Alteration of sulfation of glycoconjugates, but not sulfate transport and intracellular inorganic sulfate content in cystic fibrosis airway epithelial cells.

The secreted and cell surface high molecular weight glyco-conjugates (HMG) generated by primary cultures of airway epithelial cells from cystic fibrosis (CF) patients are oversulfated. To determine whether this abnormality is maintained in transformed CF airway epithelial cells and whether differences in transport or intracellular accumulation of sulfate can explain this alteration, we assessed sulfate metabolism in paired CF and normal cell lines as well as primary cultures of CF and normal cells. Both 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-inhibitable and -resistant [35S]sulfate efflux and influx were identical for each pair of CF and normal cell lines. Furthermore, cell content of inorganic sulfate was not significantly different in CF and normal cells. However, compared with primary CF cells that oversulfate HMG transformed CF cells oversulfated cell surface HMG but not HMG released into culture medium. Our results suggest that plasma membrane sulfate transport is not altered in CF airway epithelial cells and the abnormal sulfation of HMG may be due to perturbation in intracellular sulfate activation or transfer of activated sulfate to HMG. The relationship of this abnormality to CF transmembrane conductance regulator mutations remains to be determined.

Differential effects of ozone on lung epithelial lining fluid volume and protein content.

Urea dilution has been used to estimate the volume of epithelial lining fluid (ELF) in the respiratory tract. However, ELF volume may be overestimated as the result of rapid net diffusion of urea from tissues into the bronchoalveolar lavage (BAL) fluid. This study established a protocol for rat BAL in a manner that minimizes this problem and then used this procedure to examine the edemagenic effects of ozone (O3) exposure on ELF volume and the concentrations of ELF protein and albumin. One passage lavage with variable dwell times up to 30 s showed no difference in recovered urea, protein, and albumin and ELF volume between 0 and 4 s, but a progressive increase of each thereafter. The calculated concentrations of protein and albumin in ELF did not vary significantly with dwell time. By increasing the number of lavage passages from one to three, the amounts of recovered urea, protein, and albumin and estimated ELF volume were increased with each passage. Again, the calculated concentrations of protein and albumin in ELF did not vary appreciably. When a single lavage passage and no added dwell time were used, it was observed that exposure of rats to 2 but not 0.5 and 1 ppm O3 increased urea, protein, and albumin in the BAL immediately after 6 h exposure. In addition, at 18 h postexposure to 1 ppm O3, ELF volume increased only 21%, but protein and albumin concentrations in ELF were 2.3- and 4.5-fold of control values, respectively. A higher O3 concentration (2 ppm) moderately increased ELF volume (+83%) and exerted even greater effects on concentrations of ELF protein (7.8-fold) and albumin (19-fold) while lower O3 dosage (0.5 ppm) had no significant effect. SDS-PAGE analysis showed that small serum proteins including albumin were greatly enriched in lung BAL fluid of 1 ppm O3-exposed rats. These results demonstrate that movement of water and protein into the airspaces after O3 exposure is not strictly coupled, and that protein recovery by BAL should cautiously be used to indicate airspace edema as a result of O3 injury.

Outcome measures for clinical trials in cystic fibrosis. Summary of a Cystic Fibrosis Foundation consensus conference.

The CFF Consensus Conference concluded with a summary of those outcome measures that would be most useful in studies of patients 6 years of age and older and those measures that would be most useful in studies of the younger population (< 6 years of age) (Table). These measures were further divided into biologic markers most appropriate for initial (phase I and phase II) clinical trials and those especially useful in large, multicenter (phase III) pivotal trials. There is an ongoing need to improve the accuracy and validity of currently available measures of biologic activity and clinical efficacy in CF, especially in the younger population. The conference participants recommended that the following eight issues be addressed as soon as possible: (1) definition of pulmonary exacerbation, (2) broadly applicable methods of testing pulmonary function in small children (ideally a single test for all ages), (3) a comprehensive severity-of-disease score for young children, (4) reliable methods of quantifying chest x-ray and CT scan changes in young patients, (5) simple, inexpensive measures of lung inflammation, (6) a centralized, uniform approach to the establishment of data monitoring committees, (7) a quality of well-being scale for small children, and (8) reliable, reproducible aerosol delivery systems with defined characteristics. In addition, participants recommended that better methods be developed for assessing patients' adherence to research protocols.

Increased adherence of Staphylococcus aureus from cystic fibrosis lungs to airway epithelial cells.

Airway colonization by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF). To assess the pathogenesis of selective colonization with this organism, we compared the capacity of S. aureus isolated from the respiratory tract of CF and non-CF patients to adhere to epithelial cells from the upper and lower airways of CF and control subjects. Bacterial adherence to bronchial epithelial cell lines was significantly greater for CF than for non-CF isolates (p < 0.001). Of 17 CF S. aureus isolates 12 adhered at a level > 1 bacterium per cell; this was true for only 1 of 14 non-CF isolates. CF S. aureus isolates also bound more avidly than non-CF isolates to ciliated (p < 0.05) and squamous nasal cells (p < 0.02) and buccal epithelial cells (p < 0.005) freshly harvested by scraping. Each S. aureus isolate bound with equal avidity to epithelial cells from CF patients and healthy individuals. Adherence was not related to sex, age, severity of pulmonary disease, presence of other microorganisms in the airways, or genotype of the CF hosts. Binding of S. aureus was blocked by proteinase treatment of organisms, suggesting that adherence is mediated by one or more peptide adhesins. We propose that the high prevalence of adherent S. aureus is due either to selection of adherent strains by CF airways or to induction of an adherent phenotype by factors residing at the CF airways surface.

Sulfate concentrations and transport in human bronchial epithelial cells.

Inorganic sulfate concentrations in the cytoplasm of human bronchial epithelial cells exceeded levels in the bathing medium under all circumstances tested. Cell sulfate concentrations were directly related to medium sulfate concentrations and inversely related to medium chloride concentrations. In physiological media there was a sulfate compartment of approximately 0.3 mM that exchanged very slowly with extracellular sulfate. In media lacking chloride, sulfate was accumulated by the cells to a level as high as 2 mM. Sulfate uptake was markedly inhibited by external chloride and by stilbene sulfonic acid derivatives but was not affected by sodium in the medium. Efflux of 35SO4(2-) was stimulated by both chloride and sulfate in the bathing medium but inhibited by stilbenes. The following compounds had no effect on sulfate movements: phorbol esters, adenosine 3',5'-cyclic monophosphate derivatives, and okadaic acid. Changes in medium tonicity were likewise without effect. Our results suggest that human bronchial epithelial cells maintain a steady-state disequilibrium for inorganic sulfate. Furthermore, sulfate appears to exist in at least two compartments in the cells: one that is slowly exchangeable with sulfate in the medium and another exchangeable compartment that is of negligible size in physiological media but that becomes very large in media lacking chloride. Sulfate is transported by an anion exchanger of broad specificity that is not influenced by substances known to modulate chloride channels.

Development of mucociliary transport in the postnatal ferret trachea.

Little is known of the developmental aspects of mucociliary transport. Previous studies have documented that newborn ferret trachea has very few ciliated cells but numerous immature secretory cells in the epithelium and only rudimentary submucosal glands. Rapid and complete maturation occurs in the first postnatal month. This study examines mucociliary transport during this period of rapid maturation. We made direct observations of particle movement across the epithelium of ferret tracheas. No mucus transport could be demonstrated on the first day of life. Transport was discernible, although sporadic and slow, by 7 days and reached adult levels (10.7 +/- 3.7 mm/min) by 28 postnatal days. The emergence of transport capability correlated well with previously described developmental changes in ciliation, mucus secretion, and ion permeability and transport. Threshold mucus transport occurred at 1 wk of age when 20-25% of the surface cells are ciliated. The neonatal ferret appears to be a useful model for assessing integrated epithelial structure-function relationships that are important not only during early development but also during repair after airway injury involving deciliation.

