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 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.

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 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 Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

2001 ◽  
Vol 75 (16) ◽  
pp. 7703-7711 ◽  
Author(s):  
Robert W. Walters ◽  
Wouter van't Hof ◽  
Su Min P. Yi ◽  
Mary K. Schroth ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Transfer ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Wild Type ◽  
Airway Epithelia ◽  
Glycosyl Phosphatidylinositol ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACT In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl− transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis.

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 Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia

2007 ◽  
Vol 81 (18) ◽  
pp. 9891-9899 ◽  
Author(s):  
Paola D. Vermeer ◽  
Julia McHugh ◽  
Tatiana Rokhlina ◽  
Daniel W. Vermeer ◽  
Joseph Zabner ◽  
...  
Keyword(s):  
Growth Factor ◽  
Vaccinia Virus ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Electrical Conductance ◽  
Airway Epithelia ◽  
Virus Growth ◽  
Epithelial Integrity ◽  
Human Airway ◽  
Human Airway Epithelia

ABSTRACTVariola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors.

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