Airway Surface Liquid Height Research Articles

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.

Regulation of ion transporters and airway surface dynamics by lipoxin in cystic fibrosis bronchial epithelium

We have investigated the role of the endogenous anti-inflammatory lipoxin LXA4 in modulating Cl− secretion and Na+ absorption, airway surface liquid height (ASLh) and ciliary beat frequency (CBF) in CF and non-CF bronchial epithelia. CF (CuFi-1) and non-CF (NuLi-1) bronchial epithelial cell lines were grown under an air-surface liquid interface into well-differentiated epithelia. ASLh and CBF were measured using confocal fluorescence microscopy and ion transport using patch-clamp and short-circuit current techniques. LXA4 (1nM) treatment for 15 minutes, increased ASLh by 47.5±0.5 % and 103.0±3.0 % in NuLi and CuFi epithelia respectively (P<0.001, n=18). The stimulatory effect of LXA4 on ASLh was sustained over 24 hours in the CF epithelia and was inhibited by the following pre-treatments: bumetanide, amiloride, Boc-2 (LXA4 receptor antagonist), reactive blue (P2Y receptor antagonist) and extracellular hexokinase (ATP hydrolysis). LXA4 stimulated CBF, intracellular Ca2+ mobilization, Cl− secretion and inhibited Na+ absorption in the CF epithelia. These effects of lipoxin involving the FPR2 receptor, apical ATP release, purinoreceptor activation, inhibition of Na+ absorption and stimulation of Cl− secretion to enhance airway surface liquid dynamics open up a new therapeutic avenue to promote mucociliary clearance in cystic fibrosis airways.

Emerging therapies in cystic fibrosis

The life expectancy of people with cystic fibrosis (CF), a lethal inherited disease, has been greatly extended by advances in therapy. Currently, there are a number of potential drugs for treatment of CF lung disease in clinical trials. These therapies are targeted at all points in the pathogenesis of lung disease, from gene transfer to drugs that treat mucus, infection and inflammation in the airways. An exciting development is that of modulation of the abnormal protein that causes CF, the cystic fibrosis transmembrane conductance regulator (CFTR), where drugs are targeted at specific defects in CFTR transcription, processing or functioning. Inhaled therapies are being developed to augment airway surface liquid height, either by modulating the abnormal ion channel function in the airway epithelial cell or by rehydrating with osmotic agents. Anti-inflammatory therapy is also of great interest in CF and there are several candidate drugs in clinical trials. A number of antibacterial agents formulated for inhalation are at various stages of study or newly approved, which should improve options for chronic management of airway infection. Hopefully, many of these potential therapies will come to market and will further extend the life expectancy of people with CF.

AMPK Agonists Ameliorate Sodium and Fluid Transport and Inflammation in Cystic Fibrosis Airway Epithelial Cells

The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel and epithelial Na(+) channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I(sc)), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I(sc) in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.

Update in Cystic Fibrosis 2008

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and more than 1,500 distinct mutations with different functional consequences have been reported in the CFTR database (1). Multiple organs are involved in the disease process, but lung disease continues to account for the majority of morbidity and mortality in patients with CF. Knowledge about the pathophysiology of cystic fibrosis has greatly increased in recent years and CF has evolved into a model demonstrating how a better understanding of the underlying defect can lead to novel therapeutic approaches. However, complex interactions exist between CFTR and other regulatory proteins and all of the relevant components that cause disease manifestations both in the lung and in other organ systems are still incompletely understood. A large body of evidence supports the concept that airway surface liquid depletion is a key component in the development of CF lung disease. This is thought to be the consequence of an imbalance in defective chloride secretion and enhanced sodium absorption that has been consistently observed in studies of CF epithelium. The net fluid loss on the airway surface leads to the collapse of cilia and impaired mucociliary transport. However, regulation of mucociliary clearance is a dynamic process involving other epithelial channels that are thought to play an important role in stress-induced increases in airway surface liquid (ASL) height (2). These CFTR independent pathways are functional in CF and can be up-regulated by both external and internal stimuli, such as ATP, which may explain why mucociliary clearance is reduced but not absent in patients with CF (2). Improving airway surface hydration either with pharmacological agents or osmotic agents, such as hypertonic saline, has become an important therapeutic target in CF (3, 4).

What’s new in cystic fibrosis? From treating symptoms to correction of the basic defect

Chronic relentless lung infection and pancreatic insufficiency are the cardinal features of cystic fibrosis (CF), a life-shortening autosomal recessive disease. Mutations in the 'cystic fibrosis transmembrane conductance regulator' (CFTR) are currently classified into five groups according to their repercussion on CFTR protein synthesis and its chloride channel function. Stop codon mutations (class I) result in a truncated nonfunctional CFTR, class II mutations consist of aberrantly folded CFTR protein that is degraded by the cell quality control system, while class III mutations lead to defective regulation of the CFTR protein and, consequently, the absence of CFTR function. These three classes usually lead to a classic CF phenotype with pancreatic insufficiency. CFTR mutations that lead to defective chloride conductance are grouped together in class IV. Class V mutations interfere with normal transcription, thereby reducing the amount of otherwise normal CFTR. These latter two classes are mostly associated with a milder expression of the disease. In the absence of CFTR function, unrestrained Na+ absorption and the failure of active Cl- secretion lead to a decreased airway surface liquid (ASL) volume and subsequent failure of normal mucociliary clearance. This review highlights recent therapeutic strategies that either target the underlying defect or the early steps in CF pathophysiology. To date, gene therapy has failed to demonstrate a clinical benefit after repeated administration. Mutation-specific chloride channel correction pharmacotherapy is currently being developed, an example of which is PTC124, a new chemical compound that selectively induces read-through of premature stop codons. However, clinical efficacy for most of the compounds still has to be proven in large clinical trials. The positive effect of nebulised hypertonic saline on mucociliary clearance is based on the restoration of ASL height. Recent advances in the current treatment of lung infection and inflammation are highlighted in this review. Lung transplantation should be considered in terminally ill patients, but the timing of the transplantation is crucial: transplanting too early shortens survival, while transplanting too late results in patients dying on the waiting list.

A2B Adenosine Receptors Regulate the Mucus Clearance Component of the Lung's Innate Defense System

Adenosine (ADO) signaling is altered in both asthma and chronic obstructive pulmonary disease, and the A(2B) adenosine receptor (A(2B)-R) may drive pulmonary inflammation. Accordingly, it has been proposed that specific inhibition of the A(2B)-R could treat inflammatory lung diseases. However, stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by ADO may be crucial in permitting the superficial epithelium to maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Our goal was to determine which ADO receptor (ADO-R) underlies ASL volume regulation in bronchial epithelia. We used PCR techniques to determine ADO-R expression in bronchial epithelia and used nasal potential difference measurements, Ussing chambers studies, and XZ-confocal microscopy to look at Cl- secretion and ASL volume regulation. The A(2B)-R was the most highly expressed ADO-R in donor specimens of human bronchial epithelia, and inhibition of ADO-R in vivo prevented activation of CFTR. A(2B)-R was the only ADO-R detected in cultured human bronchial epithelial cells and inhibition of this receptor with specific A(2B)-R antagonists resulted in ASL height collapse and a failure to effect ASL height homeostasis. Removal of ADO with ADO deaminase and replacement with 5'N-ethylcarboxamide adenosine resulted in dose-dependent changes in ASL height, and suggested that the cell surface (ADO) may be in excess of 1 microM, which is sufficient to activate A(2B)-R. A(2B)-R are required for ASL volume homeostasis in human airways, and therapies directed at inhibiting A(2B)-R may lead to a cystic fibrosis-like phenotype with depleted ASL volume and mucus stasis.

Pharmacological Properties ofN-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine Methanesulfonate (552-02), a Novel Epithelial Sodium Channel Blocker with Potential Clinical Efficacy for Cystic Fibrosis Lung Disease

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N'-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k(off)), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k(off) value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 +/- hypertonic saline for CF lung disease.

