BAFFR, BCMA, TACI, respectively) were also evaluated in the lung and serum. The effect of anti-BAFF treatment was investigated both in vivo (i.p. administration of BAFF-R-Ig fusion protein) and in vitro (colony forming cell assay). Finally, BAFF levels were examined in the bronchoalveolar lavage (BAL) of asthmatic patients and healthy controls. Results Precursor pro and pre-B cells increase in the lung after allergen exposure, proliferate in the lung tissue in vivo, express markers of chemotaxis (CCR10 and CXCR4) and co-stimulation (CD40, CD86) and are resistant to apoptosis (Bax). Precursor B cells express receptors for BAFF at baseline, while after allergen challenge both their ligand BAFF and the BCMA receptor expression increases in B cell precursors. Blocking BAFFR in the lung in vivo decreases eosinophils and proliferating precursor B cells. Blocking BAFFR in bone marrow cultures in vitro reduces pre-B colony formation units. BAFF is increased in the BAL of severe asthmatics. Conclusion Our data support the concept of a BAFF-mediated role for B cell precursors in allergic airway inflammation. PMID:27513955

  7. Ozone and allergen exposure during postnatal development alters the frequency and airway distribution of CD25+ cells in infant rhesus monkeys

    SciTech Connect

    Miller, Lisa A. Gerriets, Joan E.; Tyler, Nancy K.; Abel, Kristina; Schelegle, Edward S.; Plopper, Charles G.; Hyde, Dallas M.

    2009-04-01

    The epidemiologic link between air pollutant exposure and asthma has been supported by experimental findings, but the mechanisms are not understood. In this study, we evaluated the impact of combined ozone and house dust mite (HDM) exposure on the immunophenotype of peripheral blood and airway lymphocytes from rhesus macaque monkeys during the postnatal period of development. Starting at 30 days of age, monkeys were exposed to 11 cycles of filtered air, ozone, HDM aerosol, or ozone + HDM aerosol. Each cycle consisted of ozone delivered at 0.5 ppm for 5 days (8 h/day), followed by 9 days of filtered air; animals received HDM aerosol during the last 3 days of each ozone exposure period. Between 2-3 months of age, animals co-exposed to ozone + HDM exhibited a decline in total circulating leukocyte numbers and increased total circulating lymphocyte frequency. At 3 months of age, blood CD4+/CD25+ lymphocytes were increased with ozone + HDM. At 6 months of age, CD4+/CD25+ and CD8+/CD25+ lymphocyte populations increased in both blood and lavage of ozone + HDM animals. Overall volume of CD25+ cells within airway mucosa increased with HDM exposure. Ozone did not have an additive effect on volume of mucosal CD25+ cells in HDM-exposed animals, but did alter the anatomical distribution of this cell type throughout the proximal and distal airways. We conclude that a window of postnatal development is sensitive to air pollutant and allergen exposure, resulting in immunomodulation of peripheral blood and airway lymphocyte frequency and trafficking.

  8. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

    PubMed Central

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

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection. PMID:27354786

  9. Effect of Polarization on Airway Epithelial Conditioning of Monocyte-Derived Dendritic Cells.

    PubMed

    Papazian, Dick; Chhoden, Tashi; Arge, Maria; Vorup-Jensen, Thomas; Nielsen, Claus H; Lund, Kaare; Würtzen, Peter A; Hansen, Soren

    2015-09-01

    Airway epithelial cells (AECs) form polarized barriers that interact with inhaled allergens and are involved in immune homeostasis. We examined how monocyte-derived dendritic cells (MDDCs) are affected by contact with the airway epithelium. In traditional setups, bronchial epithelial cell lines were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs' immunoregulatory properties; thus, previous observations obtained using traditional setups should be considered with caution. Using the optimized setup, AEC conditioning of MDDCs led to increased expression of programmed death 1 ligand 1, immunoglobulin-like transcript 3, CD40, CD80, and CD23. This increased expression was accompanied by decreased secretion of monocyte chemotactic protein 1 and eotaxin and donor-variable effects on IL-12 and IL-10 secretion. Conditioning varied between maturation states and depended partly on direct contact between AECs and MDDCs. The setup allowed MDDCs on the basal side of the epithelium to sample allergens administered to the apical side. Allergen uptake depended on polarization and the nature of the allergen. AEC conditioning led to decreased birch allergen-specific proliferation of autologous T cells and a trend toward decreased secretion of the Th2-specific cytokines IL-5 and IL-13. In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens.

  10. Tissue Inhibitor of Metalloproteinase-1 Moderates Airway Re-Epithelialization by Regulating Matrilysin Activity

    PubMed Central

    Chen, Peter; McGuire, John K.; Hackman, Robert C.; Kim, Kyoung-Hee; Black, Roy A.; Poindexter, Kurt; Yan, Wei; Liu, Phillip; Chen, Ann J.; Parks, William C.; Madtes, David K.

    2008-01-01

    Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration. PMID:18385523

  11. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  12. Upregulation of TRPM7 augments cell proliferation and interleukin-8 release in airway smooth muscle cells of rats exposed to cigarette smoke

    PubMed Central

    LIN, XIAOLING; YANG, CHENG; HUANG, LINJIE; CHEN, MING; SHI, JIANTING; OUYANG, LIHUA; TANG, TIANTIAN; ZHANG, WEI; LI, YIQUN; LIANG, RUIYUN; JIANG, SHANPING

    2016-01-01

    Proliferation and synthetic function (i.e. the capacity to release numerous chemokines and cytokines) of airway smooth muscle cells (ASMCs) are important in airway remodeling induced by cigarette smoke exposure. However, the molecular mechanism has not been clarified. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is expressed ubiquitously and is crucial for the cellular physiological function of many cell types. The present study aimed to detect the expression of TRPM7 in ASMCs from smoke-exposed rats and determine the importance of TRPM7 in proliferation and interleukin-8 (IL-8) release. ASMCs were isolated and cultured from smoke-exposed rats. Expression levels of TRPM7 were determined by reverse transcription-polymerase chain reaction, western blot analysis and immunofluorescence. TRPM7 was silenced with TRPM7-short hairpin RNA lentivirus vector. DNA synthesis, cell number and IL-8 release of ASMCs induced by cigarette smoke extract (CSE) and tumor necrosis factor-α (TNF-α) were assessed using [3H]-thymidine incorporation assay, hemocytometer and enzyme-linked immunosorbent assay, respectively. It was determined that mRNA and protein expression levels of TRPM7 were increased in ASMCs from smoke-exposed rats. Stimulation with CSE or TNF-α elevated DNA synthesis, cell number and IL-8 release were more marked in ASMCs from smoke-exposed rats. Silencing of TRPM7 reduced DNA synthesis, cell number and IL-8 release induced by CSE or TNF-α in ASMCs from smoke-exposed rats. In conclusion, expression of TRPM7 increased significantly in ASMCs from smoke-exposed rats and the upregulation of TRPM7 led to augmented cell proliferation and IL-8 release in ASMCs from rats exposed to cigarette smoke. PMID:27108806

  13. The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells.

    PubMed

    Yang, Chao-Huei; Tsao, Chiung-Fang; Ko, Wang-Sheng; Chiou, Ya-Ling

    2016-01-09

    In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%-99% after 48 h (p < 0.05) and induced G₁/G₀ cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials.

  14. 17β-Aminoestrogens induce guinea pig airway smooth muscle hyperresponsiveness through L-type Ca(2+) channels activation.

    PubMed

    Flores-Soto, Edgar; Martínez-Villa, Inocencio; Solís-Chagoyán, Héctor; Sommer, Bettina; Lemini, Cristina; Montaño, Luis M

    2015-09-01

    Therapy with estrogens is frequently used in menopausal women and as hormonal contraception. Because of its thrombotic effects, long term estrogen administration used in hormonal replacement therapy (HRT) and contraception could represent a health hazard. In this regard, 17β-aminoestrogens such as aminoestrol, butolame and pentolame have shown promising HRT potential, because they have a weak agonist estrogenic action and antithrombotic activity. Additionally, estrogens play a protective role in airway smooth muscle, but the effect of 17β-aminoestrogens on the airway smooth muscle has not been tested yet. In guinea pig tracheal smooth muscle pentolame and butolame induced hyperresponsiveness to histamine (His), carbachol (Cch) and KCl. Interestingly, aminoestrol did not show this effect at the highest concentration studied, it even lowered the contraction induced by Cch. The hyperresponsiveness induced by pentolame to His was abolished by nifedipine. In single tracheal myocytes, KCl induced an increment in the intracellular Ca(2+) concentration [Ca(2+)]i, pentolame also showed an increase in [Ca(2+)]i and the addition of KCl in the plateau of this rise further significantly augmented the [Ca(2+)]i response. Additionally, in patch clamp experiments pentolame increased the L-type Ca(2+) currents. Thus, 17β-aminoestrogens such as pentolame and butolame, but not aminoestrol, activate L-type Ca(2+) channel to induced hyperresponsiveness to Cch, His and KCl in guinea pig tracheal smooth muscle. Due to its lack of effect on airways and to its anticoagulant characteristics, aminoestrol seems to be the best alternative in the HRT among the 17β-aminoestrogens studied.

  15. Atmospheric remote sensing to detect effects of temperature inversions on sputum cell counts in airway diseases.

    PubMed

    Wallace, Julie; Nair, Parameswaran; Kanaroglou, Pavlos

    2010-08-01

    Temperature inversions result in the accumulation of air pollution, often to levels exceeding air quality criteria. The respiratory response may be detectable in sputum cell counts. This study investigates the effect of boundary layer temperature inversions on sputum cell counts. Total and differential cell counts of neutrophils, eosinophils, macrophages and lymphocytes were quantified in sputum samples of patients attending an outpatient clinic. Temperature inversions were identified using data from the Atmospheric Infrared Sounder, an atmospheric sensor on the Aqua spacecraft which was launched in 2002 by the National Aeronautics and Space Administration. On inversion days, a statistically significant increase in the percent of cells that were neutrophils was observed in stable patients. There was also a statistically significant increase in the percent of cells that were macrophages, in exacerbated patients. Multivariate linear regression models were used to assess the relationship between temperature inversions and cell counts, controlling patients' age, smoking status, medications and meteorological variables of temperature and humidity. The analyses indicate that, in the stable and exacerbated groups, percent neutrophils and macrophages increased by 12.6% and 2.5%, respectively, on inversion days. These results suggest that temperature inversions need consideration as an exacerbating factor in bronchitis and obstructive airway disease. The effects of air pollutants, nitrogen dioxide, carbon monoxide, fine particulate matter and ozone, were investigated. We identified no significant associations with any pollutant. However, we found that monthly averages of total cell counts were strongly correlated with monthly nitrogen dioxide concentrations, an association not previously identified in the literature.

  16. Regenerative potential of human airway stem cells in lung epithelial engineering.

    PubMed

    Gilpin, Sarah E; Charest, Jonathan M; Ren, Xi; Tapias, Luis F; Wu, Tong; Evangelista-Leite, Daniele; Mathisen, Douglas J; Ott, Harald C

    2016-11-01

    Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair. PMID:27622532

  17. Evaluation of airway resistance in primary small cell carcinoma of the trachea by MostGraph: a case study

    PubMed Central

    Hagiwara, Eri; Hayashi, Kentaro; Takahashi, Mai; Iida, Yuko; Hiranuma, Hisato; Nakagawa, Yoshiko; Hataoka, Tsukasa; Mizumura, Kenji; Maruoka, Shuichiro; Shimizu, Tetsuo; Takahashi, Noriaki; Hashimoto, Shu

    2016-01-01

    The case subject was a 58-year-old woman who presented to our hospital with a chief complaint of respiratory discomfort. Wheezing could be heard in both lungs; treatment was initiated with inhaled steroids for suspected bronchial asthma. However, 1 week later, the respiratory discomfort had not improved and the wheezing sound had progressed to the neck area. Upper airway obstruction was suspected; therefore, chest computed tomography (CT) was performed, revealing tracheal stenosis caused by a tumor in the upper airway. Because of the high risk of airway obstruction, tracheotomy and tracheal tumor resection were performed. Histopathological examination of the resected tumor revealed small cell lung cancer (SCLC); the stage was determined to be clinical stage IIIB (cT4N2M0), for which chemotherapy with two cycles of cisplatin plus etoposide followed by radiation therapy were administered. Pulmonary function testing revealed no change in the forced expiratory volume in 1 sec and flow volume (FV) curve before and after tumor resection, whereas airway resistance measured by MostGraph-01 showed a marked decrease following treatment. We believe that MostGraph-01 may be useful for measuring airway resistance and evaluating a tracheal tumor, and report a case using MostGraph-01.

  18. Evaluation of airway resistance in primary small cell carcinoma of the trachea by MostGraph: a case study.

    PubMed

    Hagiwara, Eri; Gon, Yasuhiro; Hayashi, Kentaro; Takahashi, Mai; Iida, Yuko; Hiranuma, Hisato; Nakagawa, Yoshiko; Hataoka, Tsukasa; Mizumura, Kenji; Maruoka, Shuichiro; Shimizu, Tetsuo; Takahashi, Noriaki; Hashimoto, Shu

    2016-08-01

    The case subject was a 58-year-old woman who presented to our hospital with a chief complaint of respiratory discomfort. Wheezing could be heard in both lungs; treatment was initiated with inhaled steroids for suspected bronchial asthma. However, 1 week later, the respiratory discomfort had not improved and the wheezing sound had progressed to the neck area. Upper airway obstruction was suspected; therefore, chest computed tomography (CT) was performed, revealing tracheal stenosis caused by a tumor in the upper airway. Because of the high risk of airway obstruction, tracheotomy and tracheal tumor resection were performed. Histopathological examination of the resected tumor revealed small cell lung cancer (SCLC); the stage was determined to be clinical stage IIIB (cT4N2M0), for which chemotherapy with two cycles of cisplatin plus etoposide followed by radiation therapy were administered. Pulmonary function testing revealed no change in the forced expiratory volume in 1 sec and flow volume (FV) curve before and after tumor resection, whereas airway resistance measured by MostGraph-01 showed a marked decrease following treatment. We believe that MostGraph-01 may be useful for measuring airway resistance and evaluating a tracheal tumor, and report a case using MostGraph-01. PMID:27621904

  19. Evaluation of airway resistance in primary small cell carcinoma of the trachea by MostGraph: a case study

    PubMed Central

    Hagiwara, Eri; Hayashi, Kentaro; Takahashi, Mai; Iida, Yuko; Hiranuma, Hisato; Nakagawa, Yoshiko; Hataoka, Tsukasa; Mizumura, Kenji; Maruoka, Shuichiro; Shimizu, Tetsuo; Takahashi, Noriaki; Hashimoto, Shu

    2016-01-01

    The case subject was a 58-year-old woman who presented to our hospital with a chief complaint of respiratory discomfort. Wheezing could be heard in both lungs; treatment was initiated with inhaled steroids for suspected bronchial asthma. However, 1 week later, the respiratory discomfort had not improved and the wheezing sound had progressed to the neck area. Upper airway obstruction was suspected; therefore, chest computed tomography (CT) was performed, revealing tracheal stenosis caused by a tumor in the upper airway. Because of the high risk of airway obstruction, tracheotomy and tracheal tumor resection were performed. Histopathological examination of the resected tumor revealed small cell lung cancer (SCLC); the stage was determined to be clinical stage IIIB (cT4N2M0), for which chemotherapy with two cycles of cisplatin plus etoposide followed by radiation therapy were administered. Pulmonary function testing revealed no change in the forced expiratory volume in 1 sec and flow volume (FV) curve before and after tumor resection, whereas airway resistance measured by MostGraph-01 showed a marked decrease following treatment. We believe that MostGraph-01 may be useful for measuring airway resistance and evaluating a tracheal tumor, and report a case using MostGraph-01. PMID:27621904

  20. Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys

    PubMed Central

    Schaap-Nutt, Anne; Scull, Margaret A.; Schmidt, Alexander C.; Murphy, Brian R.; Pickles, Raymond J.

    2010-01-01

    Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32°C and exhibited little productive replication at 37°C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates. PMID:20139039

  1. INCREASED IL-8 AND IL-6 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    INCREASED IL-6 AND IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES.
    R Silbajoris1, A G Lenz2, I Jaspers3, J M Samet1. 1NHEERL, USEPA, RTP, NC, USA; 2GSF-Institute for Inhalation Biology, Neuherberg, Germany; 3 CEMLB, UNC-CH, Chapel Hill, ...

  2. EFFECT OF INHALED ENDOTOXIN ON AIRWAY AND CIRCULATING INFLAMMATORY CELL PHAGOCYTOSIS AND CD11B EXPRESSION IN ATOPIC ASTHMATIC SUBJECTS

    EPA Science Inventory

    Effect of inhaled endotoxin on airway and circulating inflammatory cell phagocytosis and CD11b expression in atopic asthmatic subjects

    Neil E. Alexis, PhD, Marlowe W. Eldridge, MD, David B. Peden, MD, MS

    Chapel Hill and Research Triangle Park, NC

    Backgrou...

  3. ZN2+-INDUCED IL-8 EXPRESSION INVOLVES AP-1, JNK, AND ERK ACTIVITIES IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Exposure to zinc-laden particulate matter (PM) in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zin...

  4. ULTRAFINE CARBON PARTICLES INDUCE IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS THROUGH A POST-TRANSCRIPTIONAL MECHANISM

    EPA Science Inventory

    Ultrafine carbon particles induce IL-8 expression in human airway
    epithelial cells through a post-transcritpional mechanism
    Epidemiological studies suggest that ultrafine particles contribute to
    particulate matter (PM) - induced adverse health effects. IL-8 is an
    i...

  5. IN VITRO EFFECTS OF PARTICULATE MATTER ON AIRWAY EPITHELIAL CELLS ISOLATED FROM CONCENTRATED AIR PARTICLES-EXPOSED SPONTANEOUS HYPERTENSIVE RATS

    EPA Science Inventory

    In vitro effects of particulate matter on airway epithelial cells isolated from concentrated air particles-exposed spontaneous hypertensive rats

    Ines Pagan, Urmila Kodavanti, Paul Evansky, Daniel L Costa and Janice A Dye. U.S. Environmental Protection Agency, ORD, National...

  6. Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds

    PubMed Central

    Morris, G. E.; Bridge, J. C.; Eltboli, O. M. I.; Lewis, M. P.; Knox, A. J.; Aylott, J. W.; Brightling, C. E.; Ghaemmaghami, A. M.

    2014-01-01

    Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment. PMID:24793171

  7. Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells.

    PubMed

    Schwarzer, Christian; Fischer, Horst; Machen, Terry E

    2016-01-01

    Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10-80 fold increase, termed "swarming"), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds. PMID:27031335

  8. Airway-Resident Memory CD8 T Cells Provide Antigen-Specific Protection against Respiratory Virus Challenge through Rapid IFN-γ Production.

    PubMed

    McMaster, Sean R; Wilson, Jarad J; Wang, Hong; Kohlmeier, Jacob E

    2015-07-01

    CD8 airway resident memory T (TRM) cells are a distinctive TRM population with a high turnover rate and a unique phenotype influenced by their localization within the airways. Their role in mediating protective immunity to respiratory pathogens, although suggested by many studies, has not been directly proven. This study provides definitive evidence that airway CD8 TRM cells are sufficient to mediate protection against respiratory virus challenge. Despite being poorly cytolytic in vivo and failing to expand after encountering Ag, airway CD8 TRM cells rapidly express effector cytokines, with IFN-γ being produced most robustly. Notably, established airway CD8 TRM cells possess the ability to produce IFN-γ faster than systemic effector memory CD8 T cells. Furthermore, naive mice receiving intratracheal transfer of airway CD8 TRM cells lacking the ability to produce IFN-γ were less effective at controlling pathogen load upon heterologous challenge. This direct evidence of airway CD8 TRM cell-mediated protection demonstrates the importance of these cells as a first line of defense for optimal immunity against respiratory pathogens and suggests they should be considered in the development of future cell-mediated vaccines.

  9. The 2009 pandemic A/Wenshan/01/2009 H1N1 induces apoptotic cell death in human airway epithelial cells.

    PubMed

    Yang, Ning; Hong, Xiaoxu; Yang, Penghui; Ju, Xiangwu; Wang, Yuguo; Tang, Jun; Li, Chenggang; Fan, Quanshui; Zhang, Fuqiang; Chen, Zhongwei; Xing, Li; Zhao, Zhongpeng; Gao, Xiao; Liao, Guoyang; Li, Qihan; Wang, Xiliang; Li, Dangsheng; Jiang, Chengyu

    2011-08-01

    In 2009, a novel swine-origin H1N1 influenza virus emerged in Mexico and quickly spread to other countries, including China. This 2009 pandemic H1N1 can cause human respiratory disease, but its pathogenesis remains poorly understood. Here, we studied the infection and pathogenesis of a new 2009 pandemic strain, A/Wenshan/01/2009 H1N1, in China in human airway epithelial cell lines compared with contemporary seasonal H1N1 influenza virus. Our results showed that viral infection by the A/Wenshan H1N1 induced significant apoptotic cell death in both the human nasopharyngeal carcinoma cell line CNE-2Z and the human lung adenocarcinoma cell line A549. The A/Wenshan H1N1 virus enters both of these cell types more efficiently than the seasonal influenza virus. Viral entry in both cell lines was shown to be mediated by clathrin- and dynamin-dependent endocytosis. Therefore, we discovered that the 2009 pandemic H1N1 strain, A/Wenshan/01/2009, can induce apoptotic cell death in epithelial cells of the human respiratory tract, suggesting a molecular pathogenesis for the 2009 pandemic H1N1. PMID:21816972

  10. Transient junction anisotropies orient annular cell polarization in the Drosophila airway tubes.

    PubMed

    Hosono, Chie; Matsuda, Ryo; Adryan, Boris; Samakovlis, Christos

    2015-12-01

    In contrast to planes, three-dimensional (3D) structures such as tubes are physically anisotropic. Tubular organs exhibit a striking orientation of landmarks according to the physical anisotropy of the 3D shape, in addition to planar cell polarization. However, the influence of 3D tissue topography on the constituting cells remains underexplored. Here, we identify a regulatory network polarizing cellular biochemistry according to the physical anisotropy of the 3D tube geometry (tube cell polarization) by a genome-wide, tissue-specific RNAi screen. During Drosophila airway remodelling, each apical cellular junction is equipotent to establish perpendicular actomyosin cables, irrespective of the longitudinal or transverse tube axis. A dynamic transverse enrichment of atypical protein kinase C (aPKC) shifts the balance and transiently targets activated small GTPase RhoA, myosin phosphorylation and Rab11 vesicle trafficking to longitudinal junctions. We propose that the PAR complex translates tube physical anisotropy into longitudinal junctional anisotropy, where cell-cell communication aligns the contractile cytoskeleton of neighbouring cells.

  11. 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.

  12. CD11b+ and Sca-1+ Cells Exert the Main Beneficial Effects of Systemically Administered Bone Marrow-Derived Mononuclear Cells in a Murine Model of Mixed Th2/Th17 Allergic Airway Inflammation

    PubMed Central

    Cruz, Fernanda F.; Borg, Zachary D.; Goodwin, Meagan; Coffey, Amy L.; Wagner, Darcy E.; Rocco, Patricia R.M.

