J. H. Widdicombe

Loss of CFTR chloride channels alters salt absorption by cystic fibrosis airway epithelia in vitro.

Cystic fibrosis (CF) is caused by the loss of functional CFTR Cl- channels. However, it is not understood how this defect disrupts salt and liquid movement in the airway or whether it alters the NaCl concentration in the thin liquid film covering the airway surface. Using a new approach, we found that CF airway surface liquid had a higher NaCl concentration than normal. Both CF and non-CF epithelia absorbed salt and liquid; however, expression of CFTR Cl- channels was required for maximal absorption. Thus, loss of CFTR elevates the salt concentration in CF airway surface liquid and in sweat by related mechanisms; the elevated NaCl concentration is due to a block in transcellular Cl- movement. The high NaCl may predispose CF airways to bacterial infections by inhibiting endogenous antibacterial defenses.

Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis

Transport of lung liquid is essential for both normal pulmonary physiologic processes and for resolution of pathologic processes. The large internal surface area of the lung is lined by alveolar epithelial type I (TI) and type II (TII) cells; TI cells line >95% of this surface, TII cells <5%. Fluid transport is regulated by ion transport, with water movement following passively. Current concepts are that TII cells are the main sites of ion transport in the lung. TI cells have been thought to provide only passive barrier, rather than active, functions. Because TI cells line most of the internal surface area of the lung, we hypothesized that TI cells could be important in the regulation of lung liquid homeostasis. We measured both Na+ and K+ (Rb+) transport in TI cells isolated from adult rat lungs and compared the results to those of concomitant experiments with isolated TII cells. TI cells take up Na+ in an amiloride-inhibitable fashion, suggesting the presence of Na+ channels; TI cell Na+ uptake, per microgram of protein, is ≈2.5 times that of TII cells. Rb+ uptake in TI cells was ≈3 times that in TII cells and was inhibited by 10−4 M ouabain, the latter observation suggesting that TI cells exhibit Na+-, K+-ATPase activity. By immunocytochemical methods, TI cells contain all three subunits (α, β, and γ) of the epithelial sodium channel ENaC and two subunits of Na+-, K+-ATPase. By Western blot analysis, TI cells contain ≈3 times the amount of αENaC/μg protein of TII cells. Taken together, these studies demonstrate that TI cells not only contain molecular machinery necessary for active ion transport, but also transport ions. These results modify some basic concepts about lung liquid transport, suggesting that TI cells may contribute significantly in maintaining alveolar fluid balance and in resolving airspace edema.

A comparative study of mammalian tracheal mucous glands

We have compared the distribution, numbers and volume of mucous glands in the tracheas of 11 mammalian species. No glands were present in the rabbit. The mouse only contained glands at the border between the trachea and larynx. In the rat, glands were commonest in the cephalad third of the trachea, but on average were much scarcer than in the larger species. Between species, there was a significant correlation between airway diameter and gland volume per unit surface area, suggesting that the rate of deposition of inhaled particles may increase in large airways. In the ventral portion of the trachea of about half the species, the glands were concentrated between the cartilaginous rings; in others they were evenly distributed over and between the rings. In most species in which the trachealis muscle attached to the internal surface of the cartilaginous rings, the glands were external to the muscle. In all species in which the muscle attached to the external surface of the cartilaginous rings, the glands were internal to the muscle. In the ox, goat, dog and sheep, the volume of glands per unit tracheal surface area was markedly greater in the ventral than the dorsal aspect of the trachea. The reverse was true of the pig. In humans, gland density in the 2 regions was similar. The frequency of gland openings was determined in the ox, goat, pig, dog and sheep tracheas, and ranged from 0.3 per mm2 in the dorsal portion of the sheep trachea to 1.5 per mm2 in the ventral portion of the ox trachea. For these 5 species, the volume of gland acini per unit luminal surface area varied linearly with the numbers of gland openings, with the volume of individual glands being constant at ∼ 120 nl.

Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions

SummaryThe understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (Isc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells.

Interleukin-13–Induced Mucous Metaplasia Increases Susceptibility of Human Airway Epithelium to Rhinovirus Infection

Infection of airway epithelium by rhinovirus is the most common cause of asthma exacerbations. Even in mild asthma, airway epithelium exhibits mucous metaplasia, which increases with increasing severity of the disease. We previously showed that squamous cultures of human airway epithelium manifest rhinoviral infection at levels many times higher than in well-differentiated cultures of a mucociliary phenotype. Here we tested the hypothesis that mucous metaplasia is also associated with increased levels of rhinoviral infection. Mucous metaplasia was induced with IL-13, which doubled the numbers of goblet cells. In both control (mucociliary) and IL-13- treated (mucous metaplastic) cultures, goblet cells were preferentially infected by rhinovirus. IL-13 doubled the numbers of infected cells by increasing the numbers of infected goblet cells. Furthermore, IL-13 increased both the maturity of goblet cells and the probability that a goblet cell would be infected. The infection of cells other than goblet cells was unaltered by IL-13. Treatment with IL-13 did not alter the levels of rhinovirus receptor ICAM-1, nor did the proliferative effects of IL-13 enhance infection, because rhinovirus did not colocalize with dividing cells. However, the induction of mucous metaplasia caused changes in the apical membrane structure, notably a marked decrease in overall ciliation, and an increase in the overall flatness of the apical surface. We conclude that mucous metaplasia in asthma increases the susceptibility of airway epithelium to infection by rhinovirus because of changes in the overall architecture of the apical surface.

