KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response

2001 ◽  
Vol 6 (2) ◽  
pp. 81-89 ◽  
Author(s):  
LAWRENCE S. PRINCE ◽  
PHILIP H. KARP ◽  
THOMAS O. MONINGER ◽  
MICHAEL J. WELSH
Keyword(s):  
Keratinocyte Growth Factor ◽  
Primary Cultures ◽  
Airway Epithelia ◽  
Altered Expression ◽  
Human Airway ◽  
Gene Transcripts ◽  
Induced Genes ◽  
Well Differentiated ◽  
Cf Transmembrane Conductance Regulator ◽  
Human Airway Epithelia

Keratinocyte growth factor (KGF) regulates several functions in adult and developing lung epithelia; it causes proliferation, stimulates secretion of fluid and electrolytes, enhances repair, and may minimize injury. To gain insight into the molecular processes influenced by KGF, we applied KGF to primary cultures of well-differentiated human airway epithelia and used microarray hybridization to assess the abundance of gene transcripts. Of 7,069 genes tested, KGF changed expression levels of 910. Earlier studies showed that KGF causes epithelial proliferation, and as expected, treatment altered expression of numerous genes involved in cell proliferation. We found that KGF stimulated transepithelial Cl−transport, but the number of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) transcripts fell. Although transcripts for ClC-1 and ClC-7 Cl−channels increased, KGF failed to augment transepithelial Cl−transport in CF epithelia, suggesting that KGF-stimulated Cl−transport in differentiated airway epithelia depends on the CFTR Cl−channel. Interestingly, KGF decreased transcripts for many interferon (IFN)-induced genes. IFN causes trafficking of Stat dimers to the nucleus, where they activate transcription of IFN-induced genes. We found that KGF prevented the IFN-stimulated trafficking of Stat1 from the cytosol to the nucleus, suggesting a molecular mechanism for KGF-mediated suppression of the IFN-signaling pathway. These results suggest that in addition to stimulating proliferation and repair of damaged airway epithelia, KGF stimulates Cl−transport and may dampen the response of epithelial cells to inflammatory mediators.

scholarly journals Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2

2004 ◽  
Vol 287 (2) ◽  
pp. L428-L437 ◽  
Author(s):  
Hong Peng Jia ◽  
Joel N. Kline ◽  
Andrea Penisten ◽  
Michael A. Apicella ◽  
Theresa L. Gioannini ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Airway Epithelial Cells ◽  
Response To Treatment ◽  
Airway Epithelial ◽  
Airway Epithelia ◽  
Human Airway ◽  
Tnf Α ◽  
Pulmonary Macrophages ◽  
Well Differentiated ◽  
Human Airway Epithelia

The expression of inducible antimicrobial peptides, such as human β-defensin-2 (HBD-2) by epithelia, comprises a component of innate pulmonary defenses. We hypothesized that HBD-2 induction in airway epithelia is linked to pattern recognition receptors such as the Toll-like receptors (TLRs). We found that primary cultures of well-differentiated human airway epithelia express the mRNA for TLR-4, but little or no MD-2 mRNA, and display little HBD-2 expression in response to treatment with purified endotoxin ± LPS binding protein (LBP) and soluble CD14. Expression of endogenous MD-2 by transduction of airway epithelial cells with an adenoviral vector encoding MD-2 or extracellular addition of recombinant MD-2 both increased the responses of airway epithelia to endotoxin + LBP and sCD14 by >100-fold, as measured by NF-κB-luciferase activity and HBD-2 mRNA expression. MD-2 mRNA could be induced in airway epithelia by exposure of these cells to specific bacterial or host products (e.g., killed Haemophilus influenzae, the P6 outer membrane protein from H. influenzae, or TNF-α + IFN-γ). These findings suggest that MD-2, either coexpressed with TLR-4 or secreted when produced in excess of TLR-4 from neighboring cells, is required for airway epithelia to respond sensitively to endotoxin. The regulation of MD-2 expression in airway epithelia and pulmonary macrophages may serve as a means to modify endotoxin responsiveness in the airway.

Basolateral chloride current in human airway epithelia

2007 ◽  
Vol 293 (4) ◽  
pp. L991-L999 ◽  
Author(s):  
Omar A. Itani ◽  
Fred S. Lamb ◽  
James E. Melvin ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Signaling Pathways ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl− absorption. However, the pathways for basolateral Cl− exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl− conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl− conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl− current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl− > Br− >> I−. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca2+ and inducing cell swelling had no effect. The basolateral Cl− current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl− currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl− conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl− conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl− movement.

scholarly journals Measles Virus Preferentially Transduces the Basolateral Surface of Well-Differentiated Human Airway Epithelia

2002 ◽  
Vol 76 (5) ◽  
pp. 2403-2409 ◽  
Author(s):  
Patrick L. Sinn ◽  
Greg Williams ◽  
Sompong Vongpunsawad ◽  
Roberto Cattaneo ◽  
Paul B. McCray
Keyword(s):  
Vaccine Strain ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Egfp Expression ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

