Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population. It is characterized by a general exocrinopathy, the pulmonary syndrome of which (mucus hypersecretion and infection) still leads to death. The most characteristic feature of CF is the persistent colonization by Pseudomonas aeruginosa of the airways of CF patients. CF is caused by mutations in a gene encoding a membrane protein called CF transmembrane conductance regulator (CFTR), which was proved to possess a cyclic adenosine monophosphate (cAMP)-dependent chloride channel activity (1).

Using immunocytochemistry and in situ hybridization techniques, it was shown that the serous component of the submucosal bronchotracheal glands expresses a very high level of CFTR in comparison with the other bronchial epithelial cell types (2). Human tracheal gland serous (HTGS) cells actively participate in the local defense of the airways by secreting antibacterial proteins such as lactoferrin, lysozyme, and the secretory leukocyte protease inhibitor (SLPI) (3). Because HTGS cells (1) strongly express CFTR and (2) participate in the antibacterial defense of the lung, they are considered one of the principal target pulmonary cell types of the disease CF (4).

We successfully developed techniques for culturing HTGS cells from normal and CF patients (5). In culture, these cells were shown to be of the serous type (5) and to have retained most of their in vivo epithelial and secretory characteristics, such as secretion of the three specific serous secretory markers: SLPI, lysozyme, and lactoferrin. These cells were also shown to express high levels of CFTR (8, 9). However, there are enormous limitations due to the rarity of available CF lung tissue and to difficulties in performing CF-HTG cell cultures from highly infected surgical pieces.

Recent advances in molecular biology and gene transfer technology render strategies possible in the gene therapy of this disease (10). One of the most promising transfer vectors is currently a recombinant adenovirus, defective for the early replicative E1 gene. Adenovirus-mediated transfer of CFTR complementary DNA (cDNA) into isolated epithelial CF cells in culture has demonstrated a correction of cAMP-dependent chloride conductance dysfunction (11, 12), and this CFTR cDNA-containing adenovirus was used successfully in cotton rats (10), monkeys (13, 14), and CF human volunteers (15). Several authors have tried to determine the cell types that are transfected, by using adenovirus-mediated -galactosidase (Ad--gal) gene transfer. Some described successful transfer into the surface epithelial cells (15, 16). On the contrary, others showed that surface epithelial cells are inefficiently transfected but only some duct cells and glands may be transfected (15, 17). Furthermore, none of these studies has addressed the effectiveness of the correction by CFTR gene transfer of the CF-associated defects of tracheal gland cells.

Faced with (1) the difficulty in obtaining surgical human tracheal pieces from CF patients (in sufficient size and number for reproducibility), and (2) the potential importance of the gland serous cells to be corrected, we developed an immortalized CF tracheal gland serous cell line, CF-KM4, that was shown to have retained serous secretory functions similar to those observed in the original CF-HTG cells (7). This cell line was used to study the effect of transfection with an adenovirus vector proposed in gene therapy of CF. We hereby showed an effective integration of the CFTR gene in the transfected cells, resulting in the appearance of cAMP-dependent low-conductance chloride channel activities as well as a complete correction of the defective cholinergic and adrenergic stimulation of protein secretion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

Epinephrine, isoproterenol, carbamoylcholine, adenosine-5'-triphosphate (ATP), forskolin, 3-isobutyl-1-methylxanthine (IBMX), and Dulbecco's modified Eagle's medium/ Ham's F12 mixture (DMEM/F12) were obtained from Sigma (St. Louis, MO). Ultroser G was from Biosepra (Villeneuve la Garenne, France). Anticytokeratin antibodies (KL1 clone) were obtained from Immunotech (Marseille, France), and anti-SV40 antibodies (MAB 990) were from Chemicon, Temecula, CA. The fluorescein isothiocyanate (FITC)-conjugate rabbit antimouse antibodies were from Dakopatts (Glostrup, Denmark). All other chemicals were of cell-culture grade.

Generation of a CF-HTG Cell Line

CF-HTGS cells, isolated from an individual carrying a F508/F508 mutation and undergoing pulmonary transplantation, were cultured as described in CELL CULTURE CONDITONS, below. Subcultured cells were then grown in six-well plates until 10 d after confluence, and each well was infected with 500 µl of SV40 lysate (titer: 1 to 2 × 10⁸ plaque-forming units/ml) for 30 min. Cells were then rinsed twice with phosphate-buffered saline and fresh culture medium was added. After 3 d, the contents of each dish were passaged onto 75-cm² flasks. Within 2 wk, growing cell areas were detected and isolated using cloning rings, and each isolated cell population was passaged first in 24-well plates and then in 24-cm² flasks. We ascertained that the growing cells were immortalized because of the complete inability of the nonimmortalized CF-HTGS cells to divide at confluence. As a result, we obtained 12 different cell lines, which were screened and selected for their epithelial and secretory features that were the closest to those of the native CF-HTGS cells. Our selection criteria were growth recovery after passaging, maintenance of the ability to secrete the specific secretory marker SLPI, an epithelial morphology, the presence of cytokeratin within the cells, and the stability of all of these characteristics whatever the number of passages. After selection, one of the isolated cell lines, CF-KM4, was shown to have retained all of the above criteria.

Cell Culture Conditions

CF-HTG cells as well as the non-CF-HTG cell line MM-39 and the CF-KM4 cell line were cultured as previously described (18). Briefly, cells were maintained in a DMEM/ F12 mixture supplemented with 1% Ultroser G, 0.22 g/liter sodium pyruvate, and 8 g/liter glucose. Epinephrine (2.5 µM) was routinely added to the cell-culture medium to provide optimal growth and differentiation (19). The cells were passaged using 0.025% trypsin (GIBCO, Cergy Pontoise, France) and 0.02% ethlyenediaminetetraacetic acid and used at the third passage, 8 d after confluency, because of an extended differentiated state at that moment. Type 1 collagen-coated disposable tissue-culture flasks were used. At the end of all incubations with pharmacologic agents or virus, cell viability was assessed by measurement of lactate dehydrogenase (LDH) release in the incubation medium. LDH activity was measured using the Sigma lactate dehydrogenase kit. We routinely checked for the absence of mycoplasma in the primary culture of HTGS cells and in immortalized cell lines using the DNA fluorescent dye Hoechst 33258 as described in Reference 20.

CHO cells were generously obtained from J. R. Riordan and X. B. Chang (Mayo Clinic, Scottsdale, AZ) and cultured as detailed in Reference 21.

Immunocytochemistry

CF-KM4 cells were fixed for 30 min at 20°C with methanol and thereafter immunocytochemically examined with mouse anticytokeratin antibodies (at a 1:500 dilution) and anti-SV40 large T monoclonal antibodies (at 1:1,000 dilution). Both anticytokeratin and anti-SV40 large T antibodies were revealed with a goat antimouse antibody (at 1: 1,000 dilution) conjugated to FITC.

Polymerase Chain Reaction (PCR)

DNA was extracted from cells using standard techniques (22). Approximately 200 ng of genomic DNA from each pellet of cells was amplified by PCR. The PCR experiment was performed using the following oligonucleotide synthetic primers: 5'-CACTCCTCTTCAAGACAAA-3' and 5'-CTGGATGAAGTCAAATATGG-3', both flanking the F508 mutation. The thermal cycle profile consisted of (1) denaturation for 60 s at 94°C, (2) primer annealing for 45 s at 62°C, and (3) extension for 120 s at 72°C. The amplification was performed for 28 cycles. The amplification product (20 µl) was analyzed on polyacrylamide (10%) gel electrophoresis (PAGE) (overnight at 300 V) and visualized under ultraviolet after ethidