Control of pH of airway surface liquid of the ferret trachea in vitro

We measured the pH of airway surface liquid (ASL) secreted by the ferret trachea in vitro by using a catheter-tipped pH electrode implanted in a collecting cannula close to the airway epithelium. Mucus secretion was promoted by methacholine (0.02 mmol/l) in the organ bath. The pH of the ASL was 6.85 +/- 0.03 (SE) compared with a bath value of 7.39 +/- 0.01, when the bath was bubbled with 5.65% CO2. Changing the bath CO2 from 0 to 20.93% CO2 altered the bath pH from 8.06 to 6.96, but the ASL pH only varied from 6.92 to 6.85. This homeostasis of ASL pH was not the result of the buffering powers of the ASL, because ex situ buffer curves for secreted ASL were similar to those for Krebs-Henseleit solution. Changing the luminal CO2 content by blowing gases through the trachea changed ASL pH by values similar to that ex situ. However, when external organ bath CO2 was changed, the luminal CO2 changes were proportionately far smaller. Measurement of rates of diffusion of CO2 across the tracheal wall indicated that this was not a limiting factor in the results. Similarly, measurement of metabolic rate CO2 production in the tracheal lumen indicated that this did not significantly affect the results. We conclude that the pH of ASL is significantly on the acid side of the pH or interstitial fluid and plasma and that it is maintained relatively constant despite large changes in external pH.     

Ciliated epithelial cell lifespan in the mouse trachea and lung

The steady-state turnover of epithelial cells in the lung and trachea is highly relevant to investigators who are studying endogenous stem cells, manipulating gene expression in vivo, or using viral vectors for gene therapy. However, the average lifetime of different airway epithelial cell types has not previously been assessed using currently available genetic techniques. Here, we use Cre/loxP genetic technology to indelibly label a random fraction of ciliated cells throughout the airways of a cohort of mice and follow them in vivo for up to 18 mo. We demonstrate that ciliated airway epithelial cells are a terminally differentiated population. Moreover, their average half-life of 6 mo in the trachea and 17 mo in the lung is much longer than previously available estimates, with significant numbers of labeled cells still present after 18 mo.     

X-ray microanalysis of airway surface liquid in the mouse

The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isoflurane-anesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (approximately 60-80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(-/-) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea.     

Effects of airway surface liquid pH on host defense in cystic fibrosis

Cystic fibrosis is a lethal genetic disorder characterized by viscous mucus and bacterial colonization of the airways. Airway surface liquid represents a first line of pulmonary defense. Studies in humans and animal models of cystic fibrosis indicate that the pH of airway surface liquid is reduced in the absence of cystic fibrosis transmembrane conductance regulator function. Many aspects of the innate host defense system of the airways are pH sensitive, including antimicrobial peptide/protein activity, the rheological properties of secreted mucins, mucociliary clearance, and the activity of proteases. This review will focus on how changes in airway surface liquid pH may contribute to the host defense defect in cystic fibrosis soon after birth. Understanding how changes in pH impact mucosal immunity may lead to new therapies that can modify the airway surface liquid environment, improve airway defenses, and alter the disease course.This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.     

Erythromycin increases bactericidal activity of surface liquid in human airway epithelial cells

Macrolide antibiotics have clinical benefits in patients with diffuse panbronchiolitis and in patients with cystic fibrosis. Although many mechanisms have been proposed, the precise mechanisms are still uncertain. We examined the effects of erythromycin on bactericidal activity of airway surface liquid secreted by cultured human tracheal epithelial cells. Airway surface liquid was collected by washing the surface of human tracheal epithelial cells with a sodium solution (40 meq/l). Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were incubated with airway surface liquid, and the number of surviving bacteria was examined. The number of bacteria in airway surface liquid from the cells cultured in medium alone was significantly lower than that in the sodium solution. Furthermore, the number of bacteria in airway surface liquid from the cells treated with erythromycin was significantly lower than that in airway surface liquid from the cells treated with solvent alone. The production of mRNA and protein of human beta-defensin-1 and human beta-defensin-2 was significantly increased by erythromycin. Bactericidal activity of airway surface liquid was observed at low concentrations (40 meq/l) of sodium but not at higher concentrations (> or =80 meq/l). Airway surface liquid did not contain significant amounts of antibiotics supplemented in the culture medium. Erythromycin at the levels in airway surface liquid and in culture medium did not inhibit bacterial growth. These results suggest that erythromycin may increase bactericidal activity of airway surface liquid in human airway epithelial cells through human beta-defensins production and reduce susceptibility of the airway to bacterial infection.     

