Human sweat duct cells in primary culture. Basic bioelectric properties of cultures derived from normals and patients with cystic fibrosis

Summary Human sweat duct cells from the coiled reabsorptive segment have been cultured successfully, free from fibroblasts, in a low serum, hormone-supplemented medium. Ham's F12. The cultured cells exhibited a typical epithelial cobblestone pattern and microvilli-covered luminal cells were seen joined together with typical junctional complexes. In cultures derived from normals and patients with cystic fibrosis (CF), growth and morphologic characteristics were indistinguishable. When grown on a membranous support, and mounted in an Ussing chamber, vectorial electroconductive ion-transport could be identified. The epithelial preparations produced active mucosa to serosa-directed sodium flux via amiloride-sensitive, apical sodium channels and ouabain-sensitive sodium pumps located in the basolateral membrane, which also contained a potassium shunt. These findings are consistent with a polarized epithelium with properties similar to the intact organ. High transepithelial resistance and increased amiloride sensitivity were typical for cells derived from CF, indicating that principal normal as well as pathologic properties of the sweat duct are preserved in culture. Financial support was provided by CF Foundation USA (G1397-01), SLF, Ville Heyse, Haensch, Nationalforeningen, Winthertur, Novo, and Egmont Foundation.     

Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.     

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.     

Plasma membrane lipids of human diploid fibroblasts from normal individuals and patients with cystic fibrosis.

The lipid composition of isolated plasma membranes of human skin fibroblasts is described for the first time. Plasma membranes from a number of strains of fibroblasts from patients with cystic fibrosis and matched normals were isolated by a recently described procedure and analysed for major phospholipid classes, cholesterol and fatty acids. No differences in the quantities of these compounds were detected between cells of the two different origins. The fetal calf serum used to supplement the growth medium contained relatively more palmitoleate and oleate but less stearate than the membranes. There were also no consistent differences between cystic fibrosis and normal membranes in terms of the fatty acid compositions of their individual phospholipid classes. Consistent with this lack of chemical change in the lipids of membranes of cystic fibrosis cells, the degree of fluorescence polarization of diphenylhexatriene, an index of fluidity, was also unchanged.     

Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) (P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) (P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP₂, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP₃, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 (P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 (P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 (P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.     

Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.

Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.     

Structure and function of human sweat glands studied with histochemistry and cytochemistry

The basic structure and the physiological function of human sweat glands were reviewed. Histochemical and cytochemical techniques greatly contributed the elucidation of the ionic mechanism of sweat secretion. X-ray microanalysis using freeze-dried cryosections clarified the level of Na, K, and Cl in each secretory cell of the human sweat gland. Enzyme cytochemistry, immunohistochemistry and autoradiography elucidated the localization of Na,K-ATPase. These data supported the idea that human eccrine sweat is produced by the model of N-K-2Cl cotransport. Cationic colloidal gold localizes anionic sites on histological sections. Human eccrine and apocrine sweat glands showed completely different localization and enzyme sensitivity of anionic sites studied with cationic gold. Human sweat glands have many immunohistochemical markers. Some of them are specific to apocrine sweat glands, although many of them stain both eccrine and apocrine sweat glands. Histochemical techniques, especially immunohistochemistry using a confocal laser scanning microscope and in situ hybridization, will further clarify the relationship of the structure and function in human sweat glands.     

Effects of media buffer systems on growth and electrophysiologic characteristics of cultured sweat duct cells

Summary Primary cultures of human reabsorptive sweat duct cells were grown in MCDB 170 medium buffered with either HEPES, bicarbonate, or a mixture of HEPES and bicarbonate buffers. Cultures grown in MCDB media containing bicarbonate seemed to differentiate into a multilayered, keratinized epithelium and began senescing after 1 wk in culture. In contrast, cultures grown in media containing HEPES as the only buffer seemed to undergo a selection process, resulting in the outgrowth of cells that did not multilayer or keratinize extensively for up to 3 or 4 wk in culture. Despite marked differences in growth, cells grown in both bicarbonate and HEPES-buffered media retained electrophysiologic characteristics appropriate to the progenitor. Mean resting potentials were −21.8±0.8 mV (n=82), −23.3±1.3 mV (n=70) and −18.2±0.8 mV (n=82) for duct cells grown in HEPES, bicarbonate, and HEPES-bicarbonate media, respectively. Substitution of Cl⁻ with the impermeant anion gluconate in the bathing medium caused membrane potential depolarization in all media, revealing the presence of a Cl⁻ conductance. Administration of the Na⁺ conductance inhibitor amiloride hyperpolarized the mean resting potential of cells grown in HEPES medium (−6.8±0.6 mV,n=68), bicarbonate medium (−6.9±0.5 mV,n=60), and HEPES-bicarbonate medium (−5.9±0.6 mV,n=69), demonstrating expression of a Na⁺ conductance. We observed some but minimal variation with age in any of these conditions. This work was supported by grant DK41329-02 from the National Institute of Health, Bethesda, MD, and a Postdoctoral Fellowship to Dr. Bell from the National Cystic Fibrosis Foundation.     

Induction of alkaline phosphatase in cultured human fibroblasts: Comparison of normal cells and those from patients with cystic fibrosis

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP.Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

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

Summary The 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 (I_sc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells. This work is supported by grants HL41928 and DK39619 (DCG), HL24136 (CBB), and HL42368 (JHW and DCG) from the National Institutes of Health, Bethesda, MD.     

A cystic fibrosis phenotype in cells cultured from sweat gland secretory coil. Altered kinetics of 36Cl efflux

As a step toward understanding the metabolic consequences of the cystic fibrosis (CF) mutation, we have examined the kinetics of 36Cl efflux in cells cultured from sweat glands, a tissue that is affected in the disease. Epithelial cells, derived from the secretory coil of sweat glands of CF and control individuals, were cultured in serum-free medium, and primary cultures used for efflux experiments. Cell layers were equilibrated with Na36Cl in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered balanced salt solution for 45 min at 37 degrees C, washed in 0.25 M sucrose, and incubated in nonradioactive buffer for measurement of 36Cl efflux. Efflux from CF and control cells followed biphasic kinetics and was described by the equation Y = Ae-kat + Be-kbt. All efflux was inhibited at 6 degrees C. The fast component of efflux, Ae-kat, of both control and CF cells was inhibited by the anion channel blockers 4,4'-diisothiocyanato-2,2'-stilbene disulfonic acid, 9-anthracene carboxylate, and diphenylamine 2-carboxylate, implicating release through chloride channels. At 23 degrees C, the kinetics of 36Cl efflux from CF and control cells were indistinguishable, but efflux from control cells could be accelerated by cAMP analogs and isoproterenol. At 37 degrees C, 36Cl efflux was more rapid from control cells than from CF cells, but could not be stimulated further by beta-adrenergic agents. In both cases, the increased rate of efflux was due to a severalfold increase in the A parameter of the fast component. These differential responses constitute a "CF phenotype" of secretory sweat gland cells in culture that may be useful for further investigation of the metabolic defect in cystic fibrosis.     

The effects of thermally-induced activity in vivo upon the ultrastructure and Na, K and Cl composition of the epithelial cells of sweat glands from patients with cystic fibrosis

The secretory cells of the fundus of sweat glands from cystic fibrosis (CF) patients had higher Na and Cl contents and showed more granule depletion, cellular disruption and dilated intercellular canaliculi than normal. The cells of the coiled duct also had higher cytoplasmic levels of Na and Cl but were structurally normal. Thermal stimulation produced ultrastructural changes in the CF fundus comparable to normal, including further dilatation of the basolateral clefts, but did not induce the marked changes in the coiled duct which normally occur. The elevated Na and fall in K in the fundus and raised Na and Cl in the coiled duct upon activation, were not observed in the CF glands in which no significant changes were detected.     

Terminal glycosylation of cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (F508/F508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in 1,3/4 linkage to antennary N[emsp4 ]-acetyl glucosamine (Fuc1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fuc1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fuc1,3/4GlcNAc was detected with a specific 1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment.