High lysosomal activities in cystic fibrosis tracheal gland cells corrected by adenovirus-mediated CFTR gene transfer.

Human tracheal gland serous (HTGS) cells are now believed to be a major target of cystic fibrosis (CF) gene therapy. To evaluate the efficiency of adenovirus-mediated gene transfer in these cells we tested the adenovirus construction containing beta-galactosidase cDNA. We observed that the endogenous beta-galactosidase activity in cultured CF-HTGS cells was too strong to allow us to detect any exogenous beta-galactosidase activity. Immunohistological study on sections of human tracheal tissue confirmed the presence of beta-galactosidase in the serous component of the submucosal glands. We then looked for other lysosomal activities in normal and CF-HTGS cells. We showed that normal cells already have elevated enzyme values and that CF-HTGS cells contained 2-4-fold more beta-galactosidase, alpha-fucosidase, alpha-mannosidase and beta-glucuronidase activities than normal cells. An analysis of their kinetic constants has shown that this difference could be attributed to a lower K(m) of CF lysosomal enzymes. More importantly, these differences are eliminated after adenovirus-mediated CFTR gene transfer and not after beta-galactosidase gene transfer.

Evidence for, and characterization of, a lipopolysaccharide-inducible adenosine A2 receptor in human tracheal gland serous cells.

Human tracheal glands are considered as the principle secretory structures in the bronchotracheal tree. In earlier studies, we successfully performed primary cultures of human tracheal gland (HTG) serous cells and noted that these cells were responsive to many secretagogues including purinergic agonists but not to the inflammatory mediator adenosine. In this study, we demonstrate that adenosine was capable of including stimulation of protein secretion by HTG serous cells which had previously been cultured in pro-inflammatory conditions (induced by lipopolysaccharide (LPS)). This stimulation was inhibited by 8-phenyltheophyllline but not by dipyridamole, which is indicative of a P1 purinoceptor. This inducible receptor is the adenosine A2 subtype [rank potency order: (5'-(N-ethyl)-carboxamidoadenosine (NECA) > adenosine > N6-(phenylisopropyl)-adenosine (PIA); and stimulation of adenylyl cyclase]. The adenosine-induced protein secretion was concentration-dependent, however, increased intracellular cyclic adenosine monophosphate (cAMP) was not dependent on the concentration of adenosine. The adenosine-induced secretion and the ATP-induced secretion were shown to be additive. This study concludes that there is evidence of a LPS-inducible adenosine A2 receptor in human tracheal gland serous cells.

Intracellular free Ca2+ 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 [Ca2+]i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca2+]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 [Ca2+]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 [Ca2+]i asynchronous oscillations and the magnitude of the peak [Ca2+]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 [Ca2+]i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

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(50)s of 0.8 and 2 microM, respectively. 2-MeS-ATP also showed antagonistic properties with IC(50)s of 20 and 30 microM 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.

Altered cytokine production by cystic fibrosis tracheal gland serous cells.

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis (CF). We successfully developed techniques for culturing human tracheal gland serous cells from normal individuals (HTGS cells) and from CF patients (CF-HTGS cells) and have shown that the cultured cells have retained most of their in vivo epithelial and secretory characteristics. In order to determine to what extent the serous cells may participate in the lung defense against infection, we examined the effects of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa on HTGS and CF-HTGS cells, with special reference to tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8 secretion. HTGS cells showed a daily basal secretion of IL-6 (1.68 +/- 0.14 ng/10(6) cells) and IL-8 (9.6 +/- 1.3 ng/10(6) cells) and no constitutive secretion of TNF-alpha. Treatment with P. aeruginosa LPS resulted in a significant increase in the basal production of IL-6 (increase of 200% +/- 12%) and IL-8 (525% +/- 40%) as well as a rapid production of TNF-alpha (250 +/- 38 pg/10(6) cells). The LPS-induced secretion of IL-6 and IL-8, but not that of TNF-alpha, was inhibited by glucocorticoids. CF-HTGS cells showed a much higher basal secretion of IL-6 (13.2 +/- 0.5 ng/10(6) cells) and IL-8 (45.6 +/- 7.2 ng/10(6) cells) than normal cells. Treatment with the LPS of P. aeruginosa induced increased production of IL-6 (increase of 100% +/- 8%) and IL-8 (55% +/- 18%) but did not induce the secretion of TNF-alpha. Neither intracellular TNF-alpha nor TNF-alpha transcripts were found in CF-HTGS cells, whereas they were found in normal HTGS cells. In addition, dexamethasone was found to stimulate IL-6 and IL-8 secretion (in the presence or absence of LPS) but did not induce any secretion of TNF-alpha. All these data indicate that HTGS cells are responsive to P. aeruginosa LPS, which results in an increased secretion of IL-6, IL-8, and TNF-alpha, the secretion of which appeared to be impaired in CF-HTGS cells.

Pseudomonas aeruginosa lipopolysaccharide 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 by Pseudomonas aeruginosa in 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 with Pseudomonas aeruginosa LPS 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 by Pseudomonas aeruginosa LPS 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.

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.

A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: its usefulness in studying efficiency of gene transfer.

Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.

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.

Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells.

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.

A cystic fibrosis tracheal gland cell line, CF-KM4. Correction by adenovirus-mediated CFTR gene transfer.

Human tracheal gland serous (HTGS) cells are now considered one principal pulmonary target for the gene therapy of cystic fibrosis (CF). We developed a CF tracheal gland serous cell line, CF-KM4, obtained by the transformation of primary cultures of CF tracheal gland serous cells homozygous for the DeltaF508 mutation by using the wild-type SV40 virus. This cell line retained epithelial and secretory features of the native CF-HTGS cells in primary culture, namely, presence of cytokeratin, constitutive secretion of secretory leukocyte proteinase inhibitor, absence of responsiveness to carbachol and isoproterenol, and defective cyclic adenosine monophosphate-dependent chloride channel activity. Adenovirus-mediated CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells corrected the defective chloride channel activity as well as the responsiveness to adrenergic and cholinergic agonists. In contrast, control transfection using adenovirus-mediated beta-galactosidase gene transfer was totally ineffective. In conclusion, these results present a stable CF tracheal gland cell line that has retained its epithelial and CF-specific defective secretory characteristics which are corrected after CFTR gene transfer. This cell line therefore appears to be a useful tool for large-scale molecular and cellular pharmacologic investigations designed to test potential therapies of the disease CF.

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.