    2016-01-01

    Systemic administration of bone marrow-derived mononuclear cells (BMDMCs) or bone marrow-derived mesenchymal stromal cells (MSCs) reduces inflammation and airway hyperresponsiveness (AHR) in a murine model of Th2-mediated eosinophilic allergic airway inflammation. However, since BMDMCs are a heterogeneous population that includes MSCs, it is unclear whether the MSCs alone are responsible for the BMDMC effects. To determine which BMDMC population(s) is responsible for ameliorating AHR and lung inflammation in a model of mixed Th2-eosinophilic and Th17-neutrophilic allergic airway inflammation, reminiscent of severe clinical asthma, BMDMCs obtained from normal C57Bl/6 mice were serially depleted of CD45, CD34, CD11b, CD3, CD19, CD31, or Sca-1 positive cells. The different resulting cell populations were then assessed for ability to reduce lung inflammation and AHR in mixed Th2/Th17 allergic airway inflammation induced by mucosal sensitization to and challenge with Aspergillus hyphal extract (AHE) in syngeneic C56Bl/6 mice. BMDMCs depleted of either CD11b-positive (CD11b+) or Sca-1-positive (Sca-1+) cells were unable to ameliorate AHR or lung inflammation in this model. Depletion of the other cell types did not diminish the ameliorating effects of BMDMC administration. In conclusion, in the current model of allergic inflammation, CD11b+ cells (monocytes, macrophages, dendritic cells) and Sca-1+ cells (MSCs) are responsible for the beneficial effects of BMDMCs. Significance This study shows that bone marrow-derived mononuclear cells (BMDMCs) are as effective as bone marrow-derived mesenchymal stromal cells (MSCs) in ameliorating experimental asthma. It also demonstrates that not only MSCs present in the pool of BMDMCs are responsible for BMDMCs’ beneficial effects but also monocytes, which are the most important cell population to trigger these effects. All of this is in the setting of a clinically relevant model of severe allergic airways inflammation and thus

  13. Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

    PubMed Central

    Thevenot, Paul T.; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D.; Chustz, Regina E.; Mahne, Sarah; Kelley, Matthew A.; Hebert, Valeria Y.; Dellinger, Barry; Dugas, Tammy R.; DeMayo, Francesco J.

    2013-01-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  14. Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

    PubMed

    Wong, J Z; Woodcock-Mitchell, J; Mitchell, J; Rippetoe, P; White, S; Absher, M; Baldor, L; Evans, J; McHugh, K M; Low, R B

    1998-05-01

    In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

  15. KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms

    PubMed Central

    Coles, Garry L.; Baglia, Laurel A.; Ackerman, Kate G.

    2015-01-01

    The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that Kinesin family member 7 (Kif7) promotes Hedgehog (Hh) signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. Kif7 encodes a microtubule interacting protein that controls Hh signaling through regulation of microtubule dynamics within the primary cilium. However, whether Kif7 has a function in nonciliated cells remains largely unknown. The role Kif7 plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that Kif7 is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation in vivo and in vitro. Immunostaining and transmission electron microscopy experiments show that Kif7 dda/dda mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and Kif7 dda/dda mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that Kif7 may function as a general regulator of cellular proliferation. We ascertained that in G1, Kif7 and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that Kif7 may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein. PMID:26439735

  16. Endogenous and exogenous stem cells: a role in lung repair and use in airway tissue engineering and transplantation

    PubMed Central

    2010-01-01

    Rapid repair of the denuded alveolar surface after injury is a key to survival. The respiratory tract contains several sources of endogenous adult stem cells residing within the basal layer of the upper airways, within or near pulmonary neuroendocrine cell rests, at the bronchoalveolar junction, and within the alveolar epithelial surface, which contribute to the repair of the airway wall. Bone marrow-derived adult mesenchymal stem cells circulating in blood are also involved in tracheal regeneration. However, an organism is frequently incapable of repairing serious damage and defects of the respiratory tract resulting from acute trauma, lung cancers, and chronic pulmonary and airway diseases. Therefore, replacement of the tracheal tissue should be urgently considered. The shortage of donor trachea remains a major obstacle in tracheal transplantation. However, implementation of tissue engineering and stem cell therapy-based approaches helps to successfully solve this problem. To date, huge progress has been achieved in tracheal bioengineering. Several sources of stem cells have been used for transplantation and airway reconstitution in animal models with experimentally induced tracheal defects. Most tracheal tissue engineering approaches use biodegradable three-dimensional scaffolds, which are important for neotracheal formation by promoting cell attachment, cell redifferentiation, and production of the extracellular matrix. The advances in tracheal bioengineering recently resulted in successful transplantation of the world's first bioengineered trachea. Current trends in tracheal transplantation include the use of autologous cells, development of bioactive cell-free scaffolds capable of supporting activation and differentiation of host stem cells on the site of injury, with a future perspective of using human native sites as micro-niche for potentiation of the human body's site-specific response by sequential adding, boosting, permissive, and recruitment impulses

  17. Treatment with 8-OH-modified adenine (TLR7 ligand)-allergen conjugates decreases T helper type 2-oriented murine airway inflammation

    PubMed Central

    Nencini, Francesca; Pratesi, Sara; Petroni, Giulia; Filì, Lucia; Cardilicchia, Elisa; Casini, Andrea; Occhiato, Ernesto Giovanni; Calosi, Laura; Bani, Daniele; Romagnani, Sergio; Maggi, Enrico; Parronchi, Paola; Vultaggio, Alessandra

    2015-01-01

    A strategy to improve allergen-specific immunotherapy is to employ new adjuvants stably linked to allergens. The study is addressed to evaluate the in vivo and in vitro effects of allergens [natural Dermatophagoides pteronyssinus 2 (nDer p 2) and ovalbumin (OVA)] chemically bound to an 8-OH-modified adenine. Humoral and cellular responses were analysed in allergen-sensitized and challenged mice by using conjugates (Conj) in a therapeutic setting. The in vitro activity of the conjugates on cytokine production induced by bone marrow dendritic cells and the co-culture system was also investigated. The nDer p 2-Conj treatment in nDer p 2-primed and challenged BALB/c mice reduced the numbers of eosinophils in bronchoalveolar lavage fluid and lung, airway allergen-driven interleukin-13 (IL-13) production in lung mononuclear cells and IgE, in comparison with nDer p 2-treated mice. The increase of IgG2a paralleled that of interferon-γ (IFN-γ) and IL-10 in allergen-stimulated spleen cells. Similar effects were elicited by treatment with OVA-Conj in an OVA-driven BALB/c model. The nDer p 2-Conj or OVA-Conj redirected memory T helper type 2 cells towards the production of IL-10 and IFN-γ also in C57BL/6 mice and when subcutaneously administered. Interleukin-10, IL-12 and IL-27 were produced in vitro by Conj-stimulated bone marrow dendritic cells, whereas IL-10 and IFN-γ were up-regulated in co-cultures of CD11c+ and CD4+ T cells from Conj-treated mice stimulated with allergen. Cytofluorometric analysis indicated that the Conj expanded IFN-γ- and IL-10- producing memory T cells. The Conj effects on IL-10−/− and IL-12−/− mice confirmed the role of IL-10 and IFN-γ in inducing a protective and balanced redirection the T helper type 2-mediated airway inflammation. PMID:25930741

  18. Critical role for syndecan-4 in dendritic cell migration during development of allergic airway inflammation.

    PubMed

    Polte, Tobias; Petzold, Susanne; Bertrand, Jessica; Schütze, Nicole; Hinz, Denise; Simon, Jan C; Lehmann, Irina; Echtermeyer, Frank; Pap, Thomas; Averbeck, Marco

    2015-07-13

    Syndecan-4 (SDC4), expressed on dendritic cells (DCs) and activated T cells, plays a crucial role in DC motility and has been shown as a potential target for activated T-cell-driven diseases. In the present study, we investigate the role of SDC4 in the development of T-helper 2 cell-mediated allergic asthma. Using SDC4-deficient mice or an anti-SDC4 antibody we show that the absence or blocking of SDC4 signalling in ovalbumin-sensitized mice results in a reduced asthma phenotype compared with control animals. Most importantly, even established asthma is significantly decreased using the anti-SDC4 antibody. The disturbed SDC4 signalling leads to an impaired motility and directional migration of antigen-presenting DCs and therefore, to a modified sensitization leading to diminished airway inflammation. Our results demonstrate that SDC4 plays an important role in asthma induction and indicate SDC4 as possible target for therapeutic intervention in this disease.

  19. 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

  20. Particulate matter (PM₁₀) induces metalloprotease activity and invasion in airway epithelial cells.

    PubMed

    Morales-Bárcenas, Rocío; Chirino, Yolanda I; Sánchez-Pérez, Yesennia; Osornio-Vargas, Álvaro Román; Melendez-Zajgla, Jorge; Rosas, Irma; García-Cuellar, Claudia María

    2015-09-17

    Airborne particulate matter with an aerodynamic diameter ≤ 10 μm (PM10) is a risk factor for the development of lung diseases and cancer. The aim of this work was to identify alterations in airway epithelial (A549) cells induced by PM10 that could explain how subtoxic exposure (10 μg/cm(2)) promotes a more aggressive in vitro phenotype. Our results showed that cells exposed to PM10 from an industrial zone (IZ) and an urban commercial zone (CZ) induced an increase in protease activity and invasiveness; however, the cell mechanism is different, as only PM10 from CZ up-regulated the activity of metalloproteases MMP-2 and MMP-9 and disrupted E-cadherin/β-catenin expression after 48 h of exposure. These in vitro findings are relevant in terms of the mechanism action of PM10 in lung epithelial cells, which could be helpful in understanding the pathogenesis of some human illness associated with highly polluted cities.

  1. Role of Chitinase 3-Like-1 in Interleukin-18-Induced Pulmonary Type 1, Type 2, and Type 17 Inflammation; Alveolar Destruction; and Airway Fibrosis in the Murine Lung.

    PubMed

    Kang, Min-Jong; Yoon, Chang Min; Nam, Milang; Kim, Do-Hyun; Choi, Je-Min; Lee, Chun Geun; Elias, Jack A

    2015-12-01

    Chitinase 3-like 1 (Chi3l1), which is also called YKL-40 in humans and BRP-39 in mice, is the prototypic chitinase-like protein. Recent studies have highlighted its impressive ability to regulate the nature of tissue inflammation and the magnitude of tissue injury and fibroproliferative repair. This can be appreciated in studies that highlight its induction after cigarette smoke exposure, during which it inhibits alveolar destruction and the genesis of pulmonary emphysema. IL-18 is also known to be induced and activated by cigarette smoke, and, in murine models, the IL-18 pathway has been shown to be necessary and sufficient to generate chronic obstructive pulmonary disease-like inflammation, fibrosis, and tissue destruction. However, the relationship between Chi3l1 and IL-18 has not been defined. To address this issue we characterized the expression of Chi3l1/BRP-39 in control and lung-targeted IL-18 transgenic mice. We also characterized the effects of transgenic IL-18 in mice with wild-type and null Chi3l1 loci. The former studies demonstrated that IL-18 is a potent stimulator of Chi3l1/BRP-39 and that this stimulation is mediated via IFN-γ-, IL-13-, and IL-17A-dependent mechanisms. The latter studies demonstrated that, in the absence of Chi3l1/BRP-39, IL-18 induced type 2 and type 17 inflammation and fibrotic airway remodeling were significantly ameliorated, whereas type 1 inflammation, emphysematous alveolar destruction, and the expression of cytotoxic T lymphocyte perforin, granzyme, and retinoic acid early transcript 1 expression were enhanced. These studies demonstrate that IL-18 is a potent stimulator of Chi3l1 and that Chi3l1 is an important mediator of IL-18-induced inflammatory, fibrotic, alveolar remodeling, and cytotoxic responses.

  2. GM-CSF production from human airway smooth muscle cells is potentiated by human serum.

    PubMed Central

    Sukkar, M B; Hughes, J M; Johnson, P R; Armour, C L

    2000-01-01

    Recent evidence suggests that airway smooth muscle cells (ASMC) actively participate in the airway inflammatory process in asthma. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1) allergic asthmatic serum (AAS) modulates ASMC mediator release in response to IL-1beta and TNF-alpha, and (2) IL-1beta/TNF-alpha prime ASMC to release mediators in response to AAS. IL-5 and GM-CSF were quantified by ELISA in culture supernatants of; (1) ASMC pre-incubated with either AAS, nonallergic non-asthmatic serum (NAS) or Monomed (a serum substitute) and subsequently stimulated with IL-1beta and TNF-alpha and (2) ASMC stimulated with IL-1beta/TNF-alpha and subsequently exposed to either AAS, NAS or Monomed. IL-1beta and TNF-alpha induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or Monomed. IL-1beta and TNF-alpha, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL-1beta/TNF-alpha and serum exposure (AAS or NAS) was significantly greater than that following IL-1beta/TNF-alpha and Monomed exposure or IL-1beta/TNF-alpha exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage. PMID:11132773

  3. IL-13-producing BLT1-positive CD8 cells are increased in asthma and are associated with airway obstruction

    PubMed Central

    Dakhama, Azzeddine; Collins, Maureen L.; Ohnishi, Hiroshi; Goleva, Elena; Leung, Donald Y. M.; Alam, Rafeul; Sutherland, E. Rand; Martin, Richard J.; Gelfand, Erwin W.

    2013-01-01

    Background The role of CD8 T lymphocytes in the pathogenesis of asthma is not well understood. We investigated whether a subset of IL-13-producing BLT1-positive CD8 T lymphocytes is present in asthmatic airways and is associated with impaired lung function. Methods Bronchoalveolar lavage (BAL) cells were obtained from asthmatic (n=39) and healthy control (n=28) subjects. Cells were stimulated with phorbol ester and ionomycin in the presence of brefeldin A and stained for CD8, BLT1 and intracellular IL-13. The frequency of IL-13-producing BLT1-positive CD8 T lymphocytes was compared between the two groups and related to lung function, serum IgE levels and reticular basement membrane (RBM) thickness. Results A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 was detected in the airways of all asthmatic subjects. The frequency of this subset among recovered lymphocytes was significantly higher in the airways of asthmatic subjects compared to controls (mean ± SEM: 16.2 ± 1.4 vs. 5.3 ± 0.5, respectively, p < 0.001), and correlated positively with serum IgE levels and RBM thickness. More importantly, the frequency of CD8 T lymphocytes co-expressing BLT1 and IL-13 was inversely related to FEV1 and FEF[25-75] percent predicted values (p<0.001). Conclusions A subset of CD8 T lymphocytes expressing BLT1 and producing IL-13 is present in the airways of asthmatics. The accumulation of these cells is associated with airway obstruction, suggesting that they may play a significant pathogenic role in bronchial asthma. PMID:23573812

  4. An FGFR1-SPRY2 Signaling Axis Limits Basal Cell Proliferation in the Steady-State Airway Epithelium

    PubMed Central

    Balasooriya, Gayan I.; Johnson, Jo-Anne; Basson, M. Albert; Rawlins, Emma L.

    2016-01-01

    Summary The steady-state airway epithelium has a low rate of stem cell turnover but can nevertheless mount a rapid proliferative response following injury. This suggests a mechanism to restrain proliferation at steady state. One such mechanism has been identified in skeletal muscle in which pro-proliferative FGFR1 signaling is antagonized by SPRY1 to maintain satellite cell quiescence. Surprisingly, we found that deletion of Fgfr1 or Spry2 in basal cells of the adult mouse trachea caused an increase in steady-state proliferation. We show that in airway basal cells, SPRY2 is post-translationally modified in response to FGFR1 signaling. This allows SPRY2 to inhibit intracellular signaling downstream of other receptor tyrosine kinases and restrain basal cell proliferation. An FGFR1-SPRY2 signaling axis has previously been characterized in cell lines in vitro. We now demonstrate an in vivo biological function of this interaction and thus identify an active signaling mechanism that maintains quiescence in the airway epithelium. PMID:27046834

  5. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  6. Bisulfite and sulfite as derivatives of sulfur dioxide alters biomechanical behaviors of airway smooth muscle cells in culture.

    PubMed

    Song, Aijing; Lin, Feng; Li, Jianming; Liao, Qingfeng; Liu, Enmei; Jiang, Xuemei; Deng, Linhong

    2014-02-01

    Sulfur dioxide (SO2) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how SO2 in vivo affects airway smooth muscle (ASM) cells of which the mechanics is known to ultimately mediate airway hyperresponsiveness (AHR) - a hallmark feature of asthma. To this end, we investigated the effects of bisulfite/sulfite (1:3 M/M in neutral fluid to simulate the in vivo derivatives of inhaled SO2 in the airways), on the viability, migration, stiffness and contractility of ASM cells cultured in vitro. The results showed that bisulfite/sulfite consistently increased viability, migration, F-actin intensity and stiffness of ASM cells in similar fashion as concentration increasing from 10(-4) to 10(-1) mmol/L. However, bisulfite/sulfite increased the ASM cell contractility induced by KCl only at the concentration between 10(-4) and 10(-3) mmol/L (p < 0.05), while having no consistent effect on that induced by histamine. At the concentration of 10(0) mmol/L, bisulfite/sulfite became acutely toxic to the ASM cells. Taken together, the data suggest that SO2 derivatives at low levels in vivo may directly increase the mass, stiffness and contractility of ASM cells, which may help understand the mechanism in which specific air pollutants contribute in vivo to the pathogenesis of asthma.

  7. [Airway equipment and its maintenance for a non difficult adult airway management (endotracheal intubation and its alternative: face mask, laryngeal mask airway, laryngeal tube)].

    PubMed

    Francon, D; Estèbe, J P; Ecoffey, C

    2003-08-01

    The airway equipment for a non difficult adult airway management are described: endotracheal tubes with a specific discussion on how to inflate the balloon, laryngoscopes and blades, stylets and intubation guides, oral airways, face masks, laryngeal mask airways and laryngeal tubes. Cleaning and disinfections with the maintenance are also discussed for each type of airway management.

  8. Bidirectional counter-regulation of human lung mast cell and airway smooth muscle β2-adrenoceptors

    PubMed Central

    Newby, Chris; Amrani, Yassine; Bradding, Peter

    2015-01-01

    Human lung mast cells (HLMCs) play a central role in asthma pathogenesis through their relocation to the airway smooth muscle (ASM) bundles. β2 adrenoceptor (β2-AR)-agonists are used to relieve bronchoconstriction in asthma, but may reduce asthma control, particularly when used as monotherapy. We hypothesised that HLMC and human ASM cell (HASMC) responsiveness to β2-AR agonists would be attenuated when HLMCs are in contact with HASMCs. Cells were cultured in the presence of the short-acting β2-agonist albuterol, and the long-acting β2-agonists formoterol and olodaterol. Constitutive and FcεRI-dependent HLMC histamine release, HASMC contraction, and β2-AR phosphorylation at tyrosine 350 (Tyr350) were assessed. Constitutive HLMC histamine release was increased in HLMC-HASMC co-culture and this was enhanced by β2-AR agonists. Inhibition of FcεRI-dependent HLMC mediator release by β2-agonists was greatly reduced in HLMC-HASMC co-culture. These effects were reversed by neutralisation of stem cell factor (SCF) or cell adhesion molecule 1 (CADM1). β2-AR agonists did not prevent HASMC contraction when HLMCs were present, but this was reversed by fluticasone. β2-AR phosphorylation at Tyr350 occurred within 5 minutes in both HLMCs and HASMCs when the cells were co-cultured, and was inhibited by neutralising SCF or CADM1. HLMC interactions with HASMCs via CADM1 and Kit inhibit the potentially beneficial effects of β2-AR agonists on these cells via phosphorylation of the β2-AR. These results may explain the potentially adverse effects of β2-ARs agonists when used for asthma therapy. Targeting SCF and CADM1 may enhance β2-AR efficacy, particularly in corticosteroid-resistant patients. PMID:26608913

  9. Extracellular ATP directly gates a cation-selective channel in rabbit airway ciliated epithelial cells

    PubMed Central

    Korngreen, Alon; Ma, Weiyuan; Priel, Zvi; Silberberg, Shai D

    1998-01-01

    A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole-cell and outside-out patch configurations of the patch-clamp technique. In solutions designed to maximize currents through voltage-gated calcium channels, there were no indications of voltage-gated Ba2+ currents. Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa:PCs:PTEA of 4:1:0.1. Permeability to Cl− was negligible. Including GDP-β-S in the intracellular solution did not inhibit the effects of ATP, nor did GTP-γ-S irreversibly activate the conductance. In outside-out membrane patches, with GDP-β-S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at −120 mV. Suramin (100 μM) inhibited the whole-cell currents activated by ATP (200 μM) by 93 ± 3 %. Similar effects of suramin were observed on ATP-activated channels in outside-out membrane patches. Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At −40 mV, the time to half-maximal current activation (t½) was 46 ± 9 s during the first exposure to 200 μM ATP and decreased to 5 ± 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP-β-S in the intracellular solution. The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 μM ATP, t½ at +40 mV was 4-fold longer than t½ at −40 mV. Half-maximal activation of the conductance shifted from 210 ± 30 to 14 ± 4 μM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0.01 mM. The Hill coefficient for ATP

  10. CD4+ T cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression.

    PubMed

    Matusovsky, Oleg S; Nakada, Emily M; Kachmar, Linda; Fixman, Elizabeth D; Lauzon, Anne-Marie

    2014-07-15

    Abundant data indicate that pathogenesis in allergic airways disease is orchestrated by an aberrant T-helper 2 (Th2) inflammatory response. CD4(+) T cells have been localized to airway smooth muscle (ASM) in both human asthmatics and in rodent models of allergic airways disease, where they have been implicated in proliferative responses of ASM. Whether CD4(+) T cells also alter ASM contractility has not been addressed. We established an in vitro system to assess the ability of antigen-stimulated CD4(+) T cells to modify contractile responses of the Brown Norway rat trachealis muscle. Our data demonstrated that the unloaded velocity of shortening (Vmax) of ASM was significantly increased upon 24 h co-incubation with antigen-stimulated CD4(+) T cells, while stress did not change. Enhanced Vmax was dependent upon contact between the CD4(+) T cells and the ASM and correlated with increased levels of the fast (+)insert smooth muscle myosin heavy chain isoform. The levels of myosin light chain kinase and myosin light chain phosphorylation were also increased within the muscle. The alterations in mechanics and in the levels of contractile proteins were transient, both declining to control levels after 48 h of co-incubation. More permanent alterations in muscle phenotype might be attainable when several inflammatory cells and mediators interact together or after repeated antigenic challenges. Further studies will await new tissue culture methodologies that preserve the muscle properties over longer periods of time. In conclusion, our data suggest that inflammatory cells promote ASM hypercontractility in airway hyper-responsiveness and asthma.

  11. Effects of air pollution-related heavy metals on the viability and inflammatory responses of human airway epithelial cells.

    PubMed

    Honda, Akiko; Tsuji, Kenshi; Matsuda, Yugo; Hayashi, Tomohiro; Fukushima, Wataru; Sawahara, Takahiro; Kudo, Hitomi; Murayama, Rumiko; Takano, Hirohisa

    2015-01-01

    Various metals produced from human activity are ubiquitously detected in ambient air. The metals may lead to induction and/or exacerbation of respiratory diseases, but the significant metals and factors contributing to such diseases have not been identified. To compare the effects of each metal and different oxidation states of metals on human airway, we examined the viability and production of interleukin (IL)-6 and IL-8 using BEAS-2B cell line, derived from human airway epithelial cells. Airway epithelial cells were exposed to Mn(2+), V(4+), V(5+), Cr(3+), Cr(6+), Zn(2+), Ni(2+), and Pb(2+) at a concentration of 0.5, 5, 50, or 500 μmol/L for 24 hours. Mn and V decreased the cell viability in a concentration-dependent manner, and V(5+) tended to have a greater effect than V(4+). The Cr decreased the cell viability, and (Cr(+6)) at concentrations of 50 and 500 μmol/L was more toxic than (Cr(+3)). Zn at a concentration of 500 μmol/L greatly decreased the cell viability, whereas Ni at the same concentration increased it. Pb produced fewer changes. Mn and Ni at a concentration of 500 μmol/L induced the significant production of IL-6 and IL-8. However, most of the metals including (V(+4), V(+5)), (Cr(+3), Cr(+6)), Zn, and Pb inhibited the production of both IL-6 and IL-8. The present results indicate that various heavy metals have different effects on toxicity and the proinflammatory responses of airway epithelial cells, and those influences also depend on the oxidation states of the metals.

  12. Nerve growth factor and neurotrophin-3 mediate survival of pulmonary plasma cells during the allergic airway inflammation.

    PubMed

    Abram, Melanie; Wegmann, Michael; Fokuhl, Verena; Sonar, Sanchaita; Luger, Elke Olga; Kerzel, Sebastian; Radbruch, Andreas; Renz, Harald; Zemlin, Michael

    2009-04-15

    Allergen-specific Abs play a pivotal role in the induction and maintenance of allergic airway inflammation. During secondary immune responses, plasma cell survival and Ab production is mediated by extrinsic factors provided by the local environment (survival niches). It is unknown whether neurotrophins, a characteristic marker of allergic airway inflammation, influence plasma cell survival in the lung. Using a mouse model of allergic asthma, we found that plasma cells from the lung and spleen are distinct subpopulations exhibiting differential expression patterns of neurotrophins and their receptors (Trks). In vitro, the nerve growth factor (NGF) and neurotrophin-3 (NT3) led to a dose-dependent increase in viability of isolated pulmonary plasma cells due to up-regulation of the antiapoptotic Bcl2 pathway. In parallel, the expression of transcription factors that stimulate the production of immunoglobulins (X-box binding protein 1 and NF-kappaB subunit RelA) was enhanced in plasma cells treated with NGF and NT3. These findings were supported in vivo. When the NGF pathway was blocked by intranasal application of a selective TrkA inhibitor, sensitized mice showed reduced numbers of pulmonary plasma cells and developed lower levels of allergen-specific and total serum IgE in response to OVA inhalation. This suggests that in the allergic airway inflammation, NGF/TrkA-mediated pulmonary IgE production contributes significantly to serum-IgE levels. We conclude that the neurotrophins NGF and NT3 act as survival factors for pulmonary plasma cells and thus are important regulators of the local Ab production in the allergic airway disease.

  13. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770

    PubMed Central

    Van Goor, Fredrick; Hadida, Sabine; Grootenhuis, Peter D. J.; Burton, Bill; Cao, Dong; Neuberger, Tim; Turnbull, Amanda; Singh, Ashvani; Joubran, John; Hazlewood, Anna; Zhou, Jinglan; McCartney, Jason; Arumugam, Vijayalaksmi; Decker, Caroline; Yang, Jennifer; Young, Chris; Olson, Eric R.; Wine, Jeffery J.; Frizzell, Raymond A.; Ashlock, Melissa; Negulescu, Paul

    2009-01-01

    Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a protein kinase A (PKA)-activated epithelial anion channel involved in salt and fluid transport in multiple organs, including the lung. Most CF mutations either reduce the number of CFTR channels at the cell surface (e.g., synthesis or processing mutations) or impair channel function (e.g., gating or conductance mutations) or both. There are currently no approved therapies that target CFTR. Here we describe the in vitro pharmacology of VX-770, an orally bioavailable CFTR potentiator in clinical development for the treatment of CF. In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. VX-770 also increased Cl− secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by ≈10-fold, to ≈50% of that observed in HBE isolated from individuals without CF. Furthermore, VX-770 reduced excessive Na+ and fluid absorption to prevent dehydration of the apical surface and increased cilia beating in these epithelial cultures. These results support the hypothesis that pharmacological agents that restore or increase CFTR function can rescue epithelial cell function in human CF airway. PMID:19846789

  14. Effect of guaifenesin on mucin production, rheology, and mucociliary transport in differentiated human airway epithelial cells.

    PubMed

    Seagrave, JeanClare; Albrecht, Helmut; Park, Yong Sung; Rubin, Bruce; Solomon, Gail; Kim, K Chul

    2011-12-01

    Guaifenesin is widely used to alleviate symptoms of excessive mucus accumulation in the respiratory tract. However, its mechanism of action is poorly understood. The authors hypothesized that guaifenesin improves mucociliary clearance in humans by reducing mucin release, by decreasing mucus viscoelasticity, and by increasing mucociliary transport. To test these hypotheses, human differentiated airway epithelial cells, cultured at an air-liquid interface, were treated with clinically relevant concentrations of guaifenesin by addition to the basolateral medium. To evaluate the effect on mucin secretion, the authors used an anzyme-linked immunosorbent assay (ELISA) to measure the amounts of MUC5AC protein in apical surface fluid and cell lysates. To measure mucociliary transportability, additional cultures were treated for 1 or 6 hours with guaifenesin, and the movement of cell debris was measured from video data. Further, the authors measured mucus dynamic viscoelasticity using a micro cone and plate rheometer with nondestructive creep transformation. Guaifenesin suppressed mucin production in a dose-dependent manner at clinically relevant concentrations. The reduced mucin production was associated with increased mucociliary transport and decreased viscoelasticity of the mucus. Viability of the cultures was not significantly affected. These results suggest that guaifenesin could improve mucociliary clearance in humans by reducing the release and/or production of mucins, thereby altering mucus rheology. PMID:22044398

  15. Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

    NASA Astrophysics Data System (ADS)

    Youta Dombu, Christophe; Kroubi, Maya; Zibouche, Rima; Matran, Regis; Betbeder, Didier

    2010-09-01

    A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 °C compared with 37 °C, or by NaN3 treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

  16. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation.

  17. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. PMID:25883089

  18. Comparison of gel contraction mediated by airway smooth muscle cells from patients with and without asthma

    PubMed Central

    Matsumoto, Hisako; Moir, Lyn M; Oliver, Brian G G; Burgess, Janette K; Roth, Michael; Black, Judith L; McParland, Brent E

    2007-01-01

    Backgrounds Exaggerated bronchial constriction is the most significant and life threatening response of patients with asthma to inhaled stimuli. However, few studies have investigated the contractility of airway smooth muscle (ASM) from these patients. The purpose of this study was to establish a method to measure contraction of ASM cells by embedding them into a collagen gel, and to compare the contraction between subjects with and without asthma. Methods Gel contraction to histamine was examined in floating gels containing cultured ASM cells from subjects with and without asthma following overnight incubation while unattached (method 1) or attached (method 2) to casting plates. Smooth muscle myosin light chain kinase protein levels were also examined. Results Collagen gels containing ASM cells reduced in size when stimulated with histamine in a concentration‐dependent manner and reached a maximum at a mean (SE) of 15.7 (1.2) min. This gel contraction was decreased by inhibitors for phospholipase C (U73122), myosin light chain kinase (ML‐7) and Rho kinase (Y27632). When comparing the two patient groups, the maximal decreased area of gels containing ASM cells from patients with asthma was 19 (2)% (n = 8) using method 1 and 22 (3)% (n = 6) using method 2, both of which were greater than that of cells from patients without asthma: 13 (2)% (n = 9, p = 0.05) and 10 (4)% (n = 5, p = 0.024), respectively. Smooth muscle myosin light chain kinase levels were not different between the two groups. Conclusion The increased contraction of asthmatic ASM cells may be responsible for exaggerated bronchial constriction in asthma. PMID:17412779

  19. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model.

    PubMed

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4(+)CD25(+) regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4(+) or CD8(+) cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3(DTR) mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  20. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

    PubMed Central

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4+CD25+ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4+ or CD8+ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  1. Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery.

    PubMed

    Valencia-Gattas, Monica; Conner, Gregory E; Fregien, Nevis L

    2016-01-01

    Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases. PMID:27532261

  2. Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery

    PubMed Central

    Valencia-Gattas, Monica; Conner, Gregory E.; Fregien, Nevis L.

    2016-01-01

    Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases. PMID:27532261

  3. Primary Paediatric Bronchial Airway Epithelial Cell in Vitro Responses to Environmental Exposures

    PubMed Central

    McInnes, Neil; Davidson, Matthew; Scaife, Alison; Miller, David; Spiteri, Daniella; Engelhardt, Tom; Semple, Sean; Devereux, Graham; Walsh, Garry; Turner, Steve

    2016-01-01

    The bronchial airway epithelial cell (BAEC) is the site for initial encounters between inhaled environmental factors and the lower respiratory system. Our hypothesis was that release of pro inflammatory interleukins (IL)-6 and IL-8 from primary BAEC cultured from children will be increased after in vitro exposure to common environmental factors. Primary BAEC were obtained from children undergoing clinically indicated routine general anaesthetic procedures. Cells were exposed to three different concentrations of lipopolysaccharide (LPS) or house dust mite allergen (HDM) or particulates extracted from side stream cigarette smoke (SSCS). BAEC were obtained from 24 children (mean age 7.0 years) and exposed to stimuli. Compared with the negative control, there was an increase in IL-6 and IL-8 release after exposure to HDM (p ≤ 0.001 for both comparisons). There was reduced IL-6 after higher compared to lower SSCS exposure (p = 0.023). There was no change in BAEC release of IL-6 or IL-8 after LPS exposure. BAEC from children are able to recognise and respond in vitro with enhanced pro inflammatory mediator secretion to some inhaled exposures. PMID:27023576

  4. Intracellular calcium mobilization and phospholipid degradation in sphingosylphosphorylcholine-stimulated human airway epithelial cells.

    PubMed Central

    Orlati, S; Porcelli, A M; Hrelia, S; Lorenzini, A; Rugolo, M

    1998-01-01

    Extracellular sphingosylphosphorylcholine (SPC) caused a remarkable elevation in the intracellular Ca2+ concentration ([Ca2+]i) in immortalized human airway epithelial cells (CFNP9o-). An increase in total inositol phosphates formation was determined; however, the dose responses for [Ca2+]i elevation and inositol phosphates production were slightly different and, furthermore, PMA and pertussis toxin almost completely inhibited [Ca2+]i mobilization by SPC, whereas inositol phosphates production was only partially reduced. The possible direct interaction of SPC with Ca2+ channels of intracellular stores was determined by experiments with permeabilized cells, where SPC failed to evoke Ca2+ release, whereas lysophosphatidic acid was shown to be effective. The level of phosphatidic acid was increased by SPC only in the presence of AACOCF3, a specific inhibitor of phospholipase A2 (PLA2) and blocked by both pertussis toxin and R59022, an inhibitor of diacylglycerol kinase. R59022 enhanced diacylglycerol production by SPC and also significantly reduced [Ca2+]i mobilization. Only polyunsaturated diacylglycerol and phosphatidic acid were generated by SPC. Lastly, SPC caused stimulation of arachidonic acid release, indicating the involvement of PLA2. Taken together, these data suggest that, after SPC stimulation, phospholipase C-derived diacylglycerol is phosphorylated by a diacylglycerol kinase to phosphatidic acid, which is further hydrolysed by PLA2 activity to arachidonic and lysophosphatidic acids. We propose that lysophosphatidic acid might be the intracellular messenger able to release Ca2+ from internal stores. PMID:9729473

  5. Nicotine suppresses inflammatory factors in HBE16 airway epithelial cells after exposure to cigarette smoke extract and lipopolysaccharide.

    PubMed

    Li, Qi; Zhou, Xiangdong; Kolosov, Victor P; Perelman, Juliy M

    2010-12-01

    Cigarette smoke is a major cause of chronic inflammatory pulmonary disease, leading to inflammation, mucin (MUC) production, tissue damage, and remodeling. It is also well known that the major addictive component of cigarette smoke is nicotine. This study focused on the role of nicotine in the development of inflammatory pulmonary disease induced by cigarette smoke. HBE16 human airway epithelial cells were treated with serial dilutions of cigarette smoke chloroform extract (CE), lipopolysaccharide (LPS), and nicotine. The release of MUC5AC, tumor necrosis factor (TNF)-α, interleukin (IL)-8, and IL-6 protein were assayed by enzyme-linked immunosorbent assay. The MUC5AC protein also was observed by immunofluorescence. The expression of MUC5AC, TNF-α, IL-8, and IL-6 mRNA were detected by real-time polymerase chain reaction. We found that the mRNA of the proinflammatory mediators TNF-α, IL-8, and IL-6, as well as MUC5AC was highly expressed after CE and LPS stimulation. Nicotine did not cause an excessive expression of TNF-α, IL-8, and IL-6, nor did it affect protein production from the MUC5AC gene. Nicotine not only failed to stimulate production of TNF-α, IL-8, and IL-6, but its presence was shown to suppress the activation resulting from exposure to CE and LPS (P < 0.05). Preincubation with nicotine also would reduce the level of MUC5AC protein in culture supernatants of CE- and LPS-treated cells. However, mRNA expression of MUC5AC showed no significant change in nicotine-treated cells when compared with normal control cells. This distinctive pattern implies that nicotine may have potential to suppress airway inflammation and maintain the mucus over retention in airway secretory cells to some extent, thus forming a balance between mucus hyperproduction and hypersecretion in airways exposed to smoking and LPS. PMID:21078494

  6. Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles

    SciTech Connect

    Tal, Tamara L.; Simmons, Steven O.; Silbajoris, Robert; Dailey, Lisa; Cho, Seung-Hyun; Ramabhadran, Ram; Linak, William; Reed, William; Bromberg, Philip A.; Samet, James M.

    2010-02-15

    Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

  7. 1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

    PubMed

    Davis, A Sally; Chertow, Daniel S; Kindrachuk, Jason; Qi, Li; Schwartzman, Louis M; Suzich, Jon; Alsaaty, Sara; Logun, Carolea; Shelhamer, James H; Taubenberger, Jeffery K

    2016-06-01

    The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model. PMID:27062579

  8. 1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

    PubMed

    Davis, A Sally; Chertow, Daniel S; Kindrachuk, Jason; Qi, Li; Schwartzman, Louis M; Suzich, Jon; Alsaaty, Sara; Logun, Carolea; Shelhamer, James H; Taubenberger, Jeffery K

    2016-06-01

    The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model.

  9. Arylhydrocarbon receptor (AhR) activation in airway epithelial cells induces MUC5AC via reactive oxygen species (ROS) production.

    PubMed

    Chiba, Takahito; Uchi, Hiroshi; Tsuji, Gaku; Gondo, Hisaki; Moroi, Yoichi; Furue, Masutaka

    2011-02-01

    The dioxins and dioxin-like compounds in cigarette smoke regulate various immunological responses via the arylhydrocarbon receptor (AhR). These environmental toxicants are known to cause bronchitis, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Recent studies have demonstrated that AhR activation upregulates the expression of mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) in the airway epithelial cell line. However, the mechanism for the production of mucin has not been clarified. In this study, we investigated the role and pathway of AhR in airway epithelial cells by using selective agonists and antagonists. After stimulation with or without benzopyrene (B[a]P), an AhR agonist, MUC5AC expression was measured by real-time RT-PCR. The mechanism of AhR-induced MUC5AC expression in airway epithelial cells was studied in terms of the production of cytokine and reactive oxygen species (ROS). Treatment with B[a]P increased ROS generation in NCI-H₂₉₂ cells. Furthermore, B[a]P-induced MUC5AC upregulation and mucin production were inhibited by AhR siRNA or the use of an antioxidative agent. These results suggest that the AhR-induced increase of mucin production is partially mediated by ROS generation. An antioxidant therapy approach may help to cure AhR-induced mucus hypersecretory diseases. PMID:20709182

  10. RAPID COMMUNICATION IL-4 INDUCES IL-6 AND SIGNS OF ALLERGIC-TYPE INFLAMMATION IN THE NASAL AIRWAYS OF NONALLERGIC INDIVIDUALS

    EPA Science Inventory


    In addition to its more widely recognized role in promoting IgE synthesis, we speculate that interleukin-4 (IL-4) may modulate both allergic- and nonallergic-type inflammatory processes in the airway mucosa. We examined in vivo the effect of IL-4 on granulocyte and cytokine h...

  11. Vascular endothelial growth factor as a key inducer of angiogenesis in the asthmatic airways.

    PubMed

    Meyer, Norbert; Akdis, Cezmi A

    2013-02-01

    Asthma is a chronic inflammatory disease of the airways characterized by structural airway changes, which are known as airway remodeling, including smooth muscle hypertrophy, goblet cell hyperplasia, subepithelial fibrosis, and angiogenesis. Vascular remodeling in asthmatic lungs results from increased angiogenesis, which is mainly mediated by vascular endothelial growth factor (VEGF). VEGF is a key regulator of blood vessel growth in the airways of asthma patients by promoting proliferation and differentiation of endothelial cells and inducing vascular leakage and permeability. In addition, VEGF induces allergic inflammation, enhances allergic sensitization, and has a role in Th2 type inflammatory responses. Specific inhibitors of VEGF and blockers of its receptors might be useful to control chronic airway inflammation and vascular remodeling, and might be a new therapeutic approach for chronic inflammatory airway disease like asthma.

  12. S-nitrosothiols regulate cell-surface pH buffering by airway epithelial cells during the human immune response to rhinovirus.

    PubMed

    Carraro, Silvia; Doherty, Joseph; Zaman, Khalequz; Gainov, Iain; Turner, Ronald; Vaughan, John; Hunt, John F; Márquez, Javier; Gaston, Benjamin

    2006-05-01

    Human rhinovirus infection is a common trigger for asthma exacerbations. Asthma exacerbations and rhinovirus infections are both associated with markedly decreased pH and ammonium levels in exhaled breath condensates. This observation is thought to be related, in part, to decreased activity of airway epithelial glutaminase. We studied whether direct rhinovirus infection and/or the host immune response to the infection decreased airway epithelial cell surface pH in vitro. Interferon-gamma and tumor necrosis factor-alpha, but not direct rhinovirus infection, decreased pH, an effect partly associated with decreased ammonium concentrations. This effect was 1) prevented by nitric oxide synthase inhibition; 2) independent of cyclic GMP; 3) associated with an increase in endogenous airway epithelial cell S-nitrosothiol concentration; 4) mimicked by the exogenous S-nitrosothiol, S-nitroso-N-acetyl cysteine; and 5) independent of glutaminase expression and activity. We then confirmed that decreased epithelial pH inhibits human rhinovirus replication in airway epithelial cells. These data suggest that a nitric oxide synthase-dependent host response to viral infection mediated by S-nitrosothiols, rather than direct infection itself, plays a role in decreased airway surface pH during human rhinovirus infection. This host immune response may serve to protect the lower airways from direct infection in the normal host. In patients with asthma, however, this fall in pH could be associated with the increased mucus production, augmented inflammatory cell degranulation, bronchoconstriction, and cough characteristic of an asthma exacerbation. PMID:16603595

  13. Inhibition of endotoxin-induced airway epithelial cell injury by a novel family of pyrrol derivates.

    PubMed

    Cabrera-Benítez, Nuria E; Pérez-Roth, Eduardo; Ramos-Nuez, Ángela; Sologuren, Ithaisa; Padrón, José M; Slutsky, Arthur S; Villar, Jesús

    2016-06-01

    Inflammation and apoptosis are crucial mechanisms for the development of the acute respiratory distress syndrome (ARDS). Currently, there is no specific pharmacological therapy for ARDS. We have evaluated the ability of a new family of 1,2,3,5-tetrasubstituted pyrrol compounds for attenuating lipopolysaccharide (LPS)-induced inflammation and apoptosis in an in vitro LPS-induced airway epithelial cell injury model based on the first steps of the development of sepsis-induced ARDS. Human alveolar A549 and human bronchial BEAS-2B cells were exposed to LPS, either alone or in combination with the pyrrol derivatives. Rhein and emodin, two representative compounds with proven activity against the effects of LPS, were used as reference compounds. The pyrrol compound that was termed DTA0118 had the strongest inhibitory activity and was selected as the lead compound to further explore its properties. Exposure to LPS caused an intense inflammatory response and apoptosis in both A549 and BEAS-2B cells. DTA0118 treatment downregulated Toll-like receptor-4 expression and upregulated nuclear factor-κB inhibitor-α expression in cells exposed to LPS. These anti-inflammatory effects were accompanied by a significantly lower secretion of interleukin-6 (IL-6), IL-8, and IL-1β. The observed antiapoptotic effect of DTA0118 was associated with the upregulation of antiapoptotic Bcl-2 and downregulation of proapoptotic Bax and active caspase-3 protein levels. Our findings demonstrate the potent anti-inflammatory and antiapoptotic properties of the pyrrol DTA0118 compound and suggest that it could be considered as a potential drug therapy for the acute phase of sepsis and septic ARDS. Further investigations are needed to examine and validate these mechanisms and effects in a clinically relevant animal model of sepsis and sepsis-induced ARDS. PMID:26999659

  14. Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

    PubMed

    Hendrick, Siobhán M; Mroz, Magdalena S; Greene, Catherine M; Keely, Stephen J; Harvey, Brian J

    2014-09-01

    Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 μM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid.

  15. The non-antibiotic macrolide EM900 inhibits rhinovirus infection and cytokine production in human airway epithelial cells

    PubMed Central

    Lusamba Kalonji, Nadine; Nomura, Kazuhiro; Kawase, Tetsuaki; Ota, Chiharu; Kubo, Hiroshi; Sato, Takeya; Yanagisawa, Teruyuki; Sunazuka, Toshiaki; Ōmura, Satoshi; Yamaya, Mutsuo

    2015-01-01

    The anti-inflammatory effects of macrolides may be associated with a reduced frequency of exacerbation of chronic obstructive pulmonary disease (COPD). However, because the long-term use of antibiotics may promote the growth of drug-resistant bacteria, the development of a treatment to prevent COPD exacerbation with macrolides that do not exert anti-bacterial effects is necessary. Additionally, the inhibitory effects of nonantibiotic macrolides on the replication of rhinovirus (RV), which is the major cause of COPD exacerbation, have not been demonstrated. Primary cultures of human tracheal epithelial cells and nasal epithelial cells were pretreated with the nonantibiotic macrolide EM900 for 72 h prior to infection with a major group RV type 14 rhinovirus (RV14) and were further treated with EM900 after infection. Treatment with EM900 before and after infection reduced RV14 titers in the supernatants and viral RNA within the cells. Moreover, cytokine levels, including interleukin (IL)-1β and IL-6, were reduced in the supernatants following RV14 infection. Treatment with EM900 before and after infection also reduced the mRNA and protein expression of intercellular adhesion molecule-1 (ICAM-1), which is the receptor for RV14, after infection and reduced the activation of the nuclear factor kappa-B protein p50 in nuclear extracts after infection. Pretreatment with EM900 reduced the number and fluorescence intensity of the acidic endosomes through which RV RNA enters the cytoplasm. Thus, pretreatment with EM900 may inhibit RV infection by reducing the ICAM-1 levels and acidic endosomes and thus modulate the airway inflammation associated with RV infections. PMID:26462747

  16. Beta-agonists and secretory cell number and intracellular glycoproteins in airway epithelium. The effect of isoproterenol and salbutamol.

    PubMed Central

    Jones, R.; Reid, L.

    1979-01-01

    This study describes the effect of systemic administration of the beta-adrenergic agonists isoproterenol and salbutamol on the secretory cell populations in seven regions of rat airway epithelium (three extrapulmonary and four intrapulmonary) and on the size of salivary glands and heart. Isoproterenol (a nonselective beta-adrenergic agonist) significantly increases secretory cell number in all airway regions except the midtrachea; salbutamol (a selective beta 2 agonist) increases secretory cell number only in proximal and peripheral regions. The absolute number of secretory cells is greatest in the most peripheral region after isoproterenol administration and in the most proximal region after salbutamol, although both drugs produce the greatest relative increase at the periphery. In proximal and, particularly, peripheral regions, the increase by isoproterenol (less than 3- and 14-fold, respectively) is greater than by salbutamol (less than 2- and less than 3-fold, respectively). In all airway regions, both drugs modify intracellular glycoprotein in the secretory cell population; within a given region, modification is much the same. In the most proximal region, the population of cells synthesizing only granules of neutral glycoprotein significantly increases while in other regions increase is in cells synthesizing only granules of acid. A significant shift in glycoprotein synthesis occurs whether or not the secretory cell population is increased, which suggests that existing as well as newly appearing cells modify their product. Isoproterenol significantly increases the size of the parotid and submaxillary glands; salbutamol increases the size of the parotid only. Isoproterenol significantly increases the weight of both ventricles of the heart; salbutamol has no such effect. PMID:36762

  17. Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

    SciTech Connect

    Ulanova, Marina . E-mail: marina.ulanova@normed.ca; Marcet-Palacios, Marcelo; Munoz, Samira; Asfaha, Samuel; Kim, Moo-Kyung; Schreiber, Alan D.; Befus, A. Dean

    2006-12-15

    We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in {beta}1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via {beta}1 integrins, suggesting that TNF and {beta}1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-{kappa}B. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders.

  18. Human metapneumovirus infection induces significant changes in small noncoding RNA expression in airway epithelial cells.

    PubMed

    Deng, Junfang; Ptashkin, Ryan N; Wang, Qingrong; Liu, Guangliang; Zhang, Guanping; Lee, Inhan; Lee, Yong Sun; Bao, Xiaoyong

    2014-05-20

    Small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), virus-derived sncRNAs, and more recently identified tRNA-derived RNA fragments, are critical to posttranscriptional control of genes. Upon viral infection, host cells alter their sncRNA expression as a defense mechanism, while viruses can circumvent host defenses and promote their own propagation by affecting host cellular sncRNA expression or by expressing viral sncRNAs. Therefore, characterizing sncRNA profiles in response to viral infection is an important tool for understanding host-virus interaction, and for antiviral strategy development. Human metapneumovirus (hMPV), a recently identified pathogen, is a major cause of lower respiratory tract infections in infants and children. To investigate whether sncRNAs play a role in hMPV infection, we analyzed the changes in sncRNA profiles of airway epithelial cells in response to hMPV infection using ultrahigh-throughput sequencing. Of the cloned sncRNAs, miRNA was dominant in A549 cells, with the percentage of miRNA increasing in a time-dependent manner after the infection. In addition, several hMPV-derived sncRNAs and corresponding ribonucleases for their biogenesis were identified. hMPV M2-2 protein was revealed to be a key viral protein regulating miRNA expression. In summary, this study revealed several novel aspects of hMPV-mediated sncRNA expression, providing a new perspective on hMPV-host interactions.

  19. Nickel Mobilizes Intracellular Zinc to Induce Metallothionein in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Leikauf, George D.; Pitt, Bruce R.; Wasserloos, Karla J.; Barchowsky, Aaron

    2009-01-01

    We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release. PMID:19097988

  20. Agonist binding to β-adrenergic receptors on human airway epithelial cells inhibits migration and wound repair.

    PubMed

    Peitzman, Elizabeth R; Zaidman, Nathan A; Maniak, Peter J; O'Grady, Scott M

    2015-12-15

    Human airway epithelial cells express β-adrenergic receptors (β-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the β2-AR-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the β-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by β-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that β-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-β1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with β-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in β1-integrin activation in the basal membrane. PMID:26491049

  1. Rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells

    PubMed Central

    Chattoraj, Sangbrita S.; Ganesan, Shyamala; Jones, Andrew M.; Helm, Jennifer M; Comstock, Adam T; Bright-Thomas, Rowland; LiPuma, John J.; Hershenson, Marc B.; Sajjan, Umadevi S.

    2011-01-01

    Background Intermittent viral exacerbations in cystic fibrosis (CF) patients with chronic P. aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are associated with majority of viral-related exacerbations in CF and required prolonged intravenous antibiotic treatment. These observations imply that acute RV infection may increase lower respiratory symptoms by increasing planktonic bacterial load. However, the underlying mechanisms are not known. Methods Primary CF airway epithelial cells differentiated into mucociliary phenotype were infected with mucoid PA (MPA) followed by RV and examined for bacterial density, biofilm mass, levels of chemokines and hydrogen peroxide (H2O2). Requirement of dual oxidase 2 in RV-induced generation of H2O2 in CF cells was assessed by using gene-specific siRNA. Results Super infection with RV increased chemokine responses in CF mucociliary-differentiated airway epithelial cells with pre-existing MPA infection in the form of biofilm. This was associated with the presence of planktonic bacteria at both the apical and basolateral epithelial cell surfaces. Further, RV-induced generation of H2O2 via dual oxidase 2, a component of NADPH oxidase in CF cells was sufficient for dispersal of planktonic bacteria from biofilm. Inhibition of NADPH oxidase reduced bacterial transmigration across mucociliary-differentiated CF cells and IL-8 response in MPA and RV-infected cells. Conclusion We show that acute infection with RV liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterpart stimulates increased chemokine responses in CF airway epithelial cells, which in turn may contribute to pathogenesis of CF exacerbations. PMID:21289024

  2. 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.

  3. Nuclear factor-κB mediates the phenotype switching of airway smooth muscle cells in a murine asthma model

    PubMed Central

    Qiu, Chen; Zhang, Jian; Su, Meiping; Fan, Xiujun

    2015-01-01

    Airway smooth muscle cells (ASMCs) phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to airway proliferative diseases such as allergic asthma. Nuclear Factor-κB (NF-κB) has been reported as a key regulator for the occurrence and development of asthma. However, little is known regarding its role in ASM cell phenotypic modulation. To elucidate the role of NF-κB in regulating ASM cells phenotypic modulation, we investigated the effects of NF-κB on ASM cells contractile marker protein expression, and its impact on proliferation and apoptosis. We found that chronic asthma increased the activation of NF-κB in the primary murine ASM cells with a concomitant marked decrease in the expression of contractile phenotypic marker protein including smooth muscle alpha-actin (α-SMA). Additionally, we used the normal ASM cells under different processing to build the phenotype switching when we found the activation of NF-κB. Meanwhile, the expression of α-SMA in asthma was significantly increased by the NF-κB blocker. NF-κB blocker also suppressed asthma mouse ASM cell proliferation and promoted apoptosis. These findings highlight a novel role for the NF-κB in murine ASM cell phenotypic modulation and provide a potential target for therapeutic intervention for asthma. PMID:26722396

  4. Nuclear factor-κB mediates the phenotype switching of airway smooth muscle cells in a murine asthma model.

    PubMed

    Qiu, Chen; Zhang, Jian; Su, Meiping; Fan, Xiujun

    2015-01-01

    Airway smooth muscle cells (ASMCs) phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to airway proliferative diseases such as allergic asthma. Nuclear Factor-κB (NF-κB) has been reported as a key regulator for the occurrence and development of asthma. However, little is known regarding its role in ASM cell phenotypic modulation. To elucidate the role of NF-κB in regulating ASM cells phenotypic modulation, we investigated the effects of NF-κB on ASM cells contractile marker protein expression, and its impact on proliferation and apoptosis. We found that chronic asthma increased the activation of NF-κB in the primary murine ASM cells with a concomitant marked decrease in the expression of contractile phenotypic marker protein including smooth muscle alpha-actin (α-SMA). Additionally, we used the normal ASM cells under different processing to build the phenotype switching when we found the activation of NF-κB. Meanwhile, the expression of α-SMA in asthma was significantly increased by the NF-κB blocker. NF-κB blocker also suppressed asthma mouse ASM cell proliferation and promoted apoptosis. These findings highlight a novel role for the NF-κB in murine ASM cell phenotypic modulation and provide a potential target for therapeutic intervention for asthma.

  5. Systematic Analysis of Multiwalled Carbon Nanotube-Induced Cellular Signaling and Gene Expression in Human Small Airway Epithelial Cells

    PubMed Central

    Snyder-Talkington, Brandi N.

    2013-01-01

    Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment. PMID:23377615

  6. Human mesenchymal stem cells resolve airway inflammation, hyperreactivity, and histopathology in a mouse model of occupational asthma.

    PubMed

    Martínez-González, Itziar; Cruz, Maria-Jesús; Moreno, Rafael; Morell, Ferran; Muñoz, Xavier; Aran, Josep M

    2014-10-01

    Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1 × 10(6) cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA. PMID:24798370

  7. Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, and Histopathology in a Mouse Model of Occupational Asthma

    PubMed Central

    Martínez-González, Itziar; Moreno, Rafael; Morell, Ferran; Muñoz, Xavier

    2014-01-01

    Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1×106 cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA. PMID:24798370

  8. Airway smooth muscle cells synthesize hyaluronan cable structures independent of inter-alpha-inhibitor heavy chain attachment.

    PubMed

    Lauer, Mark E; Fulop, Csaba; Mukhopadhyay, Durba; Comhair, Suzy; Erzurum, Serpil C; Hascall, Vincent C

    2009-02-20

    The covalent association of inter-alpha-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan "cable" structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan "cables" in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass.

  9. Anti-inflammatory effects of long-chain n-3 PUFA in rhinovirus-infected cultured airway epithelial cells.

    PubMed

    Saedisomeolia, Ahmad; Wood, Lisa G; Garg, Manohar L; Gibson, Peter G; Wark, Peter A B

    2009-02-01

    Long-chain n-3 PUFA (LCn-3PUFA) including DHA and EPA, are known to decrease inflammation by inhibiting arachidonic acid (AA) metabolism to eicosanoids, decreasing the production of pro-inflammatory cytokines and reducing immune cell function. The aim of this study was to determine if EPA and DHA reduced the release of inflammatory mediators from airway epithelial cells infected with rhinovirus (RV). Airway epithelial cells (Calu-3) were incubated with EPA, DHA and AA for 24 h, followed by rhinovirus infection for 48 h. IL-6, IL-8 and interferon-gamma-induced protein-10 (IP-10) released by cells were measured using ELISA. Viral replication was measured by serial titration assays. The fatty acid content of cells was analysed using GC. Cellular viability was determined by visual inspection of cells and lactate dehydrogenase release. DHA (400 microm) resulted in a significant 16% reduction in IL-6 release after RV-43 infection, 29% reduction in IL-6 release after RV-1B infection, 28% reduction in IP-10 release after RV-43 infection and 23 % reduction in IP-10 release after RV-1B infection. Cellular DHA content negatively correlated with IL-6 and IP-10 release. None of the fatty acids significantly modified rhinovirus replication. DHA supplementation resulted in increased cellular content of DHA at the cost of AA, which may explain the decreased inflammatory response of cells. EPA and AA did not change the release of inflammatory biomarkers significantly. It is concluded that DHA has a potential role in suppressing RV-induced airway inflammation.

  10. Effects of 10 Cigarette Smoke Condensates on Primary Human Airway Epithelial Cells by Comparative Gene and Cytokine Expression Studies

    PubMed Central

    Pickett, Gavin; Seagrave, JeanClare; Boggs, Susan; Polzin, Gregory; Richter, Patricia; Tesfaigzi, Yohannes

    2010-01-01

    Cigarettes vary in tobacco blend, filter ventilation, additives, and other physical and chemical properties, but little is known regarding potential differences in toxicity to a smoker’s airway epithelia. We compared changes in gene expression and cytokine production in primary normal human bronchial epithelial cells following treatment for 18 h with cigarette smoke condensates (CSCs) prepared from five commercial and four research cigarettes, at doses of ∼4 μg/ml nicotine. Nine of the CSCs were produced under a standard International Organization for Standardization smoking machine regimen and one was produced by a more intense smoking machine regimen. Isolated messenger RNA (mRNA) was analyzed by microarray hybridization, and media was analyzed for secreted cytokines and chemokines. Twenty-one genes were differentially expressed by at least 9 of the 10 CSCs by more than twofold, including genes encoding detoxifying and antioxidant proteins. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and NAD(P)H dehydrogenase, quinone 1 (NQO-1) were selected for validation with quantitative real-time PCR (qRT-PCR) and Western blot analyses. NQO-1 expression determined with microarrays, qRT-PCR, and Western blotting differed among the CSC types, with good correlation among the different assays. CYP1A1 mRNA levels varied substantially, but there was little correlation with the protein levels. For each CSC, the three most induced and three most repressed genes were identified. These genes may be useful as markers of exposure to that particular cigarette type. Furthermore, differences in interleukin-8 secretion were observed. These studies lay the foundation for future investigations to analyze differences in the responses of in vivo systems to tobacco products marketed with claims of reduced exposure or reduced harm. PMID:20015843

  11. Interleukin-21-Producing CD4(+) T Cells Promote Type 2 Immunity to House Dust Mites.

    PubMed

    Coquet, Jonathan M; Schuijs, Martijn J; Smyth, Mark J; Deswarte, Kim; Beyaert, Rudi; Braun, Harald; Boon, Louis; Karlsson Hedestam, Gunilla B; Nutt, Steven L; Hammad, Hamida; Lambrecht, Bart N

    2015-08-18

    Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

  12. Inhibition of airway surface fluid absorption by cholinergic stimulation

    PubMed Central

    Joo, Nam Soo; Krouse, Mauri E.; Choi, Jae Young; Cho, Hyung-Ju; Wine, Jeffrey J.

    2016-01-01

    In upper airways airway surface liquid (ASL) depth and clearance rates are both increased by fluid secretion. Secretion is opposed by fluid absorption, mainly via the epithelial sodium channel, ENaC. In static systems, increased fluid depth activates ENaC and decreased depth inhibits it, suggesting that secretion indirectly activates ENaC to reduce ASL depth. We propose an alternate mechanism in which cholinergic input, which causes copious airway gland secretion, also inhibits ENaC-mediated absorption. The conjoint action accelerates clearance, and the increased transport of mucus out of the airways restores ASL depth while cleansing the airways. We were intrigued by early reports of cholinergic inhibition of absorption by airways in some species. To reinvestigate this phenomenon, we studied inward short-circuit currents (Isc) in tracheal mucosa from human, sheep, pig, ferret, and rabbit and in two types of cultured cells. Basal Isc was inhibited 20–70% by the ENaC inhibitor, benzamil. Long-lasting inhibition of ENaC-dependent Isc was also produced by basolateral carbachol in all preparations except rabbit and the H441 cell line. Atropine inhibition produced a slow recovery or prevented inhibition if added before carbachol. The mechanism for inhibition was not determined and is most likely multi-factorial. However, its physiological significance is expected to be increased mucus clearance rates in cholinergically stimulated airways. PMID:26846701

  13. Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

    SciTech Connect

    Skowron-zwarg, Marie; Boland, Sonja; Caruso, Nathalie; Coraux, Christelle; Marano, Francelyne; Tournier, Frederic . E-mail: f-tournier@paris7.jussieu.fr

    2007-07-15

    Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.

  14. Utilization of modified surfactant-associated protein B for delivery of DNA to airway cells in culture.

    PubMed Central

    Baatz, J E; Bruno, M D; Ciraolo, P J; Glasser, S W; Stripp, B R; Smyth, K L; Korfhagen, T R

    1994-01-01

    Pulmonary surfactant lines the airway epithelium and creates a potential barrier to successful transfection of the epithelium in vivo. Based on the functional properties of pulmonary surfactant protein B (SP-B) and the fact that this protein is neither toxic nor immunogenic in the airway, we hypothesized that SP-B could be modified to deliver DNA to airway cells. We have modified native bovine SP-B by the covalent linkage of poly(lysine) (average molecular mass of 3.3 or 10 kDa) to the N terminus of SP-B and formed complexes between a test plasmid and the modified SP-B. Transfection efficiency was determined by transfection of pulmonary adenocarcinoma cells (H441) in culture with the test plasmid pCPA-RSV followed by measurement of activity of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfections were performed with DNA.protein complexes using poly(lysine)10kDa-SP-B ([Lys]10kDa-SP-B) or poly(lysine)3.3kDa-SP-B ([Lys]3.3kDa-SP-B), and results were compared with transfections using unmodified poly(lysine).DNA, unmodified SP-B.DNA, or DNA only. For [Lys]10kDa-SP-B.pCPA-RSV preparations, CAT activity was readily detectable above the background of [Lys]3.3kDa-SP-B or unmodified SP-B. The SP-B-poly(lysine) conjugates were effective over a broad range of protein-to-DNA molar ratios, although they were optimal at approximately 500:1-1000:1. Transfection efficiency varied with the tested cell line but was not specific to airway cells. Addition of replication-defective adenovirus to the [Lys]10kDa-SP-B.pCPA-RSV complex enhanced CAT activity about 30-fold with respect to that produced by the [Lys]10kDa-SP-B.pCPA-RSV complex alone. This increase suggests routing of the adenoviral.[Lys]10kDa-SP-B.pCPA-RSV complex through an endosomal pathway. Effects of covalent modification on the secondary structure of SP-B were examined by Fourier transform infrared spectrometry (FTIR). Results of FTIR indicated that the conformation of [Lys]10kDa-SP-B was

  15. Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility

    SciTech Connect

    Xu, Guang-Ni; Yang, Kai; Xu, Zu-Peng; Zhu, Liang; Hou, Li-Na; Qi, Hong; Chen, Hong-Zhuan Cui, Yong-Yao

    2012-07-01

    Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclin D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation. -- Highlights: ► CSE induces tracheal cell proliferation, hyper-contractility and α-SMA expression. ► Anisodamine reverses CSE-induced tracheal hyper-contractility and cell proliferation. ► ERK, PI3K, and NF-κB pathways and cyclin D1 contribute to the reversal effect.

  16. In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.

    PubMed

    Anderson, Gregory G; Moreau-Marquis, Sophie; Stanton, Bruce A; O'Toole, George A

    2008-04-01

    P. aeruginosa forms biofilms in the lungs of individuals with cystic fibrosis (CF); however, there have been no effective model systems for studying biofilm formation in the CF lung. We have developed a tissue culture system for growth of P. aeruginosa biofilms on CF-derived human airway cells that promotes the formation of highly antibiotic-resistant microcolonies, which produce an extracellular polysaccharide matrix and require the known abiotic biofilm formation genes flgK and pilB. Treatment of P. aeruginosa biofilms with tobramycin reduced the virulence of the biofilms both by reducing bacterial numbers and by altering virulence gene expression. We performed microarray analysis of these biofilms on epithelial cells after treatment with tobramycin, and we compared these results with gene expression of (i) tobramycin-treated planktonic P. aeruginosa and (ii) tobramycin-treated P. aeruginosa biofilms on an abiotic surface. Despite the conservation in functions required to form a biofilm, our results show that the responses to tobramycin treatment of biofilms grown on biotic versus abiotic surfaces are different, as exemplified by downregulation of genes involved in Pseudomonas quinolone signal biosynthesis specifically in epithelial cell-grown biofilms versus plastic-grown biofilms. We also identified the gene PA0913, which is upregulated by tobramycin specifically in biofilms grown on CF airway cells and codes for a probable magnesium transporter, MgtE. Mutation of the PA0913 gene increased the bacterial virulence of biofilms on the epithelial cells, consistent with a role for the gene in the suppression of bacterial virulence. Taken together, our data show that analysis of biofilms on airway cells provides new insights into the interaction of these microbial communities with the host.

  17. Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine.

    PubMed

    Goncz, K K; Feeney, L; Gruenert, D C

    1999-09-01

    1. Exposure to epinephrine has been shown to have a range of effects on cells and tissues. A recent study suggested that the proliferative ability of CF epithelial cells, exposed to high concentrations of epinephrine (200 - 300 microM), was reduced when compared to that of normal cells. This approach could potentially provide a means to effectively separate cells with functional cyclic AMP-dependent Cl-ion transport from those defective in this pathway. 2. The sensitivity to killing by epinephrine is reported here for four different CF cell lines, three normal cell lines, and two CF epithelial cell lines complemented with wild-type (wt) CF transmembrane conductance regulator (CFTR) cDNA. 3. While each cell line exhibited varying sensitivity to 200 microM epinephrine, no predictable pattern was observed between the expression of wt-CFTR and cell survival following epinephrine exposure. Overall, normal cell lines did exhibit a greater resistance to epinephrine-induced cell death although, the most resistant cell line was derived from CF tracheal epithelium (SigmaCFTE29o-). 4. The expression of exogenous wt-CFTR increased the survival of one cell line (CFDEo-) when compared to the parent line, but in another complemented line, survival was reduced. 5. These findings suggest that while epinephrine induces cell killing, it is not consistently effective for preferential selection of normal over CF cells. Although CFTR may play a role in the mechanism(s) of epinephrine killing, other factors such as cell density, proliferative ability, cell type origin and phenotype are involved.

  18. Distinguishing cell type using epigenotype

    NASA Astrophysics Data System (ADS)

    Wytock, Thomas; Motter, Adilson E.

    Recently, researchers have proposed that unique cell types are attractors of their epigenetic dynamics including gene expression and chromatin conformation patterns. Traditionally, cell types have been classified by their function, morphology, cytochemistry, and other macroscopically observable properties. Because these properties are the result of many proteins working together, it should be possible to predict cell types from gene expression or chromatin conformation profiles. In this talk, I present a maximum entropy approach to identify and distinguish cell type attractors on the basis of correlations within these profiles. I will demonstrate the flexibility of this method through its separate application to gene expression and chromatin conformation datasets. I show that our method out-performs other machine-learning techniques and uncorrelated benchmarks. We adapt our method to predict growth rate from gene expression in E. coli and S. cerevisiae and compare our predictions with those from metabolic models. In addition, our method identifies a nearly convex region of state-space associated with each cell type attractor basin. Estimates of the growth rate and attractor basin make it possible to rationally control gene regulatory networks independent of a model. This research was supported by NSF-GRFP, NSF-GK12, GAANN, and Northwestern's NIH-NIGMS Molecular Biophysics Training Grant.

  19. Silibinin attenuates allergic airway inflammation in mice

    SciTech Connect

    Choi, Yun Ho; Jin, Guang Yu; Guo, Hui Shu; Piao, Hong Mei; Li, Liang chang; Li, Guang Zhao; Lin, Zhen Hua; Yan, Guang Hai

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Silibinin diminishes ovalbumin-induced inflammatory reactions in the mouse lung. Black-Right-Pointing-Pointer Silibinin reduces the levels of various cytokines into the lung of allergic mice. Black-Right-Pointing-Pointer Silibinin prevents the development of airway hyperresponsiveness in allergic mice. Black-Right-Pointing-Pointer Silibinin suppresses NF-{kappa}B transcriptional activity. -- Abstract: Allergic asthma is a chronic inflammatory disease regulated by coordination of T-helper2 (Th2) type cytokines and inflammatory signal molecules. Silibinin is one of the main flavonoids produced by milk thistle, which is reported to inhibit the inflammatory response by suppressing the nuclear factor-kappa B (NF-{kappa}B) pathway. Because NF-{kappa}B activation plays a pivotal role in the pathogenesis of allergic inflammation, we have investigated the effect of silibinin on a mouse ovalbumin (OVA)-induced asthma model. Airway hyperresponsiveness, cytokines levels, and eosinophilic infiltration were analyzed in bronchoalveolar lavage fluid and lung tissue. Pretreatment of silibinin significantly inhibited airway inflammatory cell recruitment and peribronchiolar inflammation and reduced the production of various cytokines in bronchoalveolar fluid. In addition, silibinin prevented the development of airway hyperresponsiveness and attenuated the OVA challenge-induced NF-{kappa}B activation. These findings indicate that silibinin protects against OVA-induced airway inflammation, at least in part via downregulation of NF-{kappa}B activity. Our data support the utility of silibinin as a potential medicine for the treatment of asthma.

  20. The Effects of Gas Humidification with High-Flow Nasal Cannula on Cultured Human Airway Epithelial Cells

    PubMed Central

    Chidekel, Aaron; Zhu, Yan; Wang, Jordan; Mosko, John J.; Rodriguez, Elena; Shaffer, Thomas H.

    2012-01-01

    Humidification of inspired gas is important for patients receiving respiratory support. High-flow nasal cannula (HFNC) effectively provides temperature and humidity-controlled gas to the airway. We hypothesized that various levels of gas humidification would have differential effects on airway epithelial monolayers. Calu-3 monolayers were placed in environmental chambers at 37°C with relative humidity (RH) < 20% (dry), 69% (noninterventional comparator), and >90% (HFNC) for 4 and 8 hours with 10 L/min of room air. At 4 and 8 hours, cell viability and transepithelial resistance measurements were performed, apical surface fluid was collected and assayed for indices of cell inflammation and function, and cells were harvested for histology (n = 6/condition). Transepithelial resistance and cell viability decreased over time (P < 0.001) between HFNC and dry groups (P < 0.001). Total protein secretion increased at 8 hours in the dry group (P < 0.001). Secretion of interleukin (IL)-6 and IL-8 in the dry group was greater than the other groups at 8 hours (P < 0.001). Histological analysis showed increasing injury over time for the dry group. These data demonstrate that exposure to low humidity results in reduced epithelial cell function and increased inflammation. PMID:22988501

  1. Baicalin Inhibits Lipopolysaccharide-Induced Inflammation Through Signaling NF-κB Pathway in HBE16 Airway Epithelial Cells.

    PubMed

    Dong, Shou-jin; Zhong, Yun-qing; Lu, Wen-ting; Li, Guan-hong; Jiang, Hong-li; Mao, Bing

    2015-08-01

    Baicalin, a flavonoid monomer derived from Scutellaria baicalensis called Huangqin in mandarin, is the main active ingredient contributing to S. baicalensis' efficacy. It is known in China that baicalin has potential therapeutic effects on inflammatory diseases. However, its anti-inflammatory mechanism has still not been fully interpreted. We aim to investigate the anti-inflammatory effect of baicalin on lipopolysaccharide (LPS)-induced inflammation in HBE16 airway epithelial cells and also to explore the underlying signaling mechanisms. The anti-inflammatory action of baicalin was evaluated in human airway epithelial cells HBE16 treated with LPS. Airway epithelial cells HBE16 were pretreated with a range of concentrations of baicalin for 30 min and then stimulated with 10 μg/ml LPS. The secretions of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) in cell culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expressions of IL-6, IL-8, and TNF-α were tested by quantitative real-time polymerase chain reaction (real-time RT-PCR). Furthermore, Western blotting was used to determine whether the signaling pathway NF-κB was involved in the anti-inflammatory action of baicalin. The inflammatory cell model was successfully built with 10 μg/ml LPS for 24 h in our in vitro experiments. Both the secretions and the mRNA expressions of IL-6, IL-8, and TNF-α were significantly inhibited by baicalin. Moreover, the expression levels of phospho-IKKα/β and phospho-NF-κB p65 were downregulated, and the phospho-IκB-α level was upregulated by baicalin. These findings suggest that the anti-inflammatory properties of baicalin may be resulted from the inhibition of IL-6, IL-8, and TNF-α expression via preventing signaling NF-κB pathway in HBE16 airway epithelial cells. In addition, this study provides evidence to understand the therapeutic effects of baicalin on inflammatory diseases in

  2. 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.

  3. Galvanic cell type oxygen sensor

    SciTech Connect

    Fujita, Y.; Kudo, H.; Tanigawa, I.

    1985-01-22

    A galvanic cell type oxygen sensor comprising a galvanic cell comprised of a cathode made up of metal effective for the electrolytic reduction of oxygen, an anode made up of lead material and an electrolyte made up of an aqueous mixed solution of organic acid and organic acid salt, which has a long life and a high output voltage, is not at all affected by carbon dioxide and which can prevent the generation of hydrogen from the cathode, is disclosed.

  4. Effects of COREXIT dispersants on cytotoxicity parameters in a cultured human bronchial airway cells, BEAS-2B.

    PubMed

    Shi, Yongli; Roy-Engel, Astrid M; Wang, He

    2013-01-01

    The objective of this study was to assess the cytotoxicity of COREXIT dispersants EC9500A, EC9527A, and EC9580A on human airway BEAS-2B epithelial cells. Cells were exposed to dispersants for 2 or 24 h at concentrations ranging from 0 to 300 ppm. COREXIT EC9527 at 100 ppm produced 50% viability loss as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 24 h. COREXIT 9527 at 200 ppm produced 50% cell death at 2 h and 100% at 24 h. At 300 ppm COREXIT 9527 induced 100% cell death at 2 or 24 h. In the case of COREXIT 9500A 50% cell viability was noted with 200 ppm at 2 or 24 h, with a significant decrease in cell survival to 2% at 300 ppm. In contrast, no marked change in cell viability was observed in cells treated at any COREXIT 9580A concentration examined. Western blot analysis showed an increase in expression of LC3B, a marker of autophagy, in cells treated for 2 h with 300 ppm COREXIT EC9527A as well as 100 or 300 ppm Corexit EC9500A. No marked effect on LC3B expression was observed for any COREXIT 9580A concentration. Apoptosis markers as measured by cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were detectable only in cells incubated with 300 ppm COREXIT EC9527A. Although all three dispersants induced enhanced generation of reactive oxygen species (ROS) after 2-h treatment at 300 ppm, Western blot analysis revealed that 2-h incubation was not sufficient to induce a significant change in the protein expression of superoxide dismutases SOD1, SOD2, and SOD3. Data thus indicate exposure to certain dispersants may be harmful to human airway epithelial cells in a concentration-dependent manner.

  5. EFFECTS OF COREXIT DISPERSANTS ON CYTOTOXICITY PARAMETERS IN A CULTURED HUMAN BRONCHIAL AIRWAY CELLS, BEAS-2B

    PubMed Central

    Shi, Yongli; Roy-Engel, Astrid M.; Wang, He

    2013-01-01

    The objective of this study was to assess the cytotoxicity of COREXIT dispersants EC9500A, EC9527A, and EC9580A on human airway BEAS-2B epithelial cells. Cells were exposed to dispersants for 2 or 24 h at concentrations ranging from 0 to 300 ppm. COREXIT EC9527 at 100 ppm produced 50% viability loss as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 24 h. COREXIT 9527 at 200 ppm produced 50% cell death at 2 h and 100% at 24 h. At 300 ppm COREXIT 9527 induced 100% cell death at 2 or 24 h. In the case of COREXIT 9500A 50% cell viability was noted with 200 ppm at 2 or 24 h, with a significant decrease in cell survival to 2% at 300 ppm. In contrast, no marked change in cell viability was observed in cells treated at any COREXIT 9580A concentration examined. Western blot analysis showed an increase in expression of LC3B, a marker of autophagy, in cells treated for 2 h with 300 ppm COREXIT EC9527A as well as 100 or 300 ppm Corexit EC9500A. No marked effect on LC3B expression was observed for any COREXIT 9580A concentration. Apoptosis markers as measured by cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were detectable only in cells incubated with 300 ppm COREXIT EC9527A. Although all three dispersants induced enhanced generation of reactive oxygen species (ROS) after 2-h treatment at 300 ppm, Western blot analysis revealed that 2-h incubation was not sufficient to induce a significant change in the protein expression of superoxide dismutases SOD1, SOD2, and SOD3. Data thus indicate exposure to certain dispersants may be harmful to human airway epithelial cells in a concentration-dependent manner. PMID:24028667

  6. Transcriptional Regionalization of the Fruit Fly’s Airway Epithelium

    PubMed Central

    Faisal, Muhammad N.; Hoffmann, Julia; El-Kholy, Samar; Kallsen, Kimberley; Wagner, Christina; Bruchhaus, Iris; Fink, Christine; Roeder, Thomas

    2014-01-01

    Although airway epithelia are primarily devoted to gas exchange, they have to fulfil a number of different tasks including organ maintenance and the epithelial immune response to fight airborne pathogens. These different tasks are at least partially accomplished by specialized cell types in the epithelium. In addition, a proximal to distal gradient mirroring the transition from airflow conduction to real gas exchange, is also operative. We analysed the airway system of larval Drosophila melanogaster with respect to region-specific expression in the proximal to distal axis. The larval airway system is made of epithelial cells only. We found differential expression between major trunks of the airways and more distal ones comprising primary, secondary and terminal ones. A more detailed analysis was performed using DNA-microarray analysis to identify cohorts of genes that are either predominantly expressed in the dorsal trunks or in the primary/secondary/terminal branches of the airways. Among these differentially expressed genes are especially those involved in signal transduction. Wnt-signalling associated genes for example are predominantly found in secondary/terminal airways. In addition, some G-protein coupled receptors are differentially expressed between both regions of the airways, exemplified by those activated by octopamine or tyramine, the invertebrate counterparts of epinephrine and norepinephrine. Whereas the OAMB is predominantly found in terminal airway regions, the oct3βR has higher expression levels in dorsal trunks. In addition, we observed a significant association of both, genes predominantly expressed in dorsal trunks or in primary to terminal branches branches with those regulated by hypoxia. Taken together, this observed differential expression is indicative for a proximal to distal transcriptional regionalization presumably reflecting functional differences in these parts of the fly’s airway system. PMID:25020150

  7. Cigarette smoke and α,β-unsaturated aldehydes elicit VEGF release through the p38 MAPK pathway in human airway smooth muscle cells and lung fibroblasts

    PubMed Central

    Volpi, Giorgia; Facchinetti, Fabrizio; Moretto, Nadia; Civelli, Maurizio; Patacchini, Riccardo

    2011-01-01

    BACKGROUND AND PURPOSE Vascular endothelial growth factor (VEGF) is an angiogenic factor known to be elevated in the sputum of asymptomatic smokers as well as smokers with bronchitis type of chronic obstructive pulmonary disease. The aim of this study was to investigate whether acute exposure to cigarette smoke extract altered VEGF production in lung parenchymal cells. EXPERIMENTAL APPROACH We exposed human airway smooth muscle cells (ASMC), normal human lung fibroblasts (NHLF) and small airways epithelial cells (SAEC) to aqueous cigarette smoke extract (CSE) in order to investigate the effect of cigarette smoke on VEGF expression and release. KEY RESULTS Vascular endothelial growth factor release was elevated by sub-toxic concentrations of CSE in both ASMC and NHLF, but not in SAEC. CSE-evoked VEGF release was mimicked by its component acrolein at concentrations (10–100 µM) found in CSE, and prevented by the antioxidant and α,β-unsaturated aldehyde scavenger, N-acetylcysteine (NAC). Both CSE and acrolein (30 µM) induced VEGF mRNA expression in ASMC cultures, suggesting an effect at transcriptional level. Crotonaldehyde and 4-hydroxy-2-nonenal, an endogenous α,β-unsaturated aldehyde, stimulated VEGF release, as did H2O2. CSE-evoked VEGF release was accompanied by rapid and lasting phosphorylation of p38 MAPK (mitogen-activated protein kinase), which was abolished by NAC and mimicked by acrolein. Both CSE- and acrolein-evoked VEGF release were blocked by selective inhibition of p38 MAPK signalling. CONCLUSIONS AND IMPLICATIONS α,β-Unsaturated aldehydes and possibly reactive oxygen species contained in cigarette smoke stimulate VEGF expression and release from pulmonary cells through p38 MAPK signalling. PMID:21306579

  8. Linoleic acid supplementation results in increased arachidonic acid and eicosanoid production in CF airway cells and in cftr−/− transgenic mice

    PubMed Central

    Zaman, Munir M.; Martin, Camilia R.; Andersson, Charlotte; Bhutta, Abdul Q.; Cluette-Brown, Joanne E.; Laposata, Michael

    2010-01-01

    Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o− sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr−/− transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE2, and PGF2α secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr−/− Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators. PMID:20656894

  9. PROINFLAMMATORY OXIDANT HYPOCHLOROUS ACID (HOCL) INDUCES DUAL SIGNALING PATHWAYS IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    In the airway of inflammatory diseases such as bacterial infection, cystic fibrosis and COPD, high level of HOCL (local concentration of up to 5mM) can be generated through a reaction catalyzed by leukocyte granule enzyme- Myeloperoxidase (MPO). HOCL is a very potent oxidative ag...

  10. Low level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans

    EPA Science Inventory

    Background: The effects of low level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known, however much less is known about the inflammatory and immuno-modulatory effects oflow level ozone in the airways. Techniques such as induced sputum and flo...

  11. Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line.

    PubMed

    Tong, Zhenyue; Illek, Beate; Bhagwandin, Vikash J; Verghese, George M; Caughey, George H

    2004-11-01

    Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway. PMID:15246975

  12. Airway Epithelial Regulation of Allergic Sensitization in Asthma

    PubMed Central

    Poynter, Matthew E.

    2012-01-01

    While many of the contributing cell types and mediators of allergic asthma are known, less well understood are the factors that influence the development of allergic responses that lead to the development of allergic asthma. As the first airway cell type to respond to inhaled factors, the epithelium orchestrates downstream interactions between dendritic cells (DCs) and CD4+ T cells that quantitatively and qualitatively dictate the degree and type of the allergic asthma phenotype, making the epithelium of critical importance for the genesis of allergies that later manifest in allergic asthma. Amongst the molecular processes of critical importance in airway epithelium is the transcription factor, nuclear factor-kappaB (NF-κB). This review will focus primarily on the genesis of pulmonary allergies and the participation of airway epithelial NF-κB activation therein, using examples from our own work on nitrogen dioxide (NO2) exposure and genetic modulation of airway epithelial NF-κB activation. In addition, the mechanisms through which Serum Amyloid A (SAA), an NF-κB-regulated, epithelial-derived mediator, influences allergic sensitization and asthma severity will be presented. Knowledge of the molecular and cellular processes regulating allergic sensitization in the airways has the potential to provide powerful insight into the pathogenesis of allergy, as well as targets for the prevention and treatment of asthma. PMID:22579987

  13. Linking polymorphic p53 response elements with gene expression in airway epithelial cells of smokers and cancer risk.

    PubMed

    Wang, Xuting; Pittman, Gary S; Bandele, Omari J; Bischof, Jason J; Liu, Gang; Brothers, John F; Spira, Avrum; Bell, Douglas A

    2014-12-01

    Chronic cigarette smoking exposes airway epithelial cells to thousands of carcinogens, oxidants and DNA-damaging agents, creating a field of molecular injury in the airway and altering gene expression. Studies of cytologically normal bronchial epithelial cells from smokers have identified transcription-based biomarkers that may prove useful in early diagnosis of lung cancer, including a number of p53-regulated genes. The ability of p53 to regulate transcription is critical for tumor suppression, and this suggests that single-nucleotide polymorphisms (SNPs) in functional p53 binding sites (p53 response elements, or p53REs) that affect gene expression could influence susceptibility to cancer. To connect p53RE SNP genotype with gene expression and cancer risk, we identified a set of 204 SNPs in putative p53REs, and performed cis expression quantitative trait loci (eQTL) analysis, assessing associations between SNP genotypes and mRNA levels of adjacent genes in bronchial epithelial cells obtained from 44 cigarette smokers. To further test and validate these genotype-expression associations, we searched published eQTL studies from independent populations and determined that 53% (39/74) of the bronchial epithelial eQTLs were observed in at least one of other studies. SNPs in p53REs were also evaluated for effects on p53-DNA binding using a quantitative in vitro protein-DNA binding assay. Last, based on linkage disequilibrium, we found 6 p53RE SNPs associated with gene expression were identified as cancer risk SNPs by either genome-wide association studies or candidate gene studies. We provide an approach for identifying and evaluating potentially functional SNPs that may modulate the airway gene expression response to smoking and may influence susceptibility to cancers.

  14. Sex Steroids Influence Brain-Derived Neurotropic Factor Secretion From Human Airway Smooth Muscle Cells.

    PubMed

    Wang, Sheng-Yu; Freeman, Michelle R; Sathish, Venkatachalem; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S

    2016-07-01

    Brain derived neurotropic factor (BDNF) is emerging as an important player in airway inflammation, remodeling, and hyperreactivity. Separately, there is increasing evidence that sex hormones contribute to pathophysiology in the lung. BDNF and sex steroid signaling are thought to be intricately linked in the brain. There is currently little information on BDNF and sex steroid interactions in the airway but is relevant to understanding growth factor signaling in the context of asthma in men versus women. In this study, we assessed the effect of sex steroids on BDNF expression and secretion in human airway smooth muscle (ASM). Human ASM was treated with estrogen (E2 ) or testosterone (T, 10 nM each) and intracellular BDNF and secreted BDNF measured. E2 and T significantly reduced secretion of BDNF; effects prevented by estrogen and androgen receptor inhibitor, ICI 182,780 (1 μM), and flutamide (10 μM), respectively. Interestingly, no significant changes were observed in intracellular BDNF mRNA or protein expression. High affinity BDNF receptor, TrkB, was not altered by E2 or T. E2 (but not T) significantly increased intracellular cyclic AMP levels. Notably, Epac1 and Epac2 expression were significantly reduced by E2 and T. Furthermore, SNARE complex protein SNAP25 was decreased. Overall, these novel data suggest that physiologically relevant concentrations of E2 or T inhibit BDNF secretion in human ASM, suggesting a potential interaction of sex steroids with BDNF in the airway that is different from brain. The relevance of sex steroid-BDNF interactions may lie in their overall contribution to airway diseases such as asthma. PMID:26566264

  15. Interleukin-19: A Constituent of the Regulome That Controls Antigen Presenting Cells in the Lungs and Airway Responses to Microbial Products

    PubMed Central

    Hoffman, Carol; Park, Sung-Hyun; Daley, Eleen; Emson, Claire; Louten, Jennifer; Sisco, Maureen; de Waal Malefyt, Rene; Grunig, Gabriele

    2011-01-01

    Background Interleukin (IL)-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+) alveolar macrophages and lung dendritic cells. Methodology/Principal Findings IL-19-deficient (IL-19-/-) mice were studied at baseline (naïve) and following intranasal challenge with microbial products, or recombinant cytokines. Naïve IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL) due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII) in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2) expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naïve IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13. Conclusions/Significance Because MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2

  16. Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

    SciTech Connect

    Jeong, Young-Il; Kim, Seung Hyun; Ju, Jung Won; Cho, Shin Hyeong; Lee, Won Ja; Park, Jin Wook; Park, Yeong-Min; Lee, Sang Eun

    2011-04-22

    Highlights: {yields} Treatment with Clonorchis sinensis-derived total protein attenuates OVA-induced airway inflammation and AHR to methacholine. {yields} Induction of CD4{sup +}CD25{sup +}Foxp3{sup +} T cells and IL-10 along with suppression of splenocyte proliferation by C. sinensis-derived total protein. {yields} C. sinensis-derived total protein interferes with the expression of co-stimulatory molecules in DCs. -- Abstract: Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4{sup +}CD25{sup +}Foxp3{sup +} regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naive T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical

  17. An NF-κB-independent and Erk1/2-dependent mechanism controls CXCL8/IL-8 responses of airway epithelial cells to cadmium.

    PubMed

    Cormet-Boyaka, Estelle; Jolivette, Kalyn; Bonnegarde-Bernard, Astrid; Rennolds, Jessica; Hassan, Fatemat; Mehta, Payal; Tridandapani, Susheela; Webster-Marketon, Jeanette; Boyaka, Prosper N

    2012-02-01

    Airway epithelial cells in the lung are the first line of defense against pathogens and environmental pollutants. Inhalation of the environmental pollutant cadmium has been linked to the development of lung cancer and chronic obstructive pulmonary disease, which are diseases characterized by chronic inflammation. To address the role of airway epithelial cells in cadmium-induced lung inflammation, we investigated how cadmium regulates secretion of interleukin 8 (IL-8) by airway epithelial cells. We show that exposure of human airway epithelial cells to subtoxic doses of cadmium in vitro promotes a characteristic inflammatory cytokine response consisting of IL-8, but not IL-1β or tumor necrosis factor-alpha. We also found that intranasal delivery of cadmium increases lung levels of the murine IL-8 homologs macrophage inflammatory protein-2 and keracinocyte-derived chemokine and results in an influx of Gr1+ cells into the lung. We determined that inhibition of the nuclear factor-κB (NF-κB) pathway had no effect on cadmium-induced IL-8 secretion by human airway epithelial cells, suggesting that IL-8 production was mediated through an NF-κB-independent pathway. Mitogen-activated protein kinases (MAPKs) are often involved in proinflammatory signaling. Cadmium could activate the main MAPKs (i.e., p38, JNK, and Erk1/2) in human airway epithelial cells. However, only pharmacological inhibition of Erk1/2 pathway or knockdown of the expression of Erk1 and Erk2 using small interfering RNAs suppressed secretion of IL-8 induced by cadmium. Our findings identify cadmium as a potent activator of the proinflammatory cytokine IL-8 in lung epithelial cells and reveal for the first time the role of an NF-κB-independent but Erk1/2-dependent pathway in cadmium-induced lung inflammation. PMID:22094458

  18. Syncytial-Type Cell Plates

    PubMed Central

    Otegui, Marisa; Staehelin, L. Andrew

    2000-01-01

    Cell wall formation in the syncytial endosperm of Arabidopsis was studied by using high-pressure-frozen/freeze-substituted developing seeds and immunocytochemical techniques. The endosperm cellularization process begins at the late globular embryo stage with the synchronous organization of small clusters of oppositely oriented microtubules (∼10 microtubules in each set) into phragmoplast-like structures termed mini-phragmoplasts between both sister and nonsister nuclei. These mini-phragmoplasts produce a novel kind of cell plate, the syncytial-type cell plate, from Golgi-derived vesicles ∼63 nm in diameter, which fuse by way of hourglass-shaped intermediates into wide (∼45 nm in diameter) tubules. These wide tubules quickly become coated and surrounded by a ribosome-excluding matrix; as they grow, they branch and fuse with each other to form wide tubular networks. The mini-phragmoplasts formed between a given pair of nuclei produce aligned tubular networks that grow centrifugally until they merge into a coherent wide tubular network with the mini-phragmoplasts positioned along the network margins. The individual wide tubular networks expand laterally until they meet and eventually fuse with each other at the sites of the future cell corners. Transformation of the wide tubular networks into noncoated, thin (∼27 nm in diameter) tubular networks begins at multiple sites and coincides with the appearance of clathrin-coated budding structures. After fusion with the syncytial cell wall, the thin tubular networks are converted into fenestrated sheets and cell walls. Immunolabeling experiments show that the cell plates and cell walls of the endosperm differ from those of the embryo and maternal tissue in two features: their xyloglucans lack terminal fucose residues on the side chain, and callose persists in the cell walls after the cell plates fuse with the parental plasma membrane. The lack of terminal fucose residues on xyloglucans suggests that these cell wall

  19. Negative impact of DEP exposure on human airway epithelial cell adhesion, stiffness, and repair.

    PubMed

    Doornaert, Blandine; Leblond, Valerie; Galiacy, Stephane; Gras, Gabriel; Planus, Emmanuelle; Laurent, Valerie; Isabey, Daniel; Lafuma, Chantal

    2003-01-01

    Epidemiological and experimental studies suggest that diesel exhaust particles (DEPs) may be associated with increased respiratory mortality and morbidity. Several recent studies have also shown that DEPs increase the production of inflammatory cytokines by human bronchial epithelium (HBE) cells in vitro. The present study investigates the effects of DEPs on the interaction of l-HBE cells (16HBE14o-) with the cell and matrix microenvironment based on evaluation of integrin-type cell/matrix ligand expression, cytoskeleton (CSK) stiffness, and matrix remodeling via matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 expression. The results showed that DEP exposure induced: 1) a net dose-dependent decrease in CSK stiffness through actin fibers, 2) a concomitant specific reduction of both alpha(3)- and beta(1)-integrin subunits extensively expressed on the HBE cell surface, 3) a decrease in the level of CD44, which is a major HBE cell-cell and HBE cell-matrix adhesion molecule; and 4) an isolated decrease in MMP-1 expression without any change in tissue inhibitor of matrix metalloproteinase (TIMP)-1 or TIMP-2 tissue inhibitors. Restrictive modulation of cell-matrix interaction, cell-cell connection, CSK stiffness, and fibrillary collagen remodeling results in a decreased wound closure capacity and an increased deadhesion capacity. In conclusion, on the basis of these results, we can propose that, in addition to their ability to increase the production of inflammatory cytokines, DEPs could also alter the links between actin CSK and the extracellular matrix, suggesting that they might facilitate HBE cell detachment in vivo. PMID:12471014

  20. The response of guinea pig airway epithelial cells and alveolar macrophages to environmental stress

    SciTech Connect

    Cohen, D.S.; Palmer, E.; Welch, W.J.; Sheppard, D. )

    1991-08-01

    Cells lining the respiratory tract form an interface between the organism and the external environment and are repeatedly exposed to physical, chemical, and metabolic stresses. The authors examined the response of cultured guinea pig tracheal epithelial cells and alveolar macrophages to various forms of stress, including clinically and environmentally relevant metabolic stresses such as ozone and acid exposure. Classic stress treatments such as heat shock and sodium arsenite treatment induced the synthesis of 28, 32, 72, 73, 90, and 110 kD stress proteins similar to those observed in other cell types. In contrast, no significant changes in the pattern of protein synthesis were detected after exposure to ambient concentrations of ozone, although ozone exposure caused significant cytotoxicity to both cell types. Another potent oxidant, hydrogen peroxide, similarly did not induce appreciable stress protein synthesis. However, surface acidification of tracheal epithelial cells and alveolar macrophages caused the induction of 72 and 78 kD stress proteins. While stress proteins may play a role in the response of respiratory cells to certain injuries such as hyperthermia and surface acidification, they may not be important in the defense against ozone or other forms of oxidative injury.

  1. Growth of airway epithelial cells at an air-liquid interface changes both the response to particle exposure and iron homeostasis.

    EPA Science Inventory

    RATIONALE: We tested the hypothesis that 1) relative to submerged cells, airway epithelial cells grown at an air-liquid interface and allowed to differentiate would have an altered response to particle exposure and 2) that these differences would be associated with indices of iro...

  2. Growth of airway epithelial cells at an air-liquid interface changes both the response to particle exposure and iron homeostasis

    EPA Science Inventory

    We tested the hypothesis that 1) relative to submerged cells, airway epithelial cells grown at an air-liquid interface and allowed to differentiate would have an altered response to particle exposure and 2) that these differences would be associated with indices of iron homeostas...

  3. Pseudomonas aeruginosa pyocyanin causes airway goblet cell hyperplasia and metaplasia and mucus hypersecretion by inactivating the transcriptional factor FoxA2.

    PubMed

    Hao, Yonghua; Kuang, Zhizhou; Walling, Brent E; Bhatia, Shikha; Sivaguru, Mayandi; Chen, Yin; Gaskins, H Rex; Lau, Gee W

    2012-03-01

    The redox-active exotoxin pyocyanin (PCN) can be recovered in 100 µM concentrations in the sputa of bronchiectasis patients chronically infected with Pseudomonas aeruginosa (PA). However, the importance of PCN within bronchiectatic airways colonized by PA remains unrecognized. Recently, we have shown that PCN is required for chronic PA lung infection in mice, and that chronic instillation of PCN induces goblet cell hyperplasia (GCH), pulmonary fibrosis, emphysema and influx of immune cells in mouse airways. Many of these pathological features are strikingly similar to the mouse airways devoid of functional FoxA2, a transcriptional repressor of GCH and mucus biosynthesis. In this study, we postulate that PCN causes and exacerbates GCH and mucus hypersecretion in bronchiectatic airways chronically infected by PA by inactivating FoxA2. We demonstrate that PCN represses the expression of FoxA2 in mouse airways and in bronchial epithelial cells cultured at an air-liquid interface or conventionally, resulting in GCH, increased MUC5B mucin gene expression and mucus hypersecretion. Immunohistochemical and inhibitor studies indicate that PCN upregulates the expression of Stat6 and EGFR, both of which in turn repress the expression of FoxA2. These studies demonstrate that PCN induces GCH and mucus hypersecretion by inactivating FoxA2.

  4. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    SciTech Connect

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F.

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  5. The effect of Shenmai injection on the proliferation of Rat airway smooth muscle cells in asthma and underlying mechanism

    PubMed Central

    2013-01-01

    Background Over-proliferation of airway smooth muscle cell (ASMC) is one of the important contributors to airway remodeling in asthma. The aim of this study was to investigate the effect of Shenmai injection (SMI) on the proliferation of the rat ASMC in asthma. Methods Rats were randomly divided into three groups: the control group, the asthma group, and the SMI treatment group. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry staining were used to detect the mRNA and protein expression of transient receptor potential vanilloid 1 (TRPV1) and proliferating cell nuclear antigen (PCNA) in rat ASMC respectively. Intracellular Ca2+ concentration ( [Ca2+]i ) in rat ASMC were measured with Fluo-3/AM by confocal microscopy. The proliferation was detected by MTT assay. Results Compared with the control group, the asthma group showed an increased expression of TRPV1 and [Ca2+]i in rat ASMC. The expression of PCNA and absorbance of MTT assay in asthma rat ASMC was also significantly increased. SMI could significantly decrease the expression of TRPV1 channel and [Ca2+]i in the asthmatic rat ASMC. Furthermore, the expression of PCNA and absorbance of MTT assay in asthmatic rat ASMC was significantly reduced after SMI treatment. Conclusions SMI may prevent asthma-induced ASMC over-proliferation probably by inhibiting the expression of TRPV1 channel, which regulates the intracellular calcium concentration. PMID:24010863

  6. Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation.

    PubMed

    Talbot, Sébastien; Abdulnour, Raja-Elie E; Burkett, Patrick R; Lee, Seungkyu; Cronin, Shane J F; Pascal, Maud A; Laedermann, Cedric; Foster, Simmie L; Tran, Johnathan V; Lai, Nicole; Chiu, Isaac M; Ghasemlou, Nader; DiBiase, Matthew; Roberson, David; Von Hehn, Christian; Agac, Busranour; Haworth, Oliver; Seki, Hiroyuki; Penninger, Josef M; Kuchroo, Vijay K; Bean, Bruce P; Levy, Bruce D; Woolf, Clifford J

    2015-07-15

    Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8(+) sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4(+) and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma. PMID:26119026

  7. Silencing nociceptor neurons reduces allergic airway inflammation

    PubMed Central

    Talbot, Sébastien; Abdulnour, Raja-Elie E.; Burkett, Patrick R.; Lee, Seungkyu; Cronin, Shane J.F.; Pascal, Maud A.; Laedermann, Cedric; Foster, Simmie L.; Tran, Johnathan V.; Lai, Nicole; Chiu, Isaac M.; Ghasemlou, Nader; DiBiase, Matthew; Roberson, David; Von Hehn, Christian; Agac, Busranour; Haworth, Oliver; Seki, Hiroyuki; Penninger, Josef M.; Kuchroo, Vijay K.; Bean, Bruce P.; Levy, Bruce D.; Woolf, Clifford J.

    2015-01-01

    Summary Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8+ sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large pore ion channels to specifically block nociceptors, substantially reduced ovalbumin or house dust mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4+ and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma. PMID:26119026

  8. Chronic inhalation of biomass smoke is associated with DNA damage in airway cells: involvement of particulate pollutants and benzene.

    PubMed

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

    2013-04-01

    This study examined whether indoor air pollution from biomass fuel burning induces DNA damage in airway cells. For this, sputum cells were collected from 56 premenopausal rural women who cooked with biomass (wood, dung, crop residues) and 49 age-matched controls who cooked with cleaner liquefied petroleum gas. The levels of particulate matters with diameters of less than 10 and 2.5 µm (PM(10) and PM(2.5)) in indoor air were measured using a real-time aerosol monitor. Benzene exposure was monitored by measuring trans,trans-muconic acid (t,t-MA) in urine by HPLC-UV. DNA damage was examined by alkaline comet assay in sputum cells. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in sputum cells were measured by flow cytometry and spectrophotometry, respectively. Compared with controls, biomass users had 4 times higher tail percentage DNA, 37% more comet tail length and 5 times more Olive tail moment (p < 0.001) in inflammatory and epithelial cells in sputum, suggesting extensive DNA damage. In addition, women who cooked with biomass had 6 times higher levels of urinary t,t-MA and 2-fold higher levels of ROS generation concomitant with 28% depletion of SOD. Indoor air of biomass-using households had 2-4 times more PM(10) and PM(2.5) than that of controls. After controlling potential confounders, positive association was found between DNA damage parameters, particulate pollution, urinary t,t-MA and ROS. Thus, long-term exposure to biomass smoke induces DNA damage in airway cells and the effect was probably mediated, at least in part, by oxidative stress generated by inhaled particulate matter and benzene.

  9. An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells.

    PubMed

    Massoud, Amir Hossein; Charbonnier, Louis-Marie; Lopez, David; Pellegrini, Matteo; Phipatanakul, Wanda; Chatila, Talal A

    2016-09-01

    Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the gene encoding the interleukin (IL)-4 receptor alpha chain (Il4ra(R576)) promotes conversion of induced Treg (iTreg) cells toward a T helper 17 (TH17) cell fate. This skewing is mediated by the recruitment by IL-4Rα(R576) of the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which drives IL-17 expression by activating a pathway that involves extracellular-signal-regulated kinase, IL-6 and the transcription factor STAT3. Treg cell-specific deletion of genes that regulate TH17 cell differentiation, including Il6ra and RAR-related orphan receptor gamma (Rorc), but not of Il4 or Il13, prevented exacerbated airway inflammation in mice expressing Il4ra(R576) (hereafter referred to as Il4ra(R576) mice). Furthermore, treatment of Il4ra(R576) mice with a neutralizing IL-6-specific antibody prevented iTreg cell reprogramming into TH17-like cells and protected against severe airway inflammation. These findings identify a previously unknown mechanism for the development of mixed TH2-TH17 cell inflammation in genetically prone individuals and point to interventions that stabilize iTreg cells as potentially effective therapeutic strategies. PMID:27479084

  10. Preventative Effect of an Herbal Preparation (HemoHIM) on Development of Airway Inflammation in Mice via Modulation of Th1/2 Cells Differentiation

    PubMed Central

    Kim, Jong-Jin; Cho, Hyun Wook; Park, Hae-Ran; Jung, Uhee; Jo, Sung-Kee; Yee, Sung-Tae

    2013-01-01

    HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4+ T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4+ T cells displayed increased Th1 (IFN-γ+ cell) as well as decreased Th2 (IL-4+ cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance. PMID:23844220

  11. Preventative effect of an herbal preparation (HemoHIM) on development of airway inflammation in mice via modulation of Th1/2 cells differentiation.

    PubMed

    Kim, Jong-Jin; Cho, Hyun Wook; Park, Hae-Ran; Jung, Uhee; Jo, Sung-Kee; Yee, Sung-Tae

    2013-01-01

    HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4(+) T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4(+) T cells displayed increased Th1 (IFN-γ(+) cell) as well as decreased Th2 (IL-4(+) cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance. PMID:23844220

  12. Preventative effect of an herbal preparation (HemoHIM) on development of airway inflammation in mice via modulation of Th1/2 cells differentiation.

    PubMed

    Kim, Jong-Jin; Cho, Hyun Wook; Park, Hae-Ran; Jung, Uhee; Jo, Sung-Kee; Yee, Sung-Tae

    2013-01-01

    HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4(+) T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4(+) T cells displayed increased Th1 (IFN-γ(+) cell) as well as decreased Th2 (IL-4(+) cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance.

  13. Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization, Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells.

    PubMed

    Camoretti-Mercado, Blanca; Pauer, Susan H; Yong, Hwan Mee; Smith, Dan'elle C; Deshpande, Deepak A; An, Steven S; Liggett, Stephen B

    2015-01-01

    Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca2+]i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca2+]i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca2+]i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca2+]i increases from histamine, but failed to inhibit [Ca2+]i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca2+]i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca2+]i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca2+]i inhibition and TAS2R subtype, nor whether [Ca2+]i was increased by Gq- or Gi-coupled GPCRs. Selected studies revealed a correlation between [Ca2+]i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca2+]i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca2+]i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low

  14. Fluticasone propionate and pentamidine isethionate reduce airway hyperreactivity, pulmonary eosinophilia and pulmonary dendritic cell response in a guinea pig model of asthma.

    PubMed

    Lawrence, T E; Millecchia, L L; Fedan, J S

    1998-01-01

    In this study, we examined the effects of fluticasone propionate (FP) and pentamidine isethionate (PI) on antigen-induced lung inflammation and airway hyperreactivity in guinea pigs. Male guinea pigs were sensitized on days 0 and 14 with 10 micrograms of ovalbumin (OVA) plus 1 mg of Al(OH)3. On day 21, animals were challenged with a 2% OVA aerosol inhalation until they developed pulmonary obstruction. Animals were treated with aerosol inhalation of FP (2 ml of 0.5 mg/ml, five consecutive doses at 12-hr intervals with the last dose given 6 hr before OVA challenge) or PI (30 mg/ml for 30 min 1 hr before OVA challenge), and control animals received no drug before OVA challenge. Airway reactivity to methacholine (MCh) was assessed before sensitization and 18 hr after OVA challenge. At 18 hr after challenge, histological sections of trachea and lung were examined for eosinophil, dendritic cell (DC) and macrophage cell densities in the airways. In control animals, OVA evoked airway hyperreactivity to MCh in conjunction with pulmonary eosinophilia and increases in DC prevalence in the trachea and bronchi. Treatment with FP or PI abolished the OVA-induced hyperresponsiveness and significantly reduced the OVA-induced increases in eosinophils and DCs in the airways. FP and PI had no effect on saline-treated animals. Our study indicates that both inhaled FP and inhaled PI reduce antigen-induced airway hyperreactivity and pulmonary inflammation in guinea pigs. The results also suggest that the DC is a target of the anti-inflammatory effects of these drugs in the airways. PMID:9435182

  15. Indirect airway challenges.

    PubMed

    Joos, G F; O'Connor, B; Anderson, S D; Chung, F; Cockcroft, D W; Dahlén, B; DiMaria, G; Foresi, A; Hargreave, F E; Holgate, S T; Inman, M; Lötvall, J; Magnussen, H; Polosa, R; Postma, D S; Riedler, J

    2003-06-01

    Indirect challenges act by causing the release of endogenous mediators that cause the airway smooth muscle to contract. This is in contrast to the direct challenges where agonists such as methacholine or histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Direct airway challenges have been used widely and are well standardised. They are highly sensitive, but not specific to asthma and can be used to exclude current asthma in a clinic population. Indirect bronchial stimuli, in particular exercise, hyperventilation, hypertonic aerosols, as well as adenosine, may reflect more directly the ongoing airway inflammation and are therefore more specific to identify active asthma. They are increasingly used to evaluate the prevalence of bronchial hyperresponsiveness and to assess specific problems in patients with known asthma, e.g. exercise-induced bronchoconstriction, evaluation before scuba diving. Direct bronchial responsiveness is only slowly and to a modest extent, influenced by repeated administration of inhaled steroids. Indirect challenges may reflect more closely acute changes in airway inflammation and a change in responsiveness to an indirect stimulus may be a clinically relevant marker to assess the clinical course of asthma. Moreover, some of the indirect challenges, e.g. hypertonic saline and mannitol, can be combined with the assessment of inflammatory cells by induction of sputum.

  16. Bronchial biopsy evidence for leukocyte infiltration and upregulation of leukocyte-endothelial cell adhesion molecules 6 hours after local allergen challenge of sensitized asthmatic airways.

    PubMed Central

    Montefort, S; Gratziou, C; Goulding, D; Polosa, R; Haskard, D O; Howarth, P H; Holgate, S T; Carroll, M P

    1994-01-01

    We have examined the mucosal changes occurring in bronchial biopsies from six atopic asthmatics 5-6 h after local endobronchial allergen challenge and compared them with biopsies from saline-challenged segments from the same subjects at the same time point. All the subjects developed localized bronchoconstriction in the allergen-challenged segment and had a decrease in forced expiratory volume in 1 s (FEV1) (P < 0.01) and a decrease in their methacholine provocative concentration of agonist required to reduce FEV1 from baseline by 20% (P < 0.05) 24 h postchallenge. At 6 h we observed an increase in neutrophils (P = 0.03), eosinophils (P = 0.025), mast cells (P = 0.03), and CD3+ lymphocytes (P = 0.025), but not in CD4+ or CD8+ lymphocyte counts. We also detected an increase in endothelial intercellular adhesion molecule type 1 (P < 0.05) and E-selectin (P < 0.005), but not vascular cell adhesion molecule type 1 expression with a correlative increase in submucosal and epithelial LFA+ leucocytes (P < 0.01). Thus, in sensitized asthmatics, local endobronchial allergen instillation leads to an increased inflammatory cell infiltrate of the airway mucosa that involves upregulation of specific adhesion molecules expressed on the microvasculature. Images PMID:7512980

  17. Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells.

    PubMed

    Popova, Taissia G; Turell, Michael J; Espina, Virginia; Kehn-Hall, Kylene; Kidd, Jessica; Narayanan, Aarthi; Liotta, Lance; Petricoin, Emanuel F; Kashanchi, Fatah; Bailey, Charles; Popov, Serguei G

    2010-01-01

    Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK) and downstream transcriptional factors [STAT1 (Y701), ATF2 (T69/71), MSK1 (S360) and CREB (S133)]. NF-κB phosphorylation was also increased. Activation of p53 (S15, S46) correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473), along with phosphorylation of FOX 01/03 (T24/31) which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication. PMID:21072193

  18. Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck

    PubMed Central

    O'hara, Kimberley A.; Vaghjiani, Rasilaben J.; Nemec, Antonia A.; Klei, Linda R.; Barchowsky, Aaron

    2006-01-01

    Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein–DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 μM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3α and STAT3β. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression. PMID:17078813

  19. Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck.

    PubMed

    O'Hara, Kimberley A; Vaghjiani, Rasilaben J; Nemec, Antonia A; Klei, Linda R; Barchowsky, Aaron

    2007-03-01

    Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein-DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 microM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3alpha and STAT3beta. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression.

  20. The effect of ambroxol on chloride transport, CFTR and ENaC in cystic fibrosis airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Hussain, Rashida; Strid, Hilja; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2013-11-01

    Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl(-) efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) influx via the epithelial Na(+) channel (ENaC). In cells expressing wt-CFTR, ambroxol increased the Cl(-) conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl(-) transport was measured by Cl(-) efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl(-) efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α-ENaC, and of the CFTR protein. No significant difference was observed in β-ENaC after exposure to ambroxol, whereas mRNA expression of γ-ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca(2+) concentration. Upregulation of CFTR and enhanced Cl(-) efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients.

  1. The effect of ambroxol on chloride transport, CFTR and ENaC in cystic fibrosis airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Hussain, Rashida; Strid, Hilja; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2013-11-01

    Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl(-) efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) influx via the epithelial Na(+) channel (ENaC). In cells expressing wt-CFTR, ambroxol increased the Cl(-) conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl(-) transport was measured by Cl(-) efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl(-) efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α-ENaC, and of the CFTR protein. No significant difference was observed in β-ENaC after exposure to ambroxol, whereas mRNA expression of γ-ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca(2+) concentration. Upregulation of CFTR and enhanced Cl(-) efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients. PMID:23765701

  2. Educating the Educator: Teaching Airway Adjunct Techniques in Athletic Training

    ERIC Educational Resources Information Center

    Berry, David C.; Seitz, S. Robert

    2011-01-01

    The 5th edition of the "Athletic Training Education Competencies" ("Competencies") now requires athletic training educators (ATEs) to introduce into the curriculum various types of airway adjuncts including: (1) oropharyngeal airways (OPA), (2) nasopharyngeal airways (NPA), (3) supraglottic airways (SGA), and (4) suction. The addition of these…

  3. Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

    PubMed

    Zhao, Limin; Sullivan, Michelle N; Chase, Marlee; Gonzales, Albert L; Earley, Scott

    2014-06-01

    Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

  4. Arsenic promotes ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells.

    PubMed

    Bomberger, Jennifer M; Coutermarsh, Bonita A; Barnaby, Roxanna L; Stanton, Bruce A

    2012-05-18

    Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.

  5. Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing

    PubMed Central

    Lendeckel, Derik; Eymann, Christine; Emicke, Philipp; Daeschlein, Georg; Darm, Katrin; O'Neil, Serena; Beule, Achim G.; von Woedtke, Thomas; Völker, Uwe; Weltmann, Klaus-Dieter; Jünger, Michael; Hosemann, Werner; Scharf, Christian

    2015-01-01

    Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine. PMID:26539504

  6. CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells

    PubMed Central

    Chirkova, Tatiana; Lin, Songbai; Oomens, Antonius G. P.; Gaston, Kelsey A.; Boyoglu-Barnum, Seyhan; Meng, Jia; Stobart, Christopher C.; Cotton, Calvin U.; Hartert, Tina V.; Moore, Martin L.; Ziady, Assem G.

    2015-01-01

    Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection. PMID:26297201

  7. Type 2 innate lymphoid cells: at the cross-roads in allergic asthma.

    PubMed

    van Rijt, Leonie; von Richthofen, Helen; van Ree, Ronald

    2016-07-01

    Allergic asthma is a chronic inflammatory disease of the lower airways that affects millions of people worldwide. Allergic asthma is a T helper 2 cell (Th2)-mediated disease, in which Th2 cytokines interleukin (IL)-4, IL-5, and IL-13 are closely associated with the symptoms. IL-4 is needed by B cells to switch toward an IgE response, IL-5 recruits and activates eosinophils while IL-13 increases mucus production. The identification of type 2 innate lymphoid cells (ILC2), which are able to rapidly produce large amounts of IL-5 and IL-13 in response to epithelial derived cytokines, implicated a new key player besides Th2 cells. ILCs constitute a family of innate lymphocytes distinct from T and B cells. ILC2s are located in various epithelial compartments in mice and human, including the lung. The recent finding of increased numbers of ILC2s in the airways of severe asthma patients prompts further research to clarify their immunological function. Murine studies have shown that ILC2s are an early innate source of IL-5 and IL-13 after allergen exposure, which induce airway eosinophilic infiltration, mucus hyperproduction, and airway hyperresponsiveness but not allergen-specific IgE production. ILC2s contribute to the initiation as well as to the maintenance of the adaptive type 2 immune response. Here, we review the recent progress on our understanding of the role of ILC2s in the immunopathology of allergic asthma, in particular by studies using murine models which have elucidated fundamental mechanisms by which ILC2s act.

  8. 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

  9. Airway Fibrinogenolysis and the Initiation of Allergic Inflammation

    PubMed Central

    Millien, Valentine Ongeri; Lu, Wen; Mak, Garbo; Yuan, Xiaoyi; Knight, J. Morgan; Porter, Paul; Kheradmand, Farrah

    2014-01-01

    The past 15 years of allergic disease research have produced extraordinary improvements in our understanding of the pathogenesis of airway allergic diseases such as asthma. Whereas it was previously viewed as largely an immunoglobulin E-mediated process, the gradual recognition that T cells, especially Type 2 T helper (Th2) cells and Th17 cells, play a major role in asthma and related afflictions has inspired clinical trials targeting cytokine-based inflammatory pathways that show great promise. What has yet to be clarified about the pathogenesis of allergic inflammatory disorders, however, are the fundamental initiating factors, both exogenous and endogenous, that drive and sustain B- and T-cell responses that underlie the expression of chronic disease. Here we review how proteinases derived from diverse sources drive allergic responses. A central discovery supporting the proteinase hypothesis of allergic disease pathophysiology is the role played by airway fibrinogen, which in part appears to serve as a sensor of unregulated proteinase activity and which, when cleaved, both participates in a novel allergic signaling pathway through Toll-like receptor 4 and forms fibrin clots that contribute to airway obstruction. Unresolved at present is the ultimate source of airway allergenic proteinases. From among many potential candidates, perhaps the most intriguing is the possibility such enzymes derive from airway fungi. Together, these new findings expand both our knowledge of allergic disease pathophysiology and options for therapeutic intervention. PMID:25525732

  10. MATRIX METALLOPROTEINS (MMP)-MEDIATED PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZINC (ZN)

    EPA Science Inventory

    Matrix Metalloproteinase (MMP)-Mediated Phosphorylation of The Epidermal Growth Factor Receptor (EGFR) in Human Airway Epithelial Cells (HAEC) Exposed to Zinc (Zn)
    Weidong Wu, James M. Samet, Robert Silbajoris, Lisa A. Dailey, Lee M. Graves, and Philip A. Bromberg
    Center fo...