36 Feasibility of airway surface liquid (ASL) height measurement in human nasal and bronchial biopsies

36 Feasibility of airway surface liquid (ASL) height measurement in human nasal and bronchial biopsies

Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia

In the lungs, the first line of defence against bacterial infection is the thin layer of airway surface liquid (ASL) lining the airway surface. The superficial airway epithelium exhibits complex regulatory pathways that blend ion transport to adjust ASL volume to maintain proper mucociliary clearance (MCC). We hypothesized that stresses generated by airflow and transmural pressures during breathing govern ASL volume by regulating the rate of epithelial ATP release. Luminal ATP, via interactions with apical membrane P2-purinoceptors, regulates the balance of active ion secretion versus absorption to maintain ASL volume at optimal levels for MCC. In this study we tested the hypothesis that cyclic compressive stress (CCS), mimicking normal tidal breathing, regulates ASL volume in airway epithelia. Polarized tracheobronchial epithelial cultures from normal and cystic fibrosis (CF) subjects responded to a range of CCS by increasing the rate of ATP release. In normal airway epithelia, the CCS-induced increase in ASL ATP concentration was sufficient to induce purinoceptor-mediated increases in ASL height and MCC, via inhibition of epithelial Na(+)-channel-mediated Na(+) absorption and stimulation of Cl(-) secretion through CFTR and the Ca(2+)-activated chloride channels. In contrast, static, non-oscillatory stress did not stimulate ATP release, ion transport or MCC, emphasizing the importance of rhythmic mechanical stress for airway defence. In CF airway cultures, which exhibit basal ASL depletion, CCS was partially effective, producing less ASL volume secretion than in normal cultures, but a level sufficient to restore MCC. The present data suggest that CCS may (1) regulate ASL volume in the normal lung and (2) improve clearance in the lungs of CF patients, potentially explaining the beneficial role of exercise in lung defence.

Ion and liquid transport across the bronchiolar epithelium

The proper homeostasis of the airway surface liquid (ASL) depends on transepithelial ion and fluid transport and is critically important for lung defence, and more specifically for mucociliary transport. In cystic fibrosis (CF), abnormal ion and fluid transport lead to depleted ASL volume resulting in mucus plugs and recurrent lung infections. Like bronchi, human bronchioles exhibit amiloride-sensitive Na(+) absorption and cyclic-AMP and Ca(2+)-activated Cl(-) secretion. However, cyclic-AMP-stimulated Cl(-) and fluid secretion appears to be quantitatively more important in bronchioles than in bronchi. In CF bronchioles, like in CF bronchi, the ASL height is reduced because of an abnormally persistent Na(+) absorption, combined with a lacking CFTR-dependent Cl(-) secretion. The precocity and severity of the bronchiolar disease in CF could be attributed in part to the more important role of CFTR-dependent Cl(-) secretion and fluid secretion, and the lack of compensatory ATP-driven Cl(-) secretion and fluid secretion, in bronchioles compared to bronchi.

Ion and Fluid Transport Properties of Small Airways in Cystic Fibrosis

Small airways constitute a major site of pathology in cystic fibrosis (CF) and provide most of the surface area of the conducting airways of the lung. Little is known, however, about the impact of CF on ion and fluid transport in small (bronchiolar) airways. To describe the ion and fluid transport properties of CF bronchiolar epithelium. Primary cultures of human bronchial and bronchiolar (non-CF and CF) epithelial cells were obtained. The bioelectric properties were studied in Ussing chambers and the airway surface liquid (ASL) height was measured with confocal microscopy. Primary cultures of DeltaF508 CF bronchiolar epithelial cells displayed higher transepithelial resistance than non-CF cultures, whereas baseline short circuit current and amiloride-inhibitable short circuit current were similar in both preparations. The ASL height was significantly smaller in CF compared with non-CF preparations. In the presence of amiloride, addition of forskolin increased short circuit current in non-CF but not in CF bronchiolar cultures, and the ATP-induced increase in short circuit current was lower in CF than in non-CF cultures. Non-CF bronchiolar preparations displayed larger short circuit current and fluid secretion in responses to forskolin than non-CF bronchial preparations, suggesting that CFTR-dependent Cl(-) transport may play a more important role in the regulation of fluid transport in small airways than in large airways. In CF small airways, defective Cl(-) secretion combined with unregulated (persistent) Na(+) absorption results in ASLdepletion.

Soluble Mediators, Not Cilia, Determine Airway Surface Liquid Volume in Normal and Cystic Fibrosis Superficial Airway Epithelia

