Human Tracheal Gland Cells Research Articles

Mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells

Mucus hypersecretion relates to exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD) caused by rhinovirus (RV) infection. We examined the mechanisms of RV infection-induced mucin production in human tracheal surface epithelial cells and submucosal gland cells. RV14 up-regulated the mRNA expression of MUC2, MUC3, MUC5AC, MUC5B and MUC6, and increased MUC5AC and total mucin concentration in supernatants and lysates of the surface cells. An inhibitor of the nuclear factor kappaB caffeic acid phenylethyl ester, inhibitors of selective p44/42 mitogen-activated protein kinase-kinase PD98059 and U0126, and a selective Src inhibitor PP1 attenuated MUC5AC mRNA expression, and secretion and production of MUC5AC and total mucin glycoprotein in the surface cells. In the gland cells, RV14 also increased mRNA expression of MUC2, MUC5AC, MUC5B and MUC7, and the inhibitors attenuated the secretion of total mucin glycoprotein. Src-related p44/42 mitogen-activated protein kinase pathway may be associated with RV-induced mucin hypersecretion in human airways.

037 Comparative chemokine secretory profiles in normal and CF polarized epithelial cells following S. aureus infection

Introduction Staphylococcm aureus (S. aureus) is one of the most common causes of infections in human and one the first pathogens infltrating the lung in patients with cystic fibrosis. Branchial epithelial cells are actively involved in the pathogenesis of airway inflammation through the production of multiple factors such as cytokines and chemokines. Cytokines release can lead to the efflux of immune cells, involved in the innate immune response, and stimulate the adaptative immune response. The aim of our study was to analyze 1) the apical and basolateral chemokine profiles obtained by polarized normal human airway epithelial cells (MM39) and CF human airway epithelial cells KM4, 2) the effect of S. aureus interaction with polarized MM39 and KM4 on these chemokine profiles. Methods Polarized monolayer of transformed human tracheal gland cell lines were prepared by seeding 5.10 5 cells/cm 2 on the top of microporous polyester supports. The cells were maintained in a submerged System. For each experiment, confluent airway epithelial cells were apically infected with alive S. aureus . The apical and basolateral supernatants were coUected at different time points, and the chemokine profiles for each condition were analyzed. Results A number of chemokines are secreted by the MM39 and KM4 airway among them IL-8, NAP-2, MIP-1β, Eotaxin-2, RANTES. Following S. aureus contact, the secretion of these chemokines by MM39 cells is increased, while their secretion by KM4 cells is maintained in the basal level. Differences in the levels of the chemokines present in the apical or basolateral compartment of each cell line culture are observed. Looking at the time-dependent and bacteria-dependent IL-8 secretion by MM39 and KM4, we observed an increase of the apical IL-8 secretion by MM39 while the IL-8 secretion level was constant for the KM4. Conclusion Following stimulation of the cells by S. aureus , the chemokine secretion alteration by the MM39 polarized epithelium is more pronounced than for the KM4 polarized airway epithelium. Furthermore, for most of the chemokines studied, the variation is more important in the apical supernatant. This could represent an important factor to be taken into consideration in the development of the lung immune response.

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.

Role of Hyaluronan and Reactive Oxygen Species in Tissue Kallikrein-mediated Epidermal Growth Factor Receptor Activation in Human Airways

In human airways, oxidative stress-induced submucosal gland cell hypertrophy and hyperplasia, histological features of chronic bronchitis, have been linked to epidermal growth factor receptor (EGFR) activation. To explore mechanisms of oxidative stress-induced EGFR activation and signaling, primary cultures of human tracheal submucosal gland (SMG) cells were used to assess EGFR ligand release, EGFR phosphorylation, p44/42 MAPK phosphorylation, and mucin 5AC synthesis in response to reactive oxygen species generated by xanthine/xanthine oxidase (X/XO). Exposure to X/XO increased release of epidermal growth factor (EGF) from these cells, thereby activating EGFR, phosphorylating MAPK, and increasing mucin 5AC production. The importance of EGF was confirmed by transfection of small interfering RNA inhibiting pro-EGF production, which resulted in inhibition of EGFR and MAPK phosphorylation despite X/XO exposure. Blocking signaling by using specific protease inhibitors showed that tissue kallikrein (TK) processed pro-EGF in response to X/XO. Airway TK is bound and inactivated by luminal hyaluronan (HA), and treatment of submucosal gland cells with X/XO induced HA depolymerization and TK activation. These events were blocked by reactive oxygen species scavengers and addition of exogenous excess HA and TK inhibitors. Thus, HA plays a crucial role in regulating airway TK activity and thereby TK-mediated release of active EGF from human SMG cells. Sustained HA depolymerization is expected to cause TK activation, EGF release, and EGFR signaling and to lead to SMG cell hypertrophy and hyperplasia as well as mucus hypersecretion with subsequent airflow obstruction.

Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus infection and ad-mediated gene transfer.

CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.

Influence of culture conditions on the α1,2-fucosyltransferase and MUC gene expression of a transformed cell line MM-39 derived from human tracheal gland cells

Human tracheal glands cells (HTGC) in culture are able to respond to adrenergic, cholinergic and purinergic agonists by increasing their serous and mucin secretions. These secretagogues are also able to maintain an optimal responsiveness of serous cells to stimulation when they are regularly and briefly delivered to the cells, making the HTGC a suitable model to study the serous secretion (Merten, in press). Our interest has been focused on the effects of cholinergic and purinergic secretagogues associated to histamine, on the mucous function of the transformed human tracheal gland cell line MM-39, which has a mixed, both serous and mucous, phenotype. When the cells were exposed to short stimulation every 2 days for 3 weeks with 10 or 100 μM carbachol, UTP and histamine, modifications of their mucous phenotype were observed. The expression of MUC genes appeared dependent on the culture conditions. Transcripts of MUC1, MUC4, and MUC5B genes were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 10 μM, in contrast to the unstimulated expression of MUC1 and MUC4 in control cells. MUC1, MUC4, MUC7, MUC6 and MUC11 transcripts were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 100 μM. These culture conditions were also able to induce an α1,2-fucosyltransferase activity absent in the MM-39 cells cultivated with standard conditions. There was no marked effect on the α2,3-sialyltransferase activity although the expression pattern of the sialyltransferase genes was reduced to the unique presence of ST3Gal III. In conclusion, MM-39 cells exposed to repeated stimulation by secretagogues at different concentrations express different sero-mucous phenotypes.

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells.

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNFalpha. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNFalpha; a 52% increase of alpha2,3-sialyltransferase activity was also observed in TNFalpha-stimulated MM-39 cells. After metabolic radio-labelling with [(3)H]glucosamine and [(3)H]fucose, the mucins released in the culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39-1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNFalpha was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the alpha2,3-sialyltransferase activity by TNFalpha argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNFalpha. In conclusion, the influence of TNFalpha on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.

The pathogenesis and therapy of virus infection-induced senile bronchial asthma

We investigated the pathogenesis and therapy of virus infection-induced senile bronchial asthma in vitro. To examine the effects of rhinovirus infection on the production of cytokines and intercellular adhesion molecule-1 (ICAM-1), human tracheal epithelial cells and submucosal gland cells were cultured, and infected with human rhinovirus. Rhinovirus upregulated the production of interleukin (IL)-1 beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha in supernatants of epithelial cells and submucosal-gland cells, and IL-1 alpha and granulocyte macrophage colony-stimulating factor (GM-CSF) in supernatants of submucosal gland cells. Rhinovirus upregulated the expression of ICAM-1 mRNA. Rhinovirus infection also increased epithelial permeability. These events may be important for the spread of airway inflammation after rhinovirus infection. Furthermore, we studied the effects of dexamethasone and erythromycin on the modulation of virus infection and induction of cytokines and ICAM-1 in tracheal epitherial cells. Both of them reduced viral titers of rhinovirus type 14, a major group rhinovirus, and cytokine production of supernatants, and ICAM-1 mRNA expression in the cells. Because it is known that acidic conditions by proton pumps are needed for rhinovirus entry into the cells, we studied the effects of H+ ATPase inhibitor bafilomycin A1. Bafilomycin A1 reduced the virus titers of both rhinovirus type 2 and 14 in supernatants. These findings in our in vitro study suggest that dexamethasone, erythromycin and bafilomycin A1 may inhibit rhinovirus infection and modulate airway inflammation induced by rhinovirus infection.