  11. Pulmonary Kirsten Rat Sarcoma Virus Mutation Positive Mucinous Adenocarcinoma Arising in a Congenital Pulmonary Airway Malformation, Mixed Type 1 and 2.

    PubMed

    Singh, Gopal; Coffey, Amy; Neely, Robert; Lambert, Daniel; Sonett, Joshua; Borczuk, Alain C; Gorenstein, Lyall

    2016-10-01

    Congenital pulmonary airway malformation (CPAM) is a developmental abnormality of the lung, which results from an abnormality of branching during fetal development of the lung. We report the case of an 18 year-old woman who developed Kirsten rat sarcoma virus (KRAS) mutation positive mucinous adenocarcinoma of the lung (AC) in association with mixed CPAM type 1 and 2. This case is unique as KRAS mutation positive AC is present in a setting of both CPAM 1 and 2 in the same lesion. PMID:27645976

  12. IL-1β, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs.

    PubMed

    Bal, Suzanne M; Bernink, Jochem H; Nagasawa, Maho; Groot, Jelle; Shikhagaie, Medya M; Golebski, Kornel; van Drunen, Cornelis M; Lutter, Rene; Jonkers, Rene E; Hombrink, Pleun; Bruchard, Melanie; Villaudy, Julien; Munneke, J Marius; Fokkens, Wytske; Erjefält, Jonas S; Spits, Hergen; Ros, Xavier Romero

    2016-06-01

    Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation. PMID:27111145

  13. Effects of second hand smoke on airway secretion and mucociliary clearance

    PubMed Central

    Liu, Yanyan; Di, Y. Peter

    2012-01-01

    The airway acts as the first defense against inhaled pathogens and particulate matter from the environment. One major way for the airway to clear inhaled foreign objects is through mucociliary clearance (MCC), an important component of the respiratory innate immune defense against lung disease. MCC is characterized by the upward movement of mucus by ciliary motion that requires a balance between the volume and composition of the mucus, adequate periciliary liquid (PCL) volume, and normal ciliary beat frequency (CBF). Airway surface fluid (ASL) is a thin layer liquid that consists of the highly viscous mucus upper “gel” layer, and the watery lubricating lower “sol” layer. Mucus production, secretion and clearance are considered to play a critical role in maintenance of airway health because it maintains hydration in the airway and traps particulates, bacteria, and viruses. Different types of epithelial cells, including secretory cells, and ciliated cells, contribute to the MCC function. Cigarette smoke (CS) contains chemicals and particulates that significantly affect airway secretion. Active and passive CS-induced chronic obstructive pulmonary disease (COPD) is frequently associated with hyperplasia of goblet cells and submucosal glands (SMGs), thus increasing the secretory capacity of the airways that impairs MCC. PMID:22973232

  14. Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

    PubMed

    Kelly, Catriona; Canning, Paul; Buchanan, Paul J; Williams, Mark T; Brown, Vanessa; Gruenert, Dieter C; Elborn, J Stuart; Ennis, Madeleine; Schock, Bettina C

    2013-03-01

    The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF. PMID:23316065

  15. Wood Smoke Enhances Cigarette Smoke–Induced Inflammation by Inducing the Aryl Hydrocarbon Receptor Repressor in Airway Epithelial Cells

    PubMed Central

    Awji, Elias G.; Chand, Hitendra; Bruse, Shannon; Smith, Kevin R.; Colby, Jennifer K.; Mebratu, Yohannes; Levy, Bruce D.

    2015-01-01

    Our previous studies showed that cigarette smokers who are exposed to wood smoke (WS) are at an increased risk for chronic bronchitis and reduced lung function. The present study was undertaken to determine the mechanisms for WS-induced adverse effects. We studied the effect of WS exposure using four cohorts of mice. C57Bl/6 mice were exposed for 4 or 12 weeks to filtered air, to 10 mg/m3 WS for 2 h/d, to 250 mg/m3 cigarette smoke (CS) for 6 h/d, or to CS followed by WS (CW). Inflammation was absent in the filtered air and WS groups, but enhanced by twofold in the bronchoalveolar lavage of the CW compared with CS group as measured by neutrophil numbers and levels of the neutrophil chemoattractant, keratinocyte-derived chemokine. The levels of the anti-inflammatory lipoxin, lipoxin A4, were reduced by threefold along with cyclo-oxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in airway epithelial cells and PGE2 levels in the bronchoalveolar lavage of CW compared with CS mice. We replicated, in primary human airway epithelial cells, the changes observed in mice. Immunoprecipitations showed that WS blocked the interaction of aryl hydrocarbon receptor (AHR) with AHR nuclear transporter to reduce expression of COX-2 and mPGES-1 by increasing expression of AHR repressor (AHRR). Collectively, these studies show that exposure to low concentrations of WS enhanced CS-induced inflammation by inducing AHRR expression to suppress AHR, COX-2, and mPGES-1 expression, and levels of PGE2 and lipoxin A4. Therefore, AHRR is a potential therapeutic target for WS-associated exacerbations of CS-induced inflammation. PMID:25137396

  16. Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

    PubMed

    Mullins, Benjamin J; Kicic, Anthony; Ling, Kak-Ming; Mead-Hunter, Ryan; Larcombe, Alexander N

    2016-01-01

    Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size.

  17. Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body.

    PubMed

    Kambe, Naotomo; Hiramatsu, Hidefumi; Shimonaka, Mika; Fujino, Hisanori; Nishikomori, Ryuta; Heike, Toshio; Ito, Mamoru; Kobayashi, Kimio; Ueyama, Yoshito; Matsuyoshi, Norihisa; Miyachi, Yoshiki; Nakahata, Tatsutoshi

    2004-02-01

    The transplantation of primitive human cells into sublethally irradiated immune-deficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient (NOD/SCID)/gamma(c)(null) (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor gamma-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result in a highly effective model for the investigation of human mast cell development and function in vivo.

  18. T cell treatment with small interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a murine model of asthma.

    PubMed

    Moriwaki, Atsushi; Inoue, Hiromasa; Nakano, Takako; Matsunaga, Yuko; Matsuno, Yukiko; Matsumoto, Takafumi; Fukuyama, Satoru; Kan-O, Keiko; Matsumoto, Koichiro; Tsuda-Eguchi, Miyuki; Nagakubo, Daisuke; Yoshie, Osamu; Yoshimura, Akihiko; Kubo, Masato; Nakanishi, Yoichi

    2011-04-01

    CD4(+) T cells, particularly T helper (Th) 2 cells, play a pivotal role in the pathophysiology of allergic asthma. Suppressor of cytokine signaling (SOCS) proteins control the balance of CD4(+) T cell differentiation. Mice that lack SOCS3 in T cells by crossing SOCS3-floxed mice with Lck-Cre-transgenic mice have reduced allergen-induced eosinophilia in the airways. Here, we studied the effects of SOCS3 silencing with small interfering (si) RNA in primary CD4(+) T cells on Th2 cell differentiation and on asthmatic responses in mice. Th2 cells were generated from ovalbumin (OVA)-specific T cell receptor-transgenic mice in vitro and transferred into recipient mice. Transfection of SOCS3-specific siRNA attenuated Th2 response in vitro. Adoptive transfer of SOCS3-siRNA T cells exhibited markedly suppressed airway hyperresponsiveness and eosinophilia after OVA challenge, with a concomitant decrease in OVA-specific CD4(+) T cell accumulation in the airways. To investigate the mechanism of this impaired CD4(+) T cell accumulation, we inactivated SOCS3 of T cells by crossing SOCS3-floxed (SOCS3(flox/flox)) mice with CD4-Cre transgenic mice. CD4-Cre × SOCS3(flox/flox) mice exhibited fewer IL-4-producing cells and more reduced eosinophil infiltration in bronchoalveolar lavage fluids than control mice in a model of OVA-induced asthma. Expression of CCR3 and CCR4 in CD4(+) T cells was decreased in CD4-Cre × SOCS3(flox/flox) mice. CCR4 expression was also decreased in CD4(+) T cells after transfer of SOCS3 siRNA-treated T cells. These findings suggest that the therapeutic modulation of SOCS3 expression in CD4(+) T cells might be effective in preventing the development of allergic asthma.

  19. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    SciTech Connect

    Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

    2011-10-07

    Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.

  20. Assays for in vitro monitoring of proliferation of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cells.

    PubMed

    Goncharova, Elena A; Lim, Poay; Goncharov, Dmitry A; Eszterhas, Andrew; Panettieri, Reynold A; Krymskaya, Vera P

    2006-01-01

    Vascular and airway remodeling, which are characterized by airway smooth muscle (ASM) and pulmonary arterial vascular smooth muscle (VSM) proliferation, contribute to the pathology of asthma, pulmonary hypertension, restenosis and atherosclerosis. To evaluate the proliferation of VSM and ASM cells in response to mitogens, we perform a [3H]thymidine incorporation assay. The proliferation protocol takes approximately 48 h and includes stimulating cells synchronized in G0/G1 phase of the cell cycle with agonists, labeling cells with [3H]thymidine and examining levels of [3H]thymidine incorporation by scintillation counting. Although using radiolabeled [3H]thymidine incorporation is a limitation, the greatest benefit of the assay is providing reliable and statistically significant data. PMID:17406550

  1. RSV-specific airway resident memory CD8+ T cells and differential disease severity after experimental human infection

    PubMed Central

    Jozwik, Agnieszka; Habibi, Maximillian S.; Paras, Allan; Zhu, Jie; Guvenel, Aleks; Dhariwal, Jaideep; Almond, Mark; Wong, Ernie H. C.; Sykes, Annemarie; Maybeno, Matthew; Del Rosario, Jerico; Trujillo-Torralbo, Maria-Belen; Mallia, Patrick; Sidney, John; Peters, Bjoern; Kon, Onn Min; Sette, Alessandro; Johnston, Sebastian L.; Openshaw, Peter J.; Chiu, Christopher

    2015-01-01

    In animal models, resident memory CD8+ T (Trm) cells assist in respiratory virus elimination but their importance in man has not been determined. Here, using experimental human respiratory syncytial virus (RSV) infection, we investigate systemic and local virus-specific CD8+ T-cell responses in adult volunteers. Having defined the immunodominance hierarchy, we analyse phenotype and function longitudinally in blood and by serial bronchoscopy. Despite rapid clinical recovery, we note surprisingly extensive lower airway inflammation with persistent viral antigen and cellular infiltrates. Pulmonary virus-specific CD8+ T cells display a CD69+CD103+ Trm phenotype and accumulate to strikingly high frequencies into convalescence without continued proliferation. While these have a more highly differentiated phenotype, they express fewer cytotoxicity markers than in blood. Nevertheless, their abundance before infection correlates with reduced symptoms and viral load, implying that CD8+ Trm cells in the human lung can confer protection against severe respiratory viral disease when humoral immunity is overcome. PMID:26687547

  2. Lentiviral Vector Gene Transfer to Porcine Airways

    PubMed Central

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF). PMID:23187455

  3. Airway inflammation and IgE production induced by dust mite allergen-specific memory/effector Th2 cell line can be effectively attenuated by IL-35.

    PubMed

    Huang, Chiung-Hui; Loo, Evelyn Xiu-Ling; Kuo, I-Chun; Soh, Gim Hooi; Goh, Denise Li-Meng; Lee, Bee Wah; Chua, Kaw Yan

    2011-07-01

    CD4(+) memory/effector T cells play a central role in orchestrating the rapid and robust immune responses upon re-encounter with specific Ags. However, the immunologic mechanism(s) underlying these responses are still not fully understood. To investigate this, we generated an allergen (major house dust mite allergen, Blo t 5)-specific murine Th2 cell line that secreted IL-4, IL-5, IL-10, and IL-13, but not IL-9 or TNF-α, upon activation by the cognate Ag. These cells also exhibited CD44(high)CD62L(-) and CD127(+) (IL-7Rα(+)) phenotypes, which are characteristics of memory/effector T cells. Experiments involving adoptive transfer of this Th2 cell line in mice, followed by three intranasal challenges with Blo t 5, induced a dexamethasone-sensitive eosinophilic airway inflammation. This was accompanied by elevation of Th2 cytokines and CC- and CXC-motif chemokines, as well as recruitment of lymphocytes and polymorphic mononuclear cells into the lungs. Moreover, Blo t 5-specific IgE was detected 4 d after the last intranasal challenge, whereas elevation of Blo t 5-specific IgG1 was found at week two. Finally, pulmonary delivery of the pVAX-IL-35 DNA construct effectively downregulated Blo t 5-specific allergic airway inflammation, and i.m. injection of pVAX-IL-35 led to long-lasting suppression of circulating Blo t 5-specific and total IgE. This model provides a robust research tool to elucidate the immunopathogenic role of memory/effector Th2 cells in allergic airway inflammation. Our results suggested that IL-35 could be a potential therapeutic target for allergic asthma through its attenuating effects on allergen-specific CD4(+) memory/effector Th2 cell-mediated airway inflammation.

  4. Tachykinin receptors mediating airway marcomolecular secretion

    SciTech Connect

    Gentry, S.E. )

    1991-01-01

    Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with {sup 3}H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers.

  5. Increased airway glucose increases airway bacterial load in hyperglycaemia.

    PubMed

    Gill, Simren K; Hui, Kailyn; Farne, Hugo; Garnett, James P; Baines, Deborah L; Moore, Luke S P; Holmes, Alison H; Filloux, Alain; Tregoning, John S

    2016-01-01

    Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection. We hypothesize that increased airway glucose caused by hyperglycaemia leads to increased bacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisation is significantly more likely when blood glucose is high. We engineered mutants in genes affecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa, strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented with glucose as the sole carbon source. The effect of glucose on growth in vivo was tested using streptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose. Bacterial burden in hyperglycaemic animals was greater than control animals when infected with wild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reduced both airway glucose and bacterial load. These data support airway glucose as a critical determinant of increased bacterial load during diabetes. PMID:27273266

  6. Increased airway glucose increases airway bacterial load in hyperglycaemia

    PubMed Central

    Gill, Simren K.; Hui, Kailyn; Farne, Hugo; Garnett, James P.; Baines, Deborah L.; Moore, Luke S.P.; Holmes, Alison H.; Filloux, Alain; Tregoning, John S.

    2016-01-01

    Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection. We hypothesize that increased airway glucose caused by hyperglycaemia leads to increased bacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisation is significantly more likely when blood glucose is high. We engineered mutants in genes affecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa, strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented with glucose as the sole carbon source. The effect of glucose on growth in vivo was tested using streptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose. Bacterial burden in hyperglycaemic animals was greater than control animals when infected with wild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reduced both airway glucose and bacterial load. These data support airway glucose as a critical determinant of increased bacterial load during diabetes. PMID:27273266

  7. Phenotypic modification of human airway epithelial cells in air-liquid interface culture induced by exposure to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

    PubMed

    Carson, Johnny L; Brighton, Luisa E; Jaspers, Ilona

    2015-04-01

    The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.

  8. Effects of Cell Type and Culture Media on Interleukin-6 Secretion in Response to Environmental Particles

    PubMed Central

    Veranth, John M.; Cutler, N. Shane; Kaser, Erin G.; Reilly, Christopher A.; Yost, Garold S.

    2008-01-01

    Cultured lung cells provide an alternative to animal exposures for comparing the effects of different types of air pollution particles. Studies of particulate matter in vitro have reported proinflammatory cytokine signaling in response to many types of environmental particles, but there have been few studies comparing identical treatments in multiple cell types or identical cells with alternative cell culture protocols. We compared soil-derived, diesel, coal fly ash, titanium dioxide, and kaolin particles along with soluble vanadium and lipopolysaccharide, applied to airway-derived cells grown in submerged culture. Cell types included A549, BEAS-2B, RAW 264.7, and primary macrophages. The cell culture models (specific combinations of cell types and culture conditions) were reproducibly different in the cytokine signaling responses to the suite of treatments. Further, Interleukin-6 (IL-6) response to the treatments changed when the same cells, BEAS-2B, were grown in KGM versus LHC-9 media or in media containing bovine serum. The effect of changing media composition was reversible over multiple changes of media type. Other variables tested included culture well size and degree of confluence. The observation that sensitivity of a cell type to environmental agonists can be manipulated by modifying culture conditions suggests a novel approach for studying biochemical mechanisms of particle toxicity. PMID:18178371

  9. TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

    SciTech Connect

    Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.; Casola, Antonella

    2011-09-30

    Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases. Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.

  10. Variable beta-glucans production by different states of Eurotium amstelodami explains differences in inflammatory responses in airway cells.

    PubMed

    Bellanger, Anne-Pauline; Millon, Laurence; Rognon, Bénédicte; Roussel, Sandrine; Botterel, Françoise; Bretagne, Stéphane; Reboux, Gabriel

    2011-09-01

    Eurotium amstelodami, a mold frequently identified in housing and farm air samples, is a suspected cause of respiratory diseases such as allergic alveolitis, atopic asthma, and organic dust toxic syndrome. This fungus is present in the air in three different states (ascospores, conidia, and hyphae). The aim of this study was to test in vitro the differential inflammatory response of airway cells exposed to 1,3 betaglucanase-treated protein extract (BGPE), from E. amstelodami ascospores, conidia, and hyphae. Confluent cells from the A549 cell line were inoculated with calibrated BGPE issued from the three fungal forms. The levels of eight cytokines and chemokines involved in inflammatory responses were measured after 8 h of exposure. Beta-d-glucan (BDG) was quantified in total fungal extract as well as in the BGPE from the three fungal states. Hyphal BGPE were the only ones to induce a marked inflammatory response and they contain higher quantities of BDG. The present study adds to the growing body of evidence that beta-glucan from fungal hyphae play a crucial role in respiratory diseases. PMID:21851418

  11. Method for 3D Airway Topology Extraction

    PubMed Central

    Grothausmann, Roman; Kellner, Manuela; Heidrich, Marko; Lorbeer, Raoul-Amadeus; Ripken, Tammo; Meyer, Heiko; Kuehnel, Mark P.; Ochs, Matthias; Rosenhahn, Bodo

    2015-01-01

    In lungs the number of conducting airway generations as well as bifurcation patterns varies across species and shows specific characteristics relating to illnesses or gene variations. A method to characterize the topology of the mouse airway tree using scanning laser optical tomography (SLOT) tomograms is presented in this paper. It is used to test discrimination between two types of mice based on detected differences in their conducting airway pattern. Based on segmentations of the airways in these tomograms, the main spanning tree of the volume skeleton is computed. The resulting graph structure is used to distinguish between wild type and surfactant protein (SP-D) deficient knock-out mice. PMID:25767561

  12. Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    PubMed Central

    Corry, Jacqueline; Johnson, Sara M.; Cornwell, Jessica

    2015-01-01

    ABSTRACT All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCE Worldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein

  13. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

    PubMed Central

    van ‘t Wout, Emily F. A.; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E.; Clarke, Hanna J.; Tommassen, Jan; Marciniak, Stefan J.; Hiemstra, Pieter S.

    2015-01-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  14. S-Nitrosoglutathione Reductase Inhibition Regulates Allergen-Induced Lung Inflammation and Airway Hyperreactivity

    PubMed Central

    Bassett, David J. P.; Bradley, Matthews O.; Jaffar, Zeina

    2013-01-01

    Allergic asthma is characterized by Th2 type inflammation, leading to airway hyperresponsivenes, mucus hypersecretion and tissue remodeling. S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of intracellular levels of S-nitrosothiols. GSNOR activity has been shown to be elevated in human asthmatic lungs, resulting in diminished S-nitrosothiols and thus contributing to increased airway hyperreactivity. Using a mouse model of allergic airway inflammation, we report that intranasal administration of a new selective inhibitor of GSNOR, SPL-334, caused a marked reduction in airway hyperreactivity, allergen-specific T cells and eosinophil accumulation, and mucus production in the lungs in response to allergen inhalation. Moreover, SPL-334 treatment resulted in a significant decrease in the production of the Th2 cytokines IL-5 and IL-13 and the level of the chemokine CCL11 (eotaxin-1) in the airways. Collectively, these observations reveal that GSNOR inhibitors are effective not only in reducing airway hyperresponsiveness but also in limiting lung inflammatory responses mediated by CD4+ Th2 cells. These findings suggest that the inhibition of GSNOR may provide a novel therapeutic approach for the treatment of allergic airway inflammation. PMID:23936192

  15. Effect of natural allergen exposure during the grass pollen season on airways inflammatory cells and asthma symptoms.

    PubMed Central

    Djukanović, R; Feather, I; Gratziou, C; Walls, A; Peroni, D; Bradding, P; Judd, M; Howarth, P H; Holgate, S T

    1996-01-01

    BACKGROUND: Bronchial challenge with allergen causes a specific form of airways inflammation consisting of an influx of neutrophils, eosinophils, and T cells. Because the relevance of the challenge model to clinical asthma is uncertain, the cellular changes that occur in the lungs of asthmatic subjects during natural seasonal allergen exposure were investigated. METHODS: Seventeen grass pollen sensitive asthmatic subjects with previously reported seasonal exacerbations of asthma kept records of symptoms and underwent fibreoptic bronchoscopy with bronchoalveolar lavage (BAL) and endobronchial biopsy before and during the peak of the grass pollen season. The BAL cells were analysed for differential cell counts and by flow cytometry for T cell subsets and surface activation markers. The biopsy samples were processed into glycol methacrylate resin and immunohistochemical analysis was performed for mast cells, activated eosinophils, T cells and interleukin 4 (IL-4), a cytokine with a pivotal role in allergen-induced inflammation. RESULTS: In the pollen season there was an increase in T lymphocyte activation in the BAL fluid as identified by increased expression of interleukin 2 receptor (IL-2R). In the submucosa these changes were paralleled by an increase in CD4+ T cells. By contrast, the numbers of metachromatic cells in BAL fluid staining with toluidine blue were reduced, possibly because of degranulation following allergen stimulation. In keeping with mast cell activation, the number of mucosal mast cells staining for secreted IL-4 increased during the season. In comparison with the period shortly before the onset of the season, all but two subjects experienced an asthma exacerbation which followed the rise in pollen counts but, compared with the period preceding the first bronchoscopic examination, asthma symptoms were not increased during the pollen season. CONCLUSIONS: The data suggest that natural allergen exposure, leading to a clinical exacerbation of asthma

  16. Transfer of allergic airway responses with antigen-primed CD4+ but not CD8+ T cells in brown Norway rats.

    PubMed Central

    Watanabe, A; Mishima, H; Renzi, P M; Xu, L J; Hamid, Q; Martin, J G

    1995-01-01

    Activated CD4+ helper T cells have been demonstrated in asthmatic airways and postulated to play a central role in eliciting allergic inflammation; direct evidence of their involvement seems to be lacking. We hypothesized that CD4+ T cells have the potential to induce allergic responses to antigen challenge, and tested this hypothesis in a model of allergic bronchoconstriction, the Brown Norway rat, using the approach of adoptive transfer. Animals were actively sensitized to either ovalbumin (OVA) or BSA and were used as donors of T cells. W3/25(CD4)+ or OX8(CD8)+ T cells were isolated from the cervical lymph nodes of sensitized donors and transferred to naive BN rats. 2 d after adoptive transfer recipient rats were challenged by OVA inhalation, and changes in lung resistance (RL), bronchoalveolar lavage (BAL) cells, and serum levels of antigen-specific IgE were studied. After OVA challenge recipients of OVA-primed W3/25+ T cells exhibited sustained increases in RL throughout the entire 8-h observation period and had significant bronchoalveolar lavage eosinophilia, which was detected by immunocytochemistry using an antimajor basic protein mAb. Recipients of BSA-primed W3/25+ T cells or OVA-primed OX8+ T cells failed to respond to inhaled OVA. OVA-specific immunoglobulin E was undetectable by ELISA or skin testing in any of the recipient rats after adoptive transfer. In conclusion, antigen-induced airway bronchoconstriction and eosinophilia were successfully transferred by antigen-specific W3/25+ T cells in Brown Norway rats. These responses were dependent on antigen-primed W3/25+ T cells and appeared to be independent of IgE-mediated mast cell activation. This study provides clear evidence for T cell mediated immune mechanisms in allergic airway responses in this experimental model. Images PMID:7657805

  17. Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness

    PubMed Central

    Wagers, Scott S.; Norton, Ryan J.; Rinaldi, Lisa M.; Bates, Jason H.T.; Sobel, Burton E.; Irvin, Charles G.

    2004-01-01

    Mechanisms underlying airway hyperresponsiveness are not yet fully elucidated. One of the manifestations of airway inflammation is leakage of diverse plasma proteins into the airway lumen. They include fibrinogen and thrombin. Thrombin cleaves fibrinogen to form fibrin, a major component of thrombi. Fibrin inactivates surfactant. Surfactant on the airway surface maintains airway patency by lowering surface tension. In this study, immunohistochemically detected fibrin was seen along the luminal surface of distal airways in a patient who died of status asthmaticus and in mice with induced allergic airway inflammation. In addition, we observed altered airway fibrinolytic system protein balance consistent with promotion of fibrin deposition in mice with allergic airway inflammation. The airways of mice were exposed to aerosolized fibrinogen, thrombin, or to fibrinogen followed by thrombin. Only fibrinogen followed by thrombin resulted in airway hyperresponsiveness compared with controls. An aerosolized fibrinolytic agent, tissue-type plasminogen activator, significantly diminished airway hyperresponsiveness in mice with allergic airway inflammation. These results are consistent with the hypothesis that leakage of fibrinogen and thrombin and their accumulation on the airway surface can contribute to the pathogenesis of airway hyperresponsiveness. PMID:15232617

  18. Dual Pili Post-translational Modifications Synergize to Mediate Meningococcal Adherence to Platelet Activating Factor Receptor on Human Airway Cells

    PubMed Central

    Schulz, Benjamin L.; Power, Peter M.; Swords, W. Edward; Weiser, Jeffery N.; Apicella, Michael A.; Edwards, Jennifer L.; Jennings, Michael P.

    2013-01-01

    Pili of pathogenic Neisseria are major virulence factors associated with adhesion, twitching motility, auto-aggregation, and DNA transformation. Pili of N. meningitidis are subject to several different post-translational modifications. Among these pilin modifications, the presence of phosphorylcholine (ChoP) and a glycan on the pilin protein are phase-variable (subject to high frequency, reversible on/off switching of expression). In this study we report the location of two ChoP modifications on the C-terminus of N. meningitidis pilin. We show that the surface accessibility of ChoP on pili is affected by phase variable changes to the structure of the pilin-linked glycan. We identify for the first time that the platelet activating factor receptor (PAFr) is a key, early event receptor for meningococcal adherence to human bronchial epithelial cells and tissue, and that synergy between the pilin-linked glycan and ChoP post-translational modifications is required for pili to optimally engage PAFr to mediate adherence to human airway cells. PMID:23696740

  19. Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation.

    PubMed

    Chenery, Alistair L; Antignano, Frann; Burrows, Kyle; Scheer, Sebastian; Perona-Wright, Georgia; Zaph, Colby

    2015-12-07

    Immunological cross talk between mucosal tissues such as the intestine and the lung is poorly defined during homeostasis and disease. Here, we show that a low-dose infection with the intestinally restricted helminth parasite Trichuris muris results in the production of Th1 cell-dependent gamma interferon (IFN-γ) and myeloid cell-derived interleukin-10 (IL-10) in the lung without causing overt airway pathology. This cross-mucosal immune response in the lung inhibits the development of papain-induced allergic airway inflammation, an innate cell-mediated type 2 airway inflammatory disease. Thus, we identify convergent and nonredundant roles of adaptive and innate immunity in mediating cross-mucosal suppression of type 2 airway inflammation during low-dose helminth-induced intestinal inflammation. These results provide further insight in identifying novel intersecting immune pathways elicited by gut-to-lung mucosal cross talk.

  20. Low-Dose Intestinal Trichuris muris Infection Alters the Lung Immune Microenvironment and Can Suppress Allergic Airway Inflammation.

    PubMed

    Chenery, Alistair L; Antignano, Frann; Burrows, Kyle; Scheer, Sebastian; Perona-Wright, Georgia; Zaph, Colby

    2016-02-01

    Immunological cross talk between mucosal tissues such as the intestine and the lung is poorly defined during homeostasis and disease. Here, we show that a low-dose infection with the intestinally restricted helminth parasite Trichuris muris results in the production of Th1 cell-dependent gamma interferon (IFN-γ) and myeloid cell-derived interleukin-10 (IL-10) in the lung without causing overt airway pathology. This cross-mucosal immune response in the lung inhibits the development of papain-induced allergic airway inflammation, an innate cell-mediated type 2 airway inflammatory disease. Thus, we identify convergent and nonredundant roles of adaptive and innate immunity in mediating cross-mucosal suppression of type 2 airway inflammation during low-dose helminth-induced intestinal inflammation. These results provide further insight in identifying novel intersecting immune pathways elicited by gut-to-lung mucosal cross talk. PMID:26644379

  1. Co-administration of vaccination with DNA encoding T cell epitope on the Der p and BCG inhibited airway remodeling in a murine model of chronic asthma.

    PubMed

    Kim, Chi Hong; Ahn, Joong Hyun; Kim, Seung Joon; Lee, Sook-Young; Kim, Young Kyoon; Kim, Kwan Hyoung; Moon, Hwa Sik; Song, Jeong Sup; Park, Sung Hak; Kwon, Soon Seog

    2006-01-01

    Therapeutic modalities of airway remodeling in asthma have proved to be unsuccessful regarding reversing the previously established chronic airway changes. Recently, the potential of plasmid DNA to inhibit the Th2 immune response has been demonstrated in animal models of asthma. Bacillus Calmette-Guerin (BCG) immunization also induced immunomodulation, which appeared to be reliant on the properties of the interferon-gamma that was produced. Mice were immunized with house dust mite extract (HDM). At the 3 week point, we injected BCG subcutaneously into mice on three successive weeks. One week after the BCG injection, we immunized mice with the DNA plasmid encoding for murine T-cell epitope on Dermatophagoide pteronyssinus 2 thrice weekly. At 9 weeks after immunization, we measured airway responsiveness. Twenty four hours later, we performed bronchoalveolar lavage and histological examinations. Co-administration of DNA vaccination and BCG resulted in a partial suppression of the overproduction of goblet cells and the thickness of the peribronchial smooth muscle in ongoing allergic responses. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was reduced, and regarding the change of cytokines, the concentration of IL-4 was also decreased, but interferon-gamma was increased in the co-administration group, opposed to the asthma group. These results suggest that co-administration of vaccination with the DNA encoding T-cell epitope and BCG are effective regarding ongoing allergic response and might constitute an ideal method for combating allergic disease in the future.

  2. Cigarette smoke causes acute airway disease and exacerbates chronic obstructive lung disease in neonatal mice.

    PubMed

    Jia, Jie; Conlon, Thomas M; Ballester Lopez, Carolina; Seimetz, Michael; Bednorz, Mariola; Zhou-Suckow, Zhe; Weissmann, Norbert; Eickelberg, Oliver; Mall, Marcus A; Yildirim, Ali Önder

    2016-09-01

    Epidemiological evidence demonstrates a strong link between postnatal cigarette smoke (CS) exposure and increased respiratory morbidity in young children. However, how CS induces early onset airway disease in young children, and how it interacts with endogenous risk factors, remains poorly understood. We, therefore, exposed 10-day-old neonatal wild-type and β-epithelial sodium ion channel (β-ENaC)-transgenic mice with cystic fibrosis-like lung disease to CS for 4 days. Neonatal wild-type mice exposed to CS demonstrated increased numbers of macrophages and neutrophils in the bronchoalveolar lavage fluid (BALF), which was accompanied by increased levels of Mmp12 and Cxcl1 BALF from β-ENaC-transgenic mice contained greater numbers of macrophages, which did not increase following acute CS exposure; however, there was significant increase in airway neutrophilia compared with filtered air transgenic and CS-exposed wild-type controls. Interestingly, wild-type and β-ENaC-transgenic mice demonstrated epithelial airway and vascular remodeling following CS exposure. Morphometric analysis of lung sections revealed that CS exposure caused increased mucus accumulation in the airway lumen of neonatal β-ENaC-transgenic mice compared with wild-type controls, which was accompanied by an increase in the number of goblet cells and Muc5ac upregulation. We conclude that short-term CS exposure 1) induces acute airway disease with airway epithelial and vascular remodeling in neonatal wild-type mice; and 2) exacerbates airway inflammation, mucus hypersecretion, and mucus plugging in neonatal β-ENaC-transgenic mice with chronic lung disease. Our results in neonatal mice suggest that young children may be highly susceptible to develop airway disease in response to tobacco smoke exposure, and that adverse effects may be aggravated in children with underlying chronic lung diseases. PMID:27448665

  3. Adam8 Limits the Development of Allergic Airway Inflammation in Mice

    PubMed Central

    Knolle, Martin D.; Nakajima, Takahiro; Hergrueter, Anja; Gupta, Kushagra; Polverino, Francesca; Craig, Vanessa J.; Fyfe, Susanne E.; Zahid, Muhammad; Permaul, Perdita; Cernadas, Manuela; Montano, Gilbert; Tesfaigzi, Yohannes; Sholl, Lynette; Kobzik, Lester; Israel, Elliot; Owen, Caroline A.

    2013-01-01

    To determine whether a disintegrin and a metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyper-responsiveness (AHR), we compared AAI and AHR in wild type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with ovalbumin (OVA) or house dust mite protein extract (HDM). OVA- and HDM-treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some TH2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma. PMID:23670189

  4. ROCK insufficiency attenuates ozone-induced airway hyperresponsiveness in mice.

    PubMed

    Kasahara, David I; Mathews, Joel A; Park, Chan Y; Cho, Youngji; Hunt, Gabrielle; Wurmbrand, Allison P; Liao, James K; Shore, Stephanie A

    2015-10-01

    Ozone causes airway hyperresponsiveness (AHR) and pulmonary inflammation. Rho kinase (ROCK) is a key regulator of smooth muscle cell contraction and inflammatory cell migration. To determine the contribution of the two ROCK isoforms ROCK1 and ROCK2 to ozone-induced AHR, we exposed wild-type, ROCK1(+/-), and ROCK2(+/-) mice to air or ozone (2 ppm for 3 h) and evaluated mice 24 h later. ROCK1 or ROCK2 haploinsufficiency did not affect airway responsiveness in air-exposed mice but significantly reduced ozone-induced AHR, with a greater reduction in ROCK2(+/-) mice despite increased bronchoalveolar lavage (BAL) inflammatory cells in ROCK2(+/-) mice. Compared with wild-type mice, ozone-induced increases in BAL hyaluronan, a matrix protein implicated in ozone-induced AHR, were lower in ROCK1(+/-) but not ROCK2(+/-) mice. Ozone-induced increases in other inflammatory moieties reported to contribute to ozone-induced AHR (IL-17A, osteopontin, TNFα) were not different in wild-type vs. ROCK1(+/-) or ROCK2(+/-) mice. We also observed a dose-dependent reduction in ozone-induced AHR after treatment with the ROCK1/ROCK2 inhibitor fasudil, even though fasudil was administered after induction of inflammation. Ozone increased pulmonary expression of ROCK2 but not ROCK1 or RhoA. A ROCK2 inhibitor, SR3677, reduced contractile forces in primary human airway smooth muscle cells, confirming a role for ROCK2 in airway smooth muscle contraction. Our results demonstrate that ozone-induced AHR requires ROCK. Whereas ROCK1-dependent changes in hyaluronan may contribute to ROCK1's role in O3-induced AHR, the role of ROCK2 is downstream of inflammation, likely at the level of airway smooth muscle contraction.

  5. Efficient intratracheal delivery of airway epithelial cells in mice and pigs

    PubMed Central

    Gui, Liqiong; Qian, Hong; Rocco, Kevin A.; Grecu, Loreta

    2014-01-01

    Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases. PMID:25416381

  6. Efficient intratracheal delivery of airway epithelial cells in mice and pigs.

    PubMed

    Gui, Liqiong; Qian, Hong; Rocco, Kevin A; Grecu, Loreta; Niklason, Laura E

    2015-01-15

    Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.

  7. Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Hanrahan, John W

    2012-01-01

    Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl− and HCO3− secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3− net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl− and HCO3− net fluxes. Ieq and HCO3− fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3− is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3−. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3− secretion, suggesting that HCO3− transport under these conditions requires catalysed synthesis of carbonic acid. Cl− was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50–70% of Cl− and fluid transport was bumetanide-insensitive, suggesting basolateral Cl− loading by a sodium–potassium–chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO3− gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO3− secretion was increased by bilateral Cl− removal and therefore did not require apical Cl−/HCO3− exchange. The results suggest a model in which most HCO3− is recycled basolaterally by exchange with Cl−, and the resulting HCO3−-dependent Cl− transport provides an osmotic driving force for

  8. Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.

    PubMed

    Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Hanrahan, John W

    2012-11-01

    Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl(-) and HCO(3)(-) secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (I(sc)) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (I(eq)) calculated under open-circuit conditions. I(sc) was equivalent to the HCO(3)(-) net flux measured using the pH-stat technique, whereas I(eq) was the sum of the Cl(-) and HCO(3)(-) net fluxes. I(eq) and HCO(3)(-) fluxes were increased by bafilomycin and ZnCl(2), suggesting that some secreted HCO(3)(-) is neutralized by parallel electrogenic H(+) secretion. I(eq) and fluid secretion were dependent on the presence of both Na(+) and HCO(3)(-). The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I(eq) and HCO(3)(-) secretion, suggesting that HCO(3)(-) transport under these conditions requires catalysed synthesis of carbonic acid. Cl(-) was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl(-) and fluid transport was bumetanide-insensitive, suggesting basolateral Cl(-) loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO(3)(-) gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO(3)(-) secretion was increased by bilateral Cl(-) removal and therefore did not require apical Cl(-)/HCO(3)(-) exchange. The results suggest a model in which most HCO(3)(-) is recycled basolaterally by exchange with Cl(-), and the resulting HCO(3)(-)-dependent Cl(-) transport

  9. Long Term and Standard Incubations of WST-1 Reagent Reflect the Same Inhibitory Trend of Cell Viability in Rat Airway Smooth Muscle Cells

    PubMed Central

    Yin, Lei-Miao; Wei, Yin; Wang, Yu; Xu, Yu-Dong; Yang, Yong-Qing

    2013-01-01

    The WST-1 assay is an efficient test for cell viability measurement and the standard incubation time is 2h. In order to test if one-time addition of WST-1 reagent can reflect the relative cell viability trend of the testing agents at different time points, the effects of 2h standard incubation time and long term incubation time (2h+24h, 2h+48h) of WST-1 were compared in the rat airway smooth muscle cells (ASM cells) after adding of the testing protein MRP-14. Our study demonstrated that the effect of different dosages of the protein after 2h WST-1 incubation on ASM cells showed a tendency of inhibition and achieved the maximal inhibition effect at 72h. The relative cell viability trend of the 2h+24h group was the same to that of the 2h WST-1 incubation, which means that 24h prolonged incubation time of WST-1 reagent could still reflect the relative cell viability trend. In conclusion, the study suggested that the WST-1 is a proper candidate reagent for continuous monitation of cell viability. PMID:23289007

  10. Dual oxidase regulates neutrophil recruitment in allergic airways.

    PubMed

    Chang, Sandra; Linderholm, Angela; Franzi, Lisa; Kenyon, Nicholas; Grasberger, Helmut; Harper, Richart

    2013-12-01

    Enhanced reactive oxygen species production in allergic airways is well described and correlates with increased airway contractions, inflammatory cell infiltration, goblet cell metaplasia, and mucus hypersecretion. There is also an abundance of interleukin-4/interleukin-13 (IL-4/IL-13)- or interleukin-5-secreting cells that are thought to be central to the pathogenesis of allergic asthma. We postulated that the dual oxidases (DUOX1 and DUOX2), members of the nicotinamide adenine dinucleotide phosphate oxidase family that release hydrogen peroxide (H2O2) in the respiratory tract, are critical proteins in the pathogenesis of allergic airways. DUOX activity is regulated by cytokines, including IL-4 and IL-13, and DUOX-mediated H2O2 influences several important features of allergic asthma: mucin production, IL-8 secretion, and wound healing. The objective of this study was to establish the contribution of DUOXs to the development of allergic asthma in a murine model. To accomplish this goal, we utilized a DUOXA-deficient mouse model (Duoxa(-/-)) that lacked maturation factors for both DUOX1 and DUOX2. Our results are the first to demonstrate evidence of DUOX protein and DUOX functional activity in murine airway epithelium. We also demonstrate that DUOXA maturation factors are required for airway-specific H2O2 production and localization of DUOX to cilia of fully differentiated airway epithelial cells. We compared wild-type and Duoxa(-/-) mice in an ovalbumin exposure model to determine the role of DUOX in allergic asthma. In comparison to DUOX-intact mice, Duoxa(-/-) mice had reduced mucous cell metaplasia and lower levels of TH2 cytokine levels in bronchoalveolar fluid. In addition, increased airway resistance in response to methacholine was observed in Duoxa(+/+) mice, as expected, but was absent in Duoxa(-/-) mice. Surprisingly, Duoxa(-/-) mice had decreased influx of neutrophils in bronchoalveolar fluid and lung tissue sections associated with a lower level of the

  11. Effects of bronchopulmonary inflammation induced by pseudomonas aeruginosa on adenovirus-mediated gene transfer to airway epithelial cells in mice.

    PubMed

    van Heeckeren, A; Ferkol, T; Tosi, M

    1998-03-01

    Cystic fibrosis (CF) patients have endobronchial inflammation caused by infection with mucoid Pseudomonas aeruginosa. Since adenovirus vectors are being studied for gene therapy for CF, we sought to determine whether bronchopulmonary inflammation would influence adenovirus-mediated gene transfer. We hypothesized that bronchopulmonary inflammation in mice inoculated with mucoid P. aeruginosa would be associated with a decrease in the efficacy of adenovirus-mediated gene transfer. Agarose beads embedded with mucoid P. aeruginosa (6 x 10(4) c.f.u. per mouse) were inoculated transtracheally into C57BL/6 mice. Control mice received sterile agarose beads. Ten days after inoculation with agarose beads, recombinant adenovirus containing the beta-galactosidase reporter gene (Ad2/beta Gal-2) was administered intranasally (1.1 x 10(9) IU per mouse), and mice were killed 3 days later. The extent of inflammation, determined by neutrophil numbers in bronchoalveolar lavage fluid and by areal lung inflammation, was significantly greater in mice inoculated with P. aeruginosa-laden agarose beads and Ad2/beta Gal-2 compared with controls. Mice that had received Pseudomonas-laden agarose beads and Ad2/beta Gal-2 had significantly fewer (P < 0.015) airway epithelial cells transduced (4.1 +/- 0.9%) compared with mice that received sterile agarose beads and Ad2/beta Gal-2 (9.4 +/- 1.4%). These results indicate that the efficacy of adenovirus-mediated gene transfer is reduced in Pseudomonas-induced bronchopulmonary inflammation.

  12. Cost of Stem Cell-Based Tissue-Engineered Airway Transplants in the United Kingdom: Case Series.

    PubMed

    Culme-Seymour, Emily J; Mason, Katrina; Vallejo-Torres, Laura; Carvalho, Carla; Partington, Leanne; Crowley, Claire; Hamilton, Nick J; Toll, Ed C; Butler, Colin R; Elliott, Martin J; Birchall, Martin A; Lowdell, Mark W; Mason, Chris

    2016-02-01

    Stem cell-based tissue-engineered tracheas are at an early stage in their product development cycle. Tens of patients have been treated worldwide in predominantly compassionate use settings, demonstrating significant promise. This potentially life-saving treatment is complex, and the cost and its implications for such treatments are yet to be fully understood. The costs are compounded by varying strategies for graft preparation and transplant, resulting in differing clinical and laboratory costs from different research groups. In this study, we present a detailed breakdown of the clinical and manufacturing costs for three of the United Kingdom (UK) patients treated with such transplants. All three patients were treated under Compassionate Use legislation, within the UK National Health Service (NHS) hospital setting. The total costs for the three UK patients treated ranged from $174,420 to $740,500. All three patients were in a state of poor health at time of treatment and had a number of complexities in addition to the restricted airway. This is the first time a cost analysis has been made for a tissue-engineered organ and provides a benchmark for future studies, as well as comparative data for use in reimbursement considerations.

  13. Cell-Type-Specific Optogenetics in Monkeys.

    PubMed

    Namboodiri, Vijay Mohan K; Stuber, Garret D

    2016-09-01

    The recent advent of technologies enabling cell-type-specific recording and manipulation of neuronal activity spurred tremendous progress in neuroscience. However, they have been largely limited to mice, which lack the richness in behavior of primates. Stauffer et al. now present a generalizable method for achieving cell-type specificity in monkeys. PMID:27610562

  14. [Morphofunctional characterization of mast cells of the skin and the mucous membrane of airways in specific hyposensitization carried out on an experimental model].

    PubMed

    Agafonov, V E; Mitereva, D E; Makarova, O V; Agafonov, E P

    2006-01-01

    The study of the morphofunctional state of the population of guinea-pig mast cells in response to the action of specific allergen was carried out. The mast cells of different guinea pigs, intact and sensitized, as well as those hyposensitized by two methods, the classical method and the method combining extracorporeal and oro- pharyngeal hyposensitization, were compared. As shown in this study, after specific hyposensization the tendency towards the normalization of the morphofunctional state of the mast-cell population in the airway walls and the skin was noted. PMID:16758902

  15. Airway gene therapy.

    PubMed

    Davies, Jane C; Alton, Eric W F W

    2005-01-01

    Given both the accessibility and the genetic basis of several pulmonary diseases, the lungs and airways initially seemed ideal candidates for gene therapy. Several routes of access are available, many of which have been refined and optimized for nongene drug delivery. Two respiratory diseases, cystic fibrosis (CF) and alpha1-antitrypsin (alpha1-AT) deficiency, are relatively common; the single gene responsible has been identified and current treatment strategies are not curative. This type of inherited disease was the obvious initial target for gene therapy, but it has become clear that nongenetic and acquired diseases, including cancer, may also be amenable to this approach. The majority of preclinical and clinical studies in the airway have involved viral vectors, although for diseases such as CF, likely to require repeated application, non-viral delivery systems have clear advantages. However, with both approaches a range of barriers to gene expression have been identified that are limiting success in the airway and alveolar region. This chapter reviews these issues, strategies aimed at overcoming them, and progress into clinical trials with non-viral vectors in a variety of pulmonary diseases.

  16. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells

    PubMed Central

    Volckens, John; Daile