A key aspect of the lung's innate defense system is the ability of the superficial epithelium to regulate airway surface liquid (ASL) volume to maintain a 7-μm periciliary liquid layer (PCL), which is required for cilia to beat and produce mucus flow. The mechanisms whereby airway epithelia regulate ASL height to ≥7 μm are poorly understood. Using bumetanide as an inhibitor of Cl− secretion, and nystatin as an activator of Na+ absorption, we found that a coordinated “blending” of both Cl− secretion and Na+ absorption must occur to effect ASL volume homeostasis. We then investigated how ASL volume status is regulated by the underlying epithelia. Cilia were not critical to this process as (a) ASL volume was normal in cultures from patients with primary ciliary dyskinesia with immotile cilia, and (b) in normal cultures that had not yet undergone ciliogenesis. However, we found that maneuvers that mimic deposition of excess ASL onto the proximal airways, which occurs during mucociliary clearance and after glandular secretion, acutely stimulated Na+ absorption, suggesting that volume regulation was sensitive to changes in concentrations of soluble mediators in the ASL rather than alterations in ciliary beating. To investigate this hypothesis further, we added potential “soluble mediators” to the ASL. ASL volume regulation was sensitive to a channel-activating protein (CAP; trypsin) and a CAP inhibitor (aprotinin), which regulated Na+ absorption via changes in epithelial Na+ channel (ENaC) activity in both normal and cystic fibrosis cultures. ATP was also found to acutely regulate ASL volume by inducing secretion in normal and cystic fibrosis (CF) cultures, while its metabolite adenosine (ADO) evoked secretion in normal cultures but stimulated absorption in CF cultures. Interestingly, the amount of ASL/Cl− secretion elicited by ATP/ADO was influenced by the level of CAP-induced Na+ absorption, suggesting that there are important interactions between the soluble regulators which finely tune ASL volume.

350. Pre-Clinical Assays for Cystic Fibrosis Gene Therapy

Cystic fibrosis (CF) has long been a model disease to study lung gene therapy and we and others have carried out a number of phase I/II clinical trials using viral or non-viral vectors carrying the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In addition to safety, measurements of potential difference (PD) across the airway epithelium, which is altered in CF due to the altered ion transport, have often been included as a primary endpoint. However, although important, the information obtained from PD measurements in pre-clinical and clinical gene therapy research has to be interpreted carefully, because changes in PD after transfection with the CFTR gene do not provide information about the number of cells transfected. In addition, it is unclear, how a change in PD after gene therapy correlates with more clinically relevant endpoints. The UK CF Gene Therapy Consortium has, therefore, devoted a significant amount of time and resources to refine further existing assays and set-up and validate a panel of new pre-clinical and clinical assays to screen non-viral gene transfer agents.The CF knockout mouse is the only available CF animal model and although the CF mouse does not develop traditional CF lung disease, it has long been demonstrated that the CF ion transport abnormalities are recapitulated in the murine nasal epithelium. Importantly, the cell composition of the murine nasal epithelium is also similar to the human lower airways. We have chosen the FABp-CF knockout mouse (Zhou 1994) as the standard model in our core facility. Fatty acid binding promoter (FABp)-mediated expression of CFTR in the gut, but not the airways, prevents the intestinal disease of the CF knockout mouse, thus easing work in a core facility setting. When choosing assays we aimed to cover several aspects of defective CFTR level and function [1. CFTR protein (vector- and epithelial cell-specific detection of CFTR mRNA and protein). 2. Ion transport (PD and short circuit current measurements). 3. Airway surface liquid (ASL) (ASL height), 4. Host bacterial interaction (Bacterial adherence to airway epithelial cells). During assay validation we compared FABP-CF mice and wild-type littermates and used the results to carry out power calculations that informed cohort size when assessing efficiency of gene transfer agents. Importantly, we learnt that n numbers have to be large to detect small, but potentially clinically relevant, changes in these assays and that care has to be taken not to underpower assays for assumed treatment effects.

Neutrophil elastase activates near-silent epithelial Na+channels and increases airway epithelial Na+transport

Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.

Measurements of airway surface liquid height and mucus transport by fluorescence microscopy, and of ion composition by X-ray microanalysis

The respiratory tract is lined by a thin layer of fluid, the airway surface liquid (ASL), which plays a critical role in lung defense. The paper describes methods to determine the height of the ASL and corresponding mucus transport rates using fluorescent probes, and methods to determine the ionic composition of the ASL by X-ray microanalysis.

Regulation of Airway Surface Liquid Volume and Mucus Transport by Active Ion Transport

Mucus clearance is an important component of the lung's innate defense against disease, and the ability of the airways to clear mucus is strongly dependent on the volume of liquid on airway surfaces. Whether airway surface liquid (ASL) volume is maintained by passive surface forces or by active ion transport is controversial yet crucial to the understanding of how this system operates in both health and disease. In support of active ion transport being the major determinant of ASL volume, we have demonstrated that normal airway epithelia sense and autoregulate ASL height (volume) by adjusting the rates of Na+ absorption and Cl- secretion to maintain mucus transport.