ABSTRACT Measles virus (MV) is typically spread by aerosol droplets and enters via the respiratory tract. The progression of MV infection has been widely studied; yet, the pathway for virus entry in polarized human airway epithelia has not been investigated. Herein we report the use of a replication-competent Edmonston vaccine strain of MV expressing enhanced green fluorescent protein (MV-eGFP) to infect primary cultures of well-differentiated human airway epithelia. Previous studies with polarized Caco-2 cells (intestine-derived human epithelia) and MDCK cells (kidney-derived canine epithelia) demonstrated that MV primarily infected and exited the apical surface. In striking contrast, our results indicate that MV preferentially transduces human airway cells from the basolateral surface; however, virus release remains in an apical direction. When MV-eGFP was applied apically or basolaterally to primary cultures of airway epithelia, discrete foci of eGFP expression appeared and grew; however, the cell layer integrity was maintained for the duration of the study (7 days). Interestingly, utilizing immunohistochemistry and confocal microscopy, we observed widespread expression of the receptor for the vaccine strain of MV (CD46) at greatest abundance on the apical surface of the differentiated human airway epithelia as well as in human tracheal tissue sections. These data suggest that the progression of MV infection through the respiratory epithelium may involve pathways other than direct binding and entry through the apical surface of airway epithelia.

scholarly journals ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia

2005 ◽  
Vol 79 (23) ◽  
pp. 14614-14621 ◽  
Author(s):  
Hong Peng Jia ◽  
Dwight C. Look ◽  
Lei Shi ◽  
Melissa Hickey ◽  
Lecia Pewe ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Receptor Expression ◽  
Pseudotyped Virus ◽  
Apical Surface ◽  
Sars Coronavirus ◽  
Airway Epithelia ◽  
S Protein ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

ABSTRACT Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

An In Vitro Model of Differentiated Human Airway Epithelia: Methods for Establishing Primary Cultures

2003 ◽  
pp. 115-137 ◽  
Author(s):  
Philip H. Karp ◽  
Thomas O. Moninger ◽  
S. Pary Weber ◽  
Tamara S. Nesselhauf ◽  
Janice L. Launspach ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Primary Cultures ◽  
Airway Epithelia ◽  
Human Airway ◽  
Vitro Model ◽  
Human Airway Epithelia

scholarly journals Azithromycin Treatment Alters Gene Expression in Inflammatory, Lipid Metabolism, and Cell Cycle Pathways in Well-Differentiated Human Airway Epithelia

PLoS ONE ◽  
2009 ◽  
Vol 4 (6) ◽  
pp. e5806 ◽  
Author(s):  
Carla Maria P. Ribeiro ◽  
Harry Hurd ◽  
Yichao Wu ◽  
Mary E. B. Martino ◽  
Lisa Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Lipid Metabolism ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

scholarly journals The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia

2011 ◽  
Vol 300 (1) ◽  
pp. L25-L31 ◽  
Author(s):  
Alejandro A. Pezzulo ◽  
Timothy D. Starner ◽  
Todd E. Scheetz ◽  
Geri L. Traver ◽  
Ann E. Tilley ◽  
...  
Keyword(s):  
Expression Profile ◽  
Primary Cultures ◽  
Liquid Interface ◽  
Transcriptional Profile ◽  
Surface Epithelium ◽  
Airway Epithelia ◽  
Human Airway ◽  
Air Liquid Interface ◽  
Human Airway Epithelia

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

scholarly journals Lentivirus Vectors Pseudotyped with Filoviral Envelope Glycoproteins Transduce Airway Epithelia from the Apical Surface Independently of Folate Receptor Alpha

2003 ◽  
Vol 77 (10) ◽  
pp. 5902-5910 ◽  
Author(s):  
Patrick L. Sinn ◽  
Melissa A. Hickey ◽  
Patrick D. Staber ◽  
Douglas E. Dylla ◽  
Scott A. Jeffers ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Folate Receptor ◽  
Primary Cultures ◽  
Apical Surface ◽  
Airway Epithelia ◽  
Gene Transfer Efficiency ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FRα), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FRα on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FRα-blocking antibodies, we demonstrated FRα-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FRα was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

scholarly journals Human Coronavirus 229E Infects Polarized Airway Epithelia from the Apical Surface

2000 ◽  
Vol 74 (19) ◽  
pp. 9234-9239 ◽  
Author(s):  
Guoshun Wang ◽  
Camille Deering ◽  
Michael Macke ◽  
Jianqiang Shao ◽  
Royce Burns ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Primary Cultures ◽  
Apical Surface ◽  
Human Coronavirus ◽  
Airway Epithelia ◽  
Human Airway ◽  
Apical Side ◽  
Tract Infections ◽  
Human Coronavirus 229E ◽  
Human Airway Epithelia

ABSTRACT Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.

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