scholarly journals Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology

2002 ◽  
Vol 76 (11) ◽  
pp. 5654-5666 ◽  
Author(s):  
Liqun Zhang ◽  
Mark E. Peeples ◽  
Richard C. Boucher ◽  
Peter L. Collins ◽  
Raymond J. Pickles
Keyword(s):  
Gene Transfer ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Transfer Vectors ◽  
Syncytial Virus

ABSTRACT Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.

scholarly journals Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells.

1996 ◽  
Vol 44 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
T Sugiyama ◽  
M Yamamoto-Hino ◽  
K Wasano ◽  
K Mikoshiba ◽  
M Hasegawa
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Specific Cell ◽  
Airway Epithelial ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Inositol 1,4,5 Trisphosphate

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphate receptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonal antibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epithelium from trachea to distal intrapulmonary airways (bronchioles) showed positive immunoreactivity for all types of IP3R. However, cell type as well as subcellular site immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in the apical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3R Type 2 was exclusively localized to the entire cytoplasm of ciliated cells from the trachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclear cytoplasm not only of ciliated cells at all airway levels but also in Clara cells of the bronchiolar epithelium. Double fluorescent staining using combinations of KM1083 and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibody confirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Clara cells. These results indicate that the expression of three types of IP3Rs in different cell types and subcellular sites may reflect diverse physiological functions of IP3Rs within airway epithelial cells. The double staining studies suggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specific cell marker for ciliated cells of the airway epithelium.

Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells

2004 ◽  
Vol 286 (4) ◽  
pp. L650-L657 ◽  
Author(s):  
Yingjian You ◽  
Tao Huang ◽  
Edward J. Richer ◽  
Jens-Erik Harboe Schmidt ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basal Body ◽  
Ciliated Cell ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Basal Bodies ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Wild Type

Factors required for commitment of an undifferentiated airway epithelial cell to a ciliated cell are unknown. Cell ultrastructure analysis indicates ciliated cell commitment activates a multistage program involving synthesis of cilia precursor proteins and assembly of macromolecular complexes. Foxj1 is an f-box transcription factor expressed in ciliated cells and shown to be required for cilia formation by gene deletion in a mouse model. To identify a specific role for foxj1 in directing the ciliated cell phenotype, we evaluated the capacity of foxj1 to induce ciliogenesis and direct cilia assembly. In a primary culture model of wild-type mouse airway epithelial cells, foxj1 expression preceded the appearance of cilia and in cultured foxj1 null cells cilia did not develop. Delivery of foxj1 to polarized epithelial cell lines and primary cultured alveolar epithelial cells failed to promote ciliogenesis. Similarly, delivery of foxj1 to wild-type airway epithelial cells did not enhance the total number of ciliated cells. In contrast, delivery of foxj1 to null cells resulted in the appearance of cilia. Analysis revealed that, in the absence of foxj1, null cells contained cilia precursor basal bodies, indicating prior commitment to ciliogenesis. However, the basal bodies were disorganized within the apical compartment and failed to dock with the apical membrane. Reconstitution of foxj1 in null cells restored normal basal body organization, resulting in axoneme growth. Thus foxj1 functions in late-stage ciliogenesis to regulate programs promoting basal body docking and axoneme formation in cells previously committed to the ciliated cell phenotype.

scholarly journals Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

1998 ◽  
Vol 72 (11) ◽  
pp. 8904-8912 ◽  
Author(s):  
S. Teramoto ◽  
J. S. Bartlett ◽  
D. McCarty ◽  
X. Xiao ◽  
R. J. Samulski ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Transfer ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Adeno Associated Virus ◽  
Differentiated Cells ◽  
Factors Influencing

ABSTRACT Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZgene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.

RSV infection of human airway epithelial cells causes production of the beta-chemokine RANTES

1997 ◽  
Vol 272 (3) ◽  
pp. L512-L520 ◽  
Author(s):  
S. Becker ◽  
W. Reed ◽  
F. W. Henderson ◽  
T. L. Noah
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Cultures ◽  
Airway Epithelial Cells ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Epithelial Cell Cultures ◽  
Rsv Infection ◽  
Syncytial Virus