Distribution of tracheal and laryngeal mucous glands in some rodents and the rabbit

We used scanning electron microscopy to count the number of mucous gland openings in the tracheae and lower portion of the larynges of the rat, guinea pig, hamster, mouse and rabbit. Cells of the airway surface epithelium were removed by protease digestion better to visualise the gland openings. The distribution of glands was further studied by conventional histology and by PAS/Alcian blue staining of whole mounts. In all rodent species, gland openings in the larynx occurred with a frequency of 1–2 per mm2. Mice had no gland openings in their tracheae, and hamsters, only a handful. Rat tracheae contained 126±42 gland openings (±S.D.; n = 6) at a frequency of ∼ 0.6 per mm2 at the top of the trachea and ∼ 0.15 per mm2 at the bottom. Guinea pig tracheae contained 153±90 gland openings (±S.D.; n = 5), with 54% being in the top 40% of the trachea. In both rat and guinea pig, tracheal glands were found in the ventral aspect between the cartilaginous rings, and were absent from the dorsal membranous portion. Gland openings in most species were simple circles of ∼ 50 μm diameter. However, glands in the rat trachea generally opened obliquely into shallow (∼ 20 μm deep) oval troughs (∼ 150×75 μm), which had their long axes oriented from head to tail. In the rabbit, there was no evidence of tracheal or laryngeal glands histologically. However, the tracheal and laryngeal surfaces contained numerous pits (∼ 30 μm diameter) distributed evenly over and between cartilages at a frequency of ∼ 4 per mm2. These may correspond to the ‘nests’ of goblet cells described by others.

Structural cues involved in endoplasmic reticulum degradation of G85E and G91R mutant cystic fibrosis transmembrane conductance regulator.

Abnormal folding of mutant cystic fibrosis transmembrane conductance regulator (CFTR) and subsequent degradation in the endoplasmic reticulum is the basis for most cases of cystic fibrosis. Structural differences between wild-type (WT) and mutant proteins, however, remain unknown. Here we examine the intracellular trafficking, degradation, and transmembrane topology of two mutant CFTR proteins, G85E and G91R, each of which contains an additional charged residue within the first putative transmembrane helix (TM1). In microinjected Xenopus laevis oocytes, these mutations markedly disrupted CFTR plasma membrane chloride channel activity. G85E and G91R mutants (but not a conservative mutant, G91A) failed to acquire complex N-linked carbohydrates, and were rapidly degraded before reaching the Golgi complex thus exhibiting a trafficking phenotype similar to DeltaF508 CFTR. Topologic analysis revealed that neither G85E nor G91R mutations disrupted CFTR NH2 terminus transmembrane topology. Instead, WT as well as mutant TM1 spanned the membrane in the predicted C-trans (type II) orientation, and residues 85E and 91R were localized within or adjacent to the plane of the lipid bilayer. To understand how these charged residues might provide structural cues for ER degradation, we examined the stability of WT, G85E, and G91R CFTR proteins truncated at codons 188, 393, 589, or 836 (after TM2, TM6, the first nucleotide binding domain, or the R domain, respectively). These results indicated that G85E and G91R mutations affected CFTR folding, not by gross disruption of transmembrane assembly, but rather through insertion of a charged residue within the plane of the bilayer, which in turn influenced higher order tertiary structure.

Cultures of bovine tracheal epithelium with differentiated ultrastructure and ion transport

SummaryTracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 µm). Short-circuit current (Isc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with an air interface, cells showed Isc equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 µm. Responses in Isc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air interface should prove useful in studies of airway epithelial physiology.

Primary cultures of the dog's tracheal epithelium: Fine structure, fluid, and electrolyte transport

SummaryCells from the dogs tracheal mucosa formed confluent epithelial sheets in culture. Typical tight junctions separated the apical from the basolateral portion of the cell membrane. The apical portion of the cell contained numerous short microvilli and a pronounced glycocalyx. The basolateral portion of the plasma membrane was unspecialized except for extensive gap junctions between cells. Freeze-fracture showed that the cultured cells lacked the basolateral square (orthogonal) arrays of the original tissue, particles previously implicated in ion transport. Formation of domes indicated the presence of active fluid absorption. Domes appeared between days 4 and 8 of culture and persisted for about 1 week. Cell sheets showed a transepithelial resistance of ≈ 400 Ω·cm2 and a short-circuit current (Isc) of ≈ 5 μA·cm−2. The effects of transport inhibitors indicated that both active Na absorption and active Cl secretion were present. Isc was increased by isoproterenol, prostaglandins E2 and F2α, vasoactive intestinal peptide, dibutyryl cyclic AMP, leukotrienes C4 and D4, and bradykinin. These changes were probably due to stimulation of active Cl secretion.

CAMP-dependent absorption of chloride across airway epithelium

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell85: 229-236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.