Cystic fibrosis: a current review.

Cystic fibrosis (CF) is the most common severe genetic disorder seen in Caucasians. Defective exocrine gland secretions result in chronic diseases of the respiratory and gastrointestinal systems. However, the CF gene recently has been located and cloned. Currently, genetic technology allows identification of sibling carriers and antenatal diagnosis within families. Oral implications associated with CF include enamel hypoplasia and tooth discoloration, salivary gland involvement, reduced incidence of dental caries, reservoir for potentially pathogenic respiratory bacteria, mouth breathing, and anterior open bite associated with nasal and sinus obstruction. Continued efforts to improve early diagnosis and treatment of CF should increase life expectancy. Affected patients are expected to seek regular dental care more frequently as they learn to view the disease as manageable.

Developmental changes of ferret tracheal mucin composition and biosynthesis.

We characterized the chemical composition of mucins secreted by ferret tracheal explants and the activities of key mucin glycosyltransferases in ferret tracheal epithelium during a period of rapid postnatal maturation of the mucin-secreting structures. Ferret tracheal explants secrete three major groups of high molecular weight glycoconjugates: (1) those susceptible to bovine testicular hyaluronidase; (2) those resistant to hyaluronidase and exhibiting high density (p greater than or equal to 1.60 g/mL); and (3) those resistant to hyaluronidase and exhibiting low density (1.45 less than or equal to p less than 1.60 g/mL). The hyaluronidase-resistant, low-density glycoconjugates have typical mucin properties and constitute 36% of total glycoconjugates released in newborns but only 8% in adult ferrets. Mucin secretory rate per unit surface area of trachea progressively decreases with age. Mucin amino acid and total carbohydrate contents do not vary; however, the sialic acid content increases, and fucose content as well as blood group A activity of the mucins decreases with age. Four glycosyltransferases involved in mucin biosynthesis [Gal beta 3GalNAc:(GlcNAc-GalNAc)beta 6 N-acetylglucosaminyl-, GalNAc:beta 3 galactosyl-, Gal:alpha 2 fucosyl-, and GalNAc alpha 2----6 neuraminyltransferase] are present in tracheal epithelium of ferrets at all ages. Activities of all but the neuraminyltransferase decrease with age. The relatively greater neuraminyltransferase activity is consistent with increased incorporation of sialic acid into secreted mucins over the same age span. Conversely, diminution of fucosyltransferase relative to galactosyltransferase activity may contribute to the lower fucose content and lower blood group A activity of mucins secreted by mature ferret tracheas.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis.

Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface.

Epithelial cell dysfunction in cystic fibrosis: implications for airways disease.

An important pathophysiologic factor in CF airways is the failure to clear poorly hydrated secretions. The water deficit in CF mucous secretions can now be ascribed to a fundamental defect of epithelial cell regulatory processes which promotes sodium reabsorption from surface liquids and interferes with chloride secretion onto the luminal surface. In addition, it is now known that CF airway epithelial cells oversulfate high molecular weight glycoconjugates, both secreted and cell surface-associated. Oversulfation of glycoconjugates may contribute to the altered clearance properties of CF airways mucus and in addition could favor colonization of airways by organisms such as P. aeruginosa.

Development, organization, and function of tight junctional complexes in the tracheal epithelium of infant ferrets.

The surface epithelium of newborn ferret airways matures rapidly in the first month of life. Prominent developmental features include a transition from predominantly non-ciliated to ciliated cells, quantitative and qualitative changes in secretion of macromolecules, and a transition from secretory to absorptive patterns of ion transport. Freeze-fracture replicas of ferret tracheal epithelium from 0 to 28 days of age exhibited progressive developmental patterns in tight junctional structure from beaded, unclosed patterns in newborns to more closed patterns at 28 days. Strand number increased while the depth of tight junctional structures and the proportion of strands exhibiting discontinuity decreased postnatally. Total transepithelial conductance, paracellular conductance, and cell size decreased over the first month. Our data suggest that changes in physiological parameters that reflect epithelial tight junction permeability can be attributed, at least in part, to maturation of this intercellular junction during the postnatal period.