Infection of airway epithelial cells with respiratory syncytial virus (RSV) results in the production of a restricted number of cytokines, which may modulate the inflammatory response to infection. To get a better understanding of epithelial cell-mediated inflammatory processes in RSV disease, the aim of the present study was to identify the production of mononuclear cell/eosinophil/mast cell inflammatory chemokines [monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein-1beta, and RANTES] during productive RSV infection in airway epithelial cells. Normal human primary bronchial epithelial cell cultures, nasal epithelial cell explants, and the BEAS-2B airway epithelial cell line were inoculated with RSV, and chemokine induction was assessed during the phase of logarithmic increase in infectious virus production. Only RANTES was found to increase in epithelial cell cultures in an infection-dependent manner. Furthermore, RANTES was released only by RSV-producing cells. To determine whether RANTES was induced by RSV infection in vivo, RANTES was measured in nasal lavage fluids (NLF) from children with RSV-positive and RSV-negative upper respiratory infection and children when they were well. RANTES was increased significantly during RSV infection (128 +/- 38 pg/ml NFL) compared with non-RSV infection (42 +/- 12 pg/ml NFL) and with asymptomatic baseline (13 +/- 4 ng/ml NFL) in the same children. Because RANTES is an effective eosinophil and memory T cell chemoattractant and activator and because eosinophil-dominated inflammation is a hallmark of asthmatic airways, RANTES may play a role in the pathogenesis of RSV-induced exacerbations of airway reactivity and wheezing.

scholarly journals Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Stefano Castellani ◽  
Sante Di Gioia ◽  
Teresa Trotta ◽  
Angela Bruna Maffione ◽  
Massimo Conese
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Propidium Iodide ◽  
Airway Epithelium ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Fold Increase ◽  
Airway Epithelial Cells ◽  
Transduction Efficiency ◽  
Airway Epithelial

Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25ߙkDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in6.5±2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness.

scholarly journals Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media

2020 ◽  
Author(s):  
Lindsay Broadbent ◽  
Sheerien Manzoor ◽  
Maria Zarcone ◽  
Judit Barabas ◽  
Mike Shields ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Nasal Epithelial Cell ◽  
Human Airway ◽  
Syncytial Virus ◽  
Human Airway Epithelial Cells

AbstractThe culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFNλ1 secreted from PneumaCult compared to Promocell cultures. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells impacts experimental results.

scholarly journals Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadzeya Marozkina ◽  
Laura Smith ◽  
Yi Zhao ◽  
Joe Zein ◽  
James F. Chmiel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Nitrogen Oxides ◽  
Airway Epithelium ◽  
Airflow Obstruction ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
Human Airway Epithelial Cells

AbstractEndothelial hemoglobin (Hb)α regulates endothelial nitric oxide synthase (eNOS) biochemistry. We hypothesized that Hb could also be expressed and biochemically active in the ciliated human airway epithelium. Primary human airway epithelial cells, cultured at air–liquid interface (ALI), were obtained by clinical airway brushings or from explanted lungs. Human airway Hb mRNA data were from publically available databases; or from RT-PCR. Hb proteins were identified by immunoprecipitation, immunoblot, immunohistochemistry, immunofluorescence and liquid chromatography- mass spectrometry. Viral vectors were used to alter Hbβ expression. Heme and nitrogen oxides were measured colorimetrically. Hb mRNA was expressed in human ciliated epithelial cells. Heme proteins (Hbα, β, and δ) were detected in ALI cultures by several methods. Higher levels of airway epithelial Hbβ gene expression were associated with lower FEV1 in asthma. Both Hbβ knockdown and overexpression affected cell morphology. Hbβ and eNOS were apically colocalized. Binding heme with CO decreased extracellular accumulation of nitrogen oxides. Human airway epithelial cells express Hb. Higher levels of Hbβ gene expression were associated with airflow obstruction. Hbβ and eNOS were colocalized in ciliated cells, and heme affected oxidation of the NOS product. Epithelial Hb expression may be relevant to human airways diseases.

Transgenic overexpression of β2-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

2000 ◽  
Vol 279 (2) ◽  
pp. L379-L389 ◽  
Author(s):  
Dennis W. McGraw ◽  
Susan L. Forbes ◽  
Judith C. W. Mak ◽  
David P. Witte ◽  
Patricia E. Carrigan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Adrenergic Receptors ◽  
Airway Epithelial Cells ◽  
Airway Responsiveness ◽  
Ozone Exposure ◽  
Clara Cell ◽  
Whole Body ◽  
Airway Epithelial

Airway epithelial cells express β2-adrenergic receptors (β2-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of β2-ARs to airway epithelium of transgenic (CCSP-β2-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that β2-AR density in CCSP-β2-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED200) was greater for CCSP-β2-AR than for NTG mice (345 ± 34 vs. 157 ± 14 mg/ml; P < 0.01). CCSP-β2-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline ( P < 0.05) but remained unchanged in the CCSP-β2-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED200for ozone-exposed CCSP-β2-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell β2-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of β-agonists results from activation of receptors on both epithelial and smooth muscle cells.

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