Characterization of a diadenosine tetraphosphate-receptor distinct from the ATP-purinoceptor in human tracheal gland cells

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are not well known. ATP binds to P2 receptors and induced secretory leucocyte protease inhibitor (SLPI) secretion through formation of cyclic adenosine monophosphate and mobilization of intracellular [Ca 2+]. Since diadenosine polyphosphates (ApnA) are also endogenous effectors of P2 receptors, we investigated their effects in a cell line (MM39) of human tracheal gland cells. Diadenosine tetraphosphates (Ap4A) induced significant stimulation (+50±12%) of SLPI secretion and to a similar extent to that of ATP (+65±10%). No significant effects were observed with diadenosine triphosphate (Ap3A), diadenosine pentaphosphate (Ap5A), ADP and 2-methylthio-adenosine triphosphate (2-MeS-ATP). Since Ap4A was weakly hydrolyzed (<2% of total), and the hydrolysis product was only inosine which is ineffective on cells, this Ap4A effect was not due to Ap4A hydrolysis in ATP and adenosine monophosphate (AMP). A mixture of Ap4A and ATP elicited only partial additive effects on SLPI secretion. ADP was shown to be a potent antagonist of ATP and Ap4A receptors, with IC 50s of 0.8 and 2 μM, respectively. 2-MeS-ATP also showed antagonistic properties with IC 50s of 20 and 30 μM for ATP- and Ap4A-receptors, respectively. Single cell intracellular calcium ([Ca 2+] i) measurements showed similar transient increases of [Ca 2+] i after ATP or Ap4A challenges. ATP desensitized the cell [Ca 2+] i responses to ATP and Ap4A, and Ap4A also desensitized the cell response to Ap4A. Nevertheless, Ap4A did not desensitize the cell [Ca 2+] i responses to ATP. In conclusion, both P2Y2-ATP-receptors and Ap4A-P2D-receptors seem to be present in tracheal gland cells. Ap4A may only bind to P2D-receptors whilst ATP may bind to both Ap4A- and ATP-receptors.

Development of Substituted Benzo[c]quinolizinium Compounds as Novel Activators of the Cystic Fibrosis Chloride Channel

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Extracellular Mg2+Inhibits both Histamine-Stimulated Ca2+-Signaling and Exocytosis in Human Tracheal Secretory Gland Cells

The effects of extracellular Mg2+concentration have been investigated on the histamine-stimulated exocytotic process of human tracheal secretory gland (HTG) cells. The exocytosis of secretory granules (SG) was observed concomitantly with dynamic changes of intracellular Ca2+([Ca2+]i) and Mg2+concentrations ([Mg2+]i). The rate of SG exocytosis was appraised by the decrease of quinacrine fluorescence emission. Dynamic changes of [Mg2+]iand [Ca2+]iin HTG cells were determined by the combined use of UV-microspectrofluorometry with Mag-Indo-1 and Indo-1 probes, respectively. High Mg2+medium significantly inhibited the histamine-stimulated secretion. The influence of the extracellular and intracellular Mg2+concentrations on [Ca2+]iwas analyzed. Basal [Mg2+]iincreased from 0.8 mM in a Mg2+-free medium to 1.7 mM in 10 mM Mg2+medium. Histamine induced a [Mg2+]iincrease which is dependent on extracellular Mg2+concentration. The histamine stimulated [Ca2+]irise was reduced in the presence of elevated Mg2+extracellular medium and inhibitory effects of extracellular Mg2+were concomitant with changes in [Mg2+]i. Our data suggest that the inhibition by extracellular Mg2+of stimulated exocytosis is dependent on both the increase of [Mg2+]iand the inhibition of cytosolic Ca2+influx.

Characterization of two distinct P2Y receptors in human tracheal gland cells

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are poorly known. ATP-binding characteristics, and ATP-induced formation of cAMP were investigated in a cell line (MM39) of human tracheal gland cells. The binding of a radiolabelled non-hydrolysable analogue of ATP Adenosine-5'-[35S]thiotriphosphate: [35S]ATP[gammaS] was rapid (within 30 min at 4 degrees C), stable and reversible. Scatchard analysis revealed two classes of [35S]ATP[gammaS]-binding sites. Low-affinity binding sites had a Kd1 of 20 +/- 5 microM (Bmax = 150 nmol/10(6) cells) and the high-affinity binding sites had a Kd2 of 2.5 +/- 0.2 microM (Bmax = 52 nmol/10(6) cells). Competition experiments showed competition with ATP, ADP and 2-methylthio-ATP but no competition with UTP, AMP and adenosine. UTP stimulates protein secretion as well as it induced [Ca2+]i mobilization but did not affect the intracellular cAMP levels. ATP also caused induced [Ca2+]i mobilization and protein secretion but also caused an increase in cyclicAMP content of the cells, reaching a maximum after 1 min. ATP-induced cAMP formation was concentration dependent and inhibited by the P2-antagonist suramin. Reverse-transcription-PCR amplification revealed the presence of the transcripts of both the P2Y2 and the UTP-specific P2Y4 receptors. In conclusion, P2Y2 receptors, UTP-P2Y4 receptors and unidentified ATP-specific receptors seem to be present in MM39 cells which appear to be coupled differently to intracellular second-messenger systems.

Neutrophil Elastase Promotes Rapid Exocytosis in Human Airway Gland Cells by Producing Cytosolic Ca2 +Oscillations

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Pseudomonas aeruginosaLipopolysaccharide Induces CF-like Alteration of Protein Secretion by Human Tracheal Gland Cells

Human tracheal gland (HTG) serous cells are now believed to play a major role in the physiopathology of cystic fibrosis. Because of the persistent inflammation and the specific infection byPseudomonas aeruginosain the lung, we looked for the action of the lipopolysaccharide (LPS) of this bacteria on human tracheal gland cells in culture by studying the secretion of the secretory leukocyte proteinase inhibitor (SLPI) which is a specific serous secretory marker of these cells. Treatment withPseudomonas aeruginosaLPS resulted in a significant dose-dependent increase in the basal production of SLPI (+250 ± 25%) whilst the SLPI transcript mRNA levels remained unchanged. This LPS-induced increase in secretion was inhibited by glucocorticoides. Furthermore, LPS treatment of HTG cells induces a loss of responsiveness to carbachol and isoproterenol but not to adenosine triphosphate. These findings indicate that HTG cells treated byPseudomonas aeruginosaLPS have the same behavior as those previously observed with CF-HTG cells. Exploration by using reverse transcriptase polymerase chain reaction amplification showed that LPS downregulated cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in HTG cells indicative of a link between CFTR function and consequent CF-like alteration in protein secretory process.

Mucins secreted by a transformed cell line derived from human tracheal gland cells.

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Spatial and Temporal Mg2+Signaling in Single Human Tracheal Gland Cells

The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2+variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2+concentrations (up to 10 mM) induced increases in intracellular Mg2+concentration [Mg2+]i. From a cytosolic basal concentration of 0.8 ± 0.3 mM in a medium free of Mg2+, an increase in extracellular Mg2+concentration from 1 to 10 mM, increased cytosolic [Mg2+]ifrom 1.4 ± 0.6 to 1.8 ± 0.8 mM after 10 min (p<0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2+]iis distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2+stores involved. The influx pathway for Mg2+in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2+]i.

A transformed human tracheal gland cell line, MM-39, that retains serous secretory functions.

Infection with the wild type SV40 virus was used to transform primary cultures of human tracheal gland serous (HTGS) cells. Over 80 different cell lines were obtained, but the majority had lost some of their epithelial and secretory features. However, one of these cell lines, MM-39, was shown to have conserved the physiologic characteristics of the genuine HTGS cells-i.e., the presence of cytokeratin, expression of cystic fibrosis transmembrane conductance regulator mRNA, a level of secretory leukocyte proteinase inhibitor secretion comparable to that of the native cells (25 +/- 3 ng/10(6) cells/h), and the responsiveness to pharmacological agonists: carbachol (+260 +/- 40%), isoproterenol (+260 +/- 40%), and adenosine 5'-triphosphate (+280 +/- 30%). These characteristics describe a transformed cell line of human tracheal gland cells which has retained the features of the native serous cells. As a result, this cell line appears to be a useful tool for large-scale physiologic and pharmacologic studies of bronchial secretion at the cellular level.

Intracellular free Ca 2+ dynamic changes to histamine are reduced in cystic fibrosis human tracheal gland cells

This study documents a difference between cystic fibrosis human (CF-HTG) and normal human (HTG) tracheal gland cells: the ability of histamine to induce an increase of intracellular free calcium concentration [Ca 2+] i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca 2+] i peak rise in response to histamine is smaller in CF-HTG cells than in HTG cells, and the percentage of CF-HTG cells that increase [Ca 2+] i is decreased compared with HTG cells. In contrast to histamine, the human neutrophil elastase (HNE) stimulation of both CF-HTG and HTG cells generated [Ca 2+] i asynchronous oscillations and the magnitude of the peak [Ca 2+] i response as well as the percentage of responding cells were similar for both groups. By videomicroscopy observations, the secretory response (exocytosis of secretion granules) of CF-HTG cells occurred with HNE, but not with histamine, thus suggesting that [Ca 2+] i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

Asynchronous Dynamic Changes of Intracellular Free Ca2+ and Possible Exocytosis in Human Tracheal Gland Cells Induced by Neutrophil Elastase

Measurements of the intracellular free calcium concentration [Ca2+]i in single cells of the human tracheal gland cell line MM 39 demonstrate dynamic changes in [Ca2+]i after their exposure to human neutrophil elastase (HNE). A heterogeneity in [Ca2+]i responses measured cell to cell in monolayer culture is evident : cells generate an initial [Ca2+]i peak rise with or without a delayed time (up to 180 sec) followed either by a rapid return to baseline, asynchronous oscillations or a sustained plateau phase. From basal concentration of 85± 15 nM, HNE (1 μM) produces a [Ca2+]i increase of 91 ± 66 nM in about 50 % of responding cells. At lower concentrations of HNE (0.1 μM, 0.01 μM), the [Ca2+]i rise remains similar, but only 30-40 % of the cells are responding. Pretreatment of cells with the recombinant elafin protein, a specific elastase inhibitor, reduces both the [Ca2+]i response to HNE and the number of responding cells. Electron microscopy observations reveal an increased number of secretory granules located beneath the cell plasma membrane after HNE treatment. These results suggest that intracellular [Ca2+]i changes may be associated to the HNE-induced exocytosis in human tracheal gland cells. These findings could have implications with regard to the pathogenesis of increased mucus secretion in human airway diseases.

Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensional collagen gel matrix.

The extracellular matrix has been demonstrated to affect the differentiation of epithelial cells. We present evidence that in a three-dimensional (3-D) type I collagen gel matrix, isolated human adult tracheal gland (HTG) cells are capable of reconstructing new functional gland-like tubules in vitro. During the first two weeks in culture, HTG cells developed globular epithelial cell aggregates in which lumina is absent. By the third week in culture, the tubulogenesis and the formation of branching structures became evident with a polarized morphology, which in many aspects resembles the in vivo morphology. A central lumen was lined by polarized secretory epithelial cells exhibiting well-developed microvilli and apical secretory granules. Furthermore, we showed that the capacity of in vitro tracheal gland differentiation was associated with the basal deposition of laminin and type IV collagen around the gland-like tubules. A cell-associated 72 kDa type IV collagenase was expressed in developing tubule cells, as shown by immunocytochemistry. The secretion of the antileucoprotease (ALP), a protein marker of tracheal gland serous cells, was bidirectional in gland-like tubules, since up to 65% of released ALP was in the basolateral direction. Taken together, these observations indicate that isolated HTG cells in a 3-D collagen matrix form functional tracheal gland-like tubules and suggest that similar new tracheobronchial gland formations may occur during the human normal gland development and remodeling.