Airway surface liquid composition in mice

Airway surface liquid (ASL) lines the conducting airways of the respiratory tract. We collected small samples of this liquid from the lower tracheae of anesthetized C57BL/6 mice and determined its ionic composition (in mM: 87.2 Na(+), 4.7 K(+), and 57.0 Cl(-)). Intravenous methacholine produced significant increases in the concentrations of Na(+), K(+), and Cl(-) within ASL. A limited analysis of liquid from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice revealed no significant differences compared with littermate controls; however, Pseudomonas aeruginosa infection led to an increase in the salt concentration of ASL in cftr(+/+) mice. Morphometric measurements of tracheal submucosal gland volume revealed significant differences between inbred mouse strains, corresponding to ease of ASL collection. We conclude that although submucosal glands may be responsible for the production of some ASL, the ionic composition of this liquid is actively regulated by the underlying epithelial cells.     

Basolateral Cl channels in primary airway epithelial cultures

Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.     

Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells

BACKGROUND: Following injury to the airway epithelium, rapid regeneration of a functional epithelium is necessary in order to restore the epithelial barrier integrity. In the perspective of airway gene/cell therapy, we analyzed the capacity of human airway epithelial cells cultured as three-dimensional (3-D) spheroid structures to be efficiently transduced on long term by a pseudotyped lentiviral vector. The capacity of the 3-D spheroid structures to repopulate a denuded tracheal basement membrane and regenerate a well-differentiated airway epithelium was also analyzed. METHODS: An HIV-1-derived VSV-G pseudotyped lentiviral vector encoding the enhanced green fluorescent protein (eGFP) was used. Airway epithelial cells were isolated from mature human fetal tracheas and airway xenografts, cultured as 3-D spheroid structures, and either transduced at multiplicity of infection (MOI) 10 and 100 or assayed in an ex vivo and in vivo model to evaluate their regeneration capacity. RESULTS: An in vivo repopulation assay in SCID-hu mice with transduced isolated fetal airway epithelial cells shows that lentiviral transduction does not alter the airway reconstitution. Transduction of the 3-D spheroid structures shows that 12% of cells were eGFP-positive for up to 80 days. In ex vivo and in vivo assays (NUDE-hu mice), the 3-D spheroid structures are able to repopulate denuded basement membrane and reconstitute a well-differentiated human airway surface epithelium. CONCLUSIONS: The efficient and long-term lentiviral transduction of 3-D spheroid structures together with their capacity to regenerate a well-differentiated mucociliary epithelium demonstrate the potential relevance of these 3-D structures in human airway gene/cell therapy.     

Role of cell surface glycosylation in mediating repair of human airway epithelial cell monolayers

Our laboratory recently demonstrated the pattern of cell surface glycosylation of nonsecretory central airway epithelium (Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, and White SR. Histochem J 31: 145-151, 1999), but the role of glycosylation in airway epithelial cell migration and repair is unknown. We examined the functional role of cell surface carbohydrates in wound repair after mechanical injury of 1HAEo(-) human airway epithelial and primary bronchial epithelial monolayers. Wound repair stimulated by epidermal growth factor was substantially attenuated by 10(-7) M tunicamycin (TM), an N-glycosylation inhibitor, but not by the inhibitors deoxymannojirimycin or castanospermine. Wound repair of 1HAEo(-) and primary airway epithelial cells was blocked completely by removal of cell surface terminal fucose residues by alpha-fucosidase. Cell adhesion to collagen matrix was prevented by TM but was only reduced ~20% from control values with prior alpha-fucosidase treatment. Cell migration in Blind Well chambers stimulated by epidermal growth factor was blocked by pretreatment with TM but alpha-fucosidase pretreatment produced no difference from control values. These data suggest that cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration and that N-glycosylation with terminal fucosylation plays a role in the complex process of repair by coordination of certain cell-cell functions.     

Quantitative characterization of domes in primary mouse mammary epithelial tumor cell cultures

Summary Dome populations from primary cultures of mouse mammary tumor cells were quantitatively studied in regard to size, distribution, density and the area occupied by light-diffusion photography and image analysis. The effects of fetal bovine serum, insulin and hydrocortisone were analyzed. Quantitative characterization documented dome diameter (mode diameter 0.26 to 0.52 mm), dome area occupied (average 23%, maximum 38.7%), and density (average 78 domes per cm², maximum 117 domes per cm²) for standard culture conditions. Insulin and hydrocortisone had a primary effect on dome density whereas 15% fetal bovine serum had a minimal effect. However, insulin and hydrocortisone had a synergistic optimal effect on dome population. Time-lapse cinematography revealed that the dome population is not static, but a very dynamic one. Domes underwent irregular pulsations of expansion and contraction. Dome enlargement was either by a series of expansions and contractions, by lateral involvement of other cells, or by coalescence of two or more domes. Domes have been considered to be the in vitro counterpart of the in vivo acinus of the mouse mammary gland. However, quantitative dome population characterization has not been available. Dome analysis by light-diffraction photography and image analysis lends itself towards correlative studies of domes and their differentiative products. Supported in part by Public Health Service Contract NO1-CP 61013 within the Virus Cancer Program of the National Cancer Institute and by Public Health Service Training Grant CA05245 from the National Cancer Institute.     

Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells

Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an alpha2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an alpha2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the alpha2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The alpha2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, alpha2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the alpha2,3-linked SA receptor.     

Glycosylation and annexin II cell surface translocation mediate airway epithelial wound repair

Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells (1HAEo(-)) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of 1HAEo(-) cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immunodetection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair.     

Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways

The composition and depth of the airway surface liquid (ASL) are key parameters in airway physiology that are thought to be important in the pathophysiology of cystic fibrosis and other diseases of the airways. We reported novel fluorescent indicator and microscopy methods to measure [Na+], [Cl-], pH, and depth of the ASL in large airways (Jayaraman, S., Y. Song, L. Vetrivel, L. Shankar, and A.S. Verkman. 2001. J. Clin. Invest. 107:317-324.). Here we report a stripped-lung preparation to measure ASL composition and depth in small distal airways. Distal ASL was stained with ion- or pH-sensitive fluorescent indicators by infusion into mouse trachea of a perfluorocarbon suspension of the indicator. After stripping the pleura and limited microdissection of the lung parenchyma, airways were exposed for measurement of ASL [Na+], [Cl-], and pH by ratio imaging microscopy, and depth by confocal microscopy. The stripped-lung preparation was validated in stability and tissue viability studies. ASL [Na+] was 122 +/- 2 mM, [Cl-] was 123 +/- 4 mM and pH was 7.28 +/- 0.07, and not dependent on airway size (<100- to >250-mum diameter), ENaC inhibition by amiloride, or CFTR inhibition by the thiazolidinone CFTRinh-172. ASL depth was 8-35 mum depending on airway size, substantially less than that in mouse trachea of approximately 55 mum, and not altered significantly by amiloride. These results establish a novel lung preparation and fluorescence approach to study distal airway physiology and provide the first data on the composition and depth of distal ASL.     

Bidirectional virus secretion and nonciliated cell tropism following Andes virus infection of primary airway epithelial cell cultures

Hantavirus pulmonary syndrome (HPS) is an acute disease resulting from infection with any one of a number of New World hantaviruses. HPS has a mortality rate of 40% and, unlike many other severe respiratory diseases, often occurs in young, healthy adults. Infection is usually initiated after inhalation of rodent excreta containing virus particles, but human-to-human transmission has been documented. Postmortem tissue samples show high levels of viral antigen within the respiratory endothelium, but it is not clear how the virus can traverse the respiratory epithelium in order to initiate infection in the microvasculature. We have utilized Andes virus infection of primary, differentiated airway epithelial cells to investigate the ability of the virus to interact with and cross the respiratory epithelium. Andes virus infects the Clara and goblet cell populations but not the ciliated cells, and this infection pattern corresponds to the expression of beta(3) integrin, the viral receptor. The virus can infect via the apical or basolateral membrane, and progeny virus particles are secreted bidirectionally. There is no obvious cytopathology associated with infection, and beta(3) integrins do not appear to be critical for respiratory epithelial cell monolayer integrity. Our data suggest that hantavirus infection of the respiratory epithelium may play an important role in the early or prodrome phase of disease as well as serving as a source of virus involved in transmission.     

Airway epithelial cell inflammatory signalling in cystic fibrosis

Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians, estimated to affect one out of 2500-4000 new-borns. In patients with CF, lack of CF transmembrane conductance regulator (CFTR) Cl(-) channel function leads to progressive pulmonary damage and ultimately to death. Severe and persistent polymorphonuclear neutrophil-dominated endobronchial inflammation and chronic bacterial infection are characteristic hallmarks of CF lung disease. Whether CFTR dysfunction results directly in an increased predisposition to infection and whether inflammation arises independent of infection remains to be established. The loss of functional CFTR in airway epithelial cells promotes depletion and increased oxidation of the airway surface liquid. Activated neutrophils present in airways produce large amounts of proteases and reactive oxygen species (ROS). Together these changes are associated with diminished mucociliary clearance of bacteria, activation of epithelial cell signalling through multiple pathways, and subsequent hyperinflammatory responses in CF airways. The NF-kappaB pathway and Ca(2+) mobilization in airway epithelial cells are believed to be of key importance for control of lung inflammation through regulated production of mediators such as interleukin-8 that participate in recruitment and activation of neutrophils, modulation of apoptosis, and control of epithelial barrier integrity. In this review, the current understanding of the molecular mechanisms by which airway epithelial cells contribute to abnormal lung inflammation in CF, as well as the anti-inflammatory strategies that can be proposed are discussed.     

Measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia

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.