Features of developing ferret tracheal epithelium: ultrastructural observations of in vivo and in vitro differentiation of ciliated cells.

Ultrastructural features of the developing, surface epithelium of ferrets from birth to 28 days of age were characterized. Progressive ciliogenesis in vivo was observed, beginning with cells covering the membranous portion of the trachea. Emerging cilia appeared in ultrathin sections and by scanning electron microscopy at sites correlating with accumulation of integral membrane particles seen in freeze-fracture preparations. Two patterns of ciliogenesis were observed: (1) the random emergence of cilia over the apical cell surface, and (2) initial emergence of cilia at the peripheral boundary of the luminal border of individual cells. Novel, ringlike structures were observed on the surfaces of nonciliated cells at all ages studied. Active ciliogenesis as well as the appearance of ring structures also were documented in the superficial epithelium from 1- to 5-day-old animals maintained in vitro for up to 4 days.

Developmental changes in glycoconjugate secretion by ferret tracheas.

Surface epithelium and submucosal glands of the ferret trachea undergo extensive postnatal development. This study examined developmental changes in rates of release and types of high molecular weight glycoconjugates secreted by explanted ferret tracheas. Digestion with bovine testicular hyaluronidase separated the high molecular weight glycoconjugates into two types, hyaluronidase-resistant mucins and hyaluronidase-susceptible glycosaminoglycans. Release rates were measured under unstimulated conditions and in the presence of known secretagogues. The unstimulated rate of release of total 3H-glycoconjugates was 4-fold higher at birth than after complete maturation. The mucin content varied from 39 to 74% of total 3H-glycoconjugates; however, no age-related pattern was observed for mucin/glycosaminoglycan ratios. The rate of release of 3H-mucins was 6-fold higher at birth than in the adult but rapidly dropped to adult levels by 28 days of age. The secretory cells in the tracheal epithelium of newborn ferrets had more abundant rough endoplasmic reticulum than did mature goblet cells, suggesting increased synthesis of secretory product. Response to methacholine and trypsin, both known stimulators of mucin release, was not observed until 28 and 54 days of age, respectively. Incorporation of 35S-sulfate into mucins relative to that for 3H-glucosamine increased with age, consistent with increasingly acidic histochemical staining properties of secretory cells. These developmental differences in rates of release, modulation of release, and relative sulfation of mucins may represent changes in secretory and synthetic mechanisms of the secretory cells.

Postnatal development of tracheal surface epithelium and submucosal glands in the ferret.

We explored the usefulness of the postnatal ferret as a model for early developmental events in the large airways, using light and scanning electron microscopy. In the first 28 postnatal days, ferret tracheal surface epithelium and glands undergo dramatic growth and development. Tracheal surface area increases 8-fold. At birth, ciliated cells are sparse (9.4 +/- 1.2% of total epithelial cells). A significant increase in ciliated cells is observed at weekly intervals and by day 28 the ciliated cell is the predominant cell type (54.2 +/- 2.8% of total epithelial cells). Secretory cells decrease from 66.4 +/- 1.0% at birth to 22.2 +/- 2.8% of total epithelial cells. Histochemical staining of the granules of the epithelial secretory cells changes from predominantly non-acidic (staining with PAS but not Alcian blue) to predominantly acidic (staining also with Alcian blue). During the same time interval, tracheal glands develop from intraepithelial cellular aggregates devoid of secretory granules at birth into complex, submucosal tubuloacinar structures composed predominantly of cells containing non-acidic secretory granules at 28 days. Therefore, infant ferrets offer an opportunity to examine the structural and functional components of the mucociliary clearance mechanism at developmental stages which occur prenatally in many laboratory animals and in humans.