Chloride secretion by cultures of pig tracheal gland cells.

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Hormonal and purinergic stimulation of bicarbonate secretion in oviducts of rhesus monkey.

Because an increase in the HCO(3)(-) concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO(3)(-) across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO(3)(-). Purinergic agents potently stimulated short-circuit current (I(sc)) with an initial transient response declining within approximately 2 min to a sustained response. The potency sequence of ATP approximately UTP > ADP >> AMP suggested that the I(sc) response was mediated mainly by P2Y(2) receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline I(sc) or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO(3)(-)-CO(2) medium, and the pH of the unbuffered luminal medium was maintained at approximately 7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the I(sc) responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO(3)(-) secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO(3)(-) concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.

Differentiated structure and function of primary cultures of monkey oviductal epithelium.

We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Omega.cm(2) (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca(i). Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.

Secretion of lactoferrin and lysozyme by cultures of human airway epithelium.

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.

Resistance of differentiated human airway epithelium to infection by rhinovirus.

Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.

Effects of media on differentiation of cultured human tracheal epithelium.

The purpose of the this study was to find media that supported high levels of differentiation in primary cultures of human tracheal epithelium. We tested six previously described, partially defined media and three nondefined media. Cells were grown with an air interface on porous-bottomed inserts, and differentiation was assessed from electrophysiological properties, levels of total protein and deoxyribonucleic acid, and histology. In all media, cells polarized and developed tight junctions, as assessed from transepithelial electrical resistance and were better differentiated at 14 d after plating than at 7 d. The partially defined media described previously by Gray et al. (Am. J. Respir. Cell. Mol. Biol. 14:104-112; 1996) and Matsui et al. (J. Clin. Invest. 102:1125-1131; 1998) and an undefined medium containing Ultroser G serum substitute produced the most highly differentiated epithelial cells, as revealed by a high short-circuit current (I(sc)) and a ciliated, pseudostratified appearance. In other media, cells tended to be either squamous or stratified squamous, with I(sc) levels <25% of those obtained with the three optimal media. Though no key factor in the composition of the partially defined media could be identified, two of the four media with high concentrations of retinoic acid produced good differentiation. In contrast, the two media with the lowest [Ca] (0.11 mM) produced poorly differentiated cells, as did the two partially defined media with low or no retinoic acid concentration.

Effects of growth surface on differentiation of cultures of human tracheal epithelium.

Optical measurements from epithelial cells grown on clear solid surfaces (e.g., coverslips, petri dishes) are often compared with other measurements (e.g., short-circuit current; I(sc)) obtained from cells grown on opaque porous surfaces (inserts). However, the relative levels of differentiation of cells grown under the two conditions are usually unknown. To address this issue, we grew primary cultures of human tracheal epithelium on solid surfaces or on porous inserts and compared their total levels of protein and deoxyribonucleic acid, electrical properties in Ussing chambers, and ultrastructure. To measure ion transport across cells grown on solid supports, cells were grown on inserts placed on parafilm. Later, separation of insert from parafilm allowed the cells' I(sc) to be measured in Ussing chambers. Four different media were used. Cells grown in one medium showed very low levels of differentiation on all growth supports. In the other media, growth on inserts markedly enhanced differentiation as compared with solid supports. Baseline I(sc) of cells grown on either clear or opaque inserts was at least 30 times greater than that of cells grown on solid supports, though I(sc) with clear inserts averaged approximately 30% lower than that with opaque inserts. We conclude that though differentiation of cells may vary slightly depending on the insert used, cells on any type of insert are much better differentiated than cells grown on solid surfaces. Thus, it is both possible and desirable to make all functional measurements on cells grown on clear porous supports.

Regulation of the depth and composition of airway surface liquid.

The airways are lined with a film of liquid about 10 microm deep that is in two layers. Around the cilia is the watery periciliary sol. Over this is a mucous blanket that traps inhaled particles. The low viscosity of the periciliary sol allows the cilia to beat and propel the mucous blanket to the mouth. In large airways, mucus comes predominantly from the mucous glands but also from goblet cells in the surface epithelium. Water is added to the airway surface by gland secretion that is driven by active Cl secretion by serous cells. During inflammation elevation of the subepithelial hydrostatic pressure may also add significant volumes of water to the airway lumen. Water is removed by active Na transport across the surface epithelium. In airway diseases, the balance is shifted from water secretion to mucus secretion. In bronchitis and asthma this is due mainly to conversion of gland serous to mucous cells. In cystic fibrosis, gland serous cells cannot secrete water because they lack functioning CFTR in their apical membranes (CFTR is the cystic fibrosis transmembrane conductance regulator, a Cl channel that is abundant in serous cells). In all three diseases, the result is secretion of excessively concentrated gland secretions that are poorly moved by the cilia and accumulate. Altered salt and water transport by the surface epithelium may also contribute to the pathology of cystic fibrosis.

Distribution and numbers of mucous glands in the horse trachea.

Horses commonly suffer from respiratory diseases associated with excess secretions in the airway lumen, some of which are presumably derived from airway mucous glands. However, these structures have been little investigated in the horse. Accordingly, we describe here the number, distribution and size of equine tracheal mucous glands, and compare the data with similar information for other mammalian species. Two types of gland acini were present. In the thick connective tissue, up to 400 microm beneath the epithelium, gland acini were grouped in thin sheets that, in cross-section, averaged 20 microm thick and were up to 4.0 mm in length. However, it is probable that most sheets had maximal diameters much less than 4.0 mm. Between 400 to 900 microm below the epithelium, the connective tissue was much more diffuse, and glands were larger and more globular. Gland volume in the ventral portion was approximately 1.7 microl/cm2 of mucosal surface, and approximately 1.1 microl/cm2 in the dorsal portion. Glands were somewhat more abundant between, rather than over, the cartilaginous rings, but the difference between the 2 locations was not marked. Mucous gland openings were small (20 microm diameter) and very unevenly distributed, generally occurring about 100 microm apart in longitudinal rows of about 5. Average frequency of openings in the ventral portions of 3 tracheas was approximately 1.0/mm2 of mucosal surface. The volume of individual glands was therefore approximately 17 nl. Although the frequency of gland openings in the horse trachea is similar to that for the tracheas of other large mammalian species, horse tracheal gland volume was only about 15% that of the other species. Therefore, the excess 'mucous' secretions seen in equine recurrent airway obstruction and other respiratory diseases are unlikely to be caused by comparatively high levels of airway mucous gland secretion. Instead, they may be caused mainly by hyperplasia of the mucus-producing cells of the surface epithelium or by vascular transudation.

Estimate of the subepithelial hydrostatic pressure that drives inflammatory transudate into the airway lumen.

Inflammatory diseases of the upper respiratory tract are characterized by flow of plasma filtrate across the epithelium into the airway lumen ("transudation"). Elsewhere, we have proposed that extravasation from microvessels causes edema, and this is associated with elevated subepithelial hydrostatic pressure that drives transudation. To test this hypothesis, we have attempted to block transudation by elevating luminal hydrostatic pressure. We measured the appearance of plasma markers into the lumen of an isolated perfused segment of rat trachea in vivo and found that stimulation of one vagal nerve caused a rapid (half-time approximately 5 min) and nonselective increase in the flow of markers from blood to airway lumen. Leukocyte migration also caused transudation that developed much more slowly (half-time = 2-3 h). In both cases, transudation was blocked by application of luminal hydrostatic pressures. The critical luminal pressure needed to block vagally induced transudation was approximately 4.5 cmH2O, and, to block epithelial transudation induced by leukocyte traffic, it was 3 cmH2O, and we conclude that these are the subepithelial pressures that drive inflammatory transudation into the airway lumen.

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 approximately 0.6 per mm2 at the top of the trachea and approximately 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 approximately 50 microm diameter. However, glands in the rat trachea generally opened obliquely into shallow (approximately 20 microm deep) oval troughs (approximately 150 x 75 microm), 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 (approximately 30 microm diameter) distributed evenly over and between cartilages at a frequency of approximately 4 per mm2. These may correspond to the 'nests' of goblet cells described by others.

Altered NaCl concentration of airway surface liquid in cystic fibrosis.

A novel isotopic technique suggests that the [Na] and [Cl] of airway surface liquid are both normally approximately 50 mM. In cystic fibrosis, lack of the functional cystic fibrosis transmembrane conductance regulator (CFTR) causes failure of transcellular Cl absorption, resulting in an elevation of [Na] and [Cl] of airway surface liquid to approximately 100 mM.

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 approximately 120 nl.

Restoration of cyclic adenosine monophosphate-stimulated chloride channel activity in human cystic fibrosis tracheobronchial submucosal gland cells by adenovirus-mediated and cationic lipid-mediated gene transfer.

In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV >> E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.

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. Cell 85: 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.

Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia.

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption (Jv) across bovine cells of 3.2 microliter. cm-2. h-1 was inhibited by approximately 78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance (Rt). Effects of P. aeruginosa were blocked by amiloride, which decreased Jv by 112% from baseline of 2.35 +/- 1.25 microliter. cm-2. h-1, increased Rt by 101% from baseline of 610 +/- 257 Omega. cm2, and decreased TEP by 91% from baseline of -55 +/- 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of approximately 2.5 +/- 0.4 microliter. cm-2. h-1 and decreased Rt without changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on Jv, TEP, or Rt. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a sigma factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.

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.

Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes.

Adeno-associated virus (AAV)-based vectors have been shown to be effective in transferring the cystic fibrosis gene (CFTR) into airway epithelial cells in animal models and in patients. However, the level of CFTR gene expression has been low because the vector cannot accommodate the CFTR gene together with a promoter. In this study, we described a strategy to reduce the size of the CFTR cDNA to allow the incorporation of an effective promoter with the CFTR gene into AAV vectors. We engineered and tested 20 CFTR mini-genes containing deletions that were targeted to regions that may contain nonessential sequences. Functional analyses showed that four of the shortened CFTRs (one with combined deletions) retained the function and the characteristics of a wild-type CFTR, as measured by open probability, time voltage dependence, and regulation by cAMP. By using an AAV vector with a P5 promoter, we transduced these short forms of CFTR genes into target cells and demonstrated high levels of CFTR expression. We also demonstrated that smaller AAV/CFTR vectors with a P5 promoter expressed the CFTR gene more efficiently than larger vectors or a vector in which CFTR gene was expressed from the AAV inverted terminal repeat sequence. The CFTR mini-gene with combined deletions was packaged into AAV virions more efficiently, generated higher titers of transducing virions, and more effectively transferred CFTR function into target cells. These new vectors should circumvent the limitations of AAV vector for CFTR expression. Our strategy also may be applicable to other genes, the sizes of which exceed the packaging limit of an AAV vector.

Regulation of the depth of surface liquid in bovine trachea.

The luminal surface of airways is lined by a thin film of airway surface liquid (ASL). Physiological regulation of the depth of ASL has not been reported previously. In this paper, we have used low-temperature scanning electron microscopy of rapidly frozen specimens of bovine tracheal epithelium to demonstrate alterations in the depth of ASL in response to the cholinergic agonist methacholine. We first established that methacholine selectively stimulated airway glands, with maximal secretion at approximately 2 min and a return to baseline within approximately 5 min. A 2-min exposure to methacholine increased the depth of ASL from 23 to 78 microns. Thereafter, depth decreased linearly with time, reaching 32 microns at 30 min. The initial increase in depth was blocked by bumetanide, an inhibitor of active chloride secretion, whereas the slow decline back to baseline was inhibited by amiloride, a blocker of active sodium absorption. We conclude that the methacholine-induced changes in ASL depth reflect transient gland secretion followed by liquid absorption across the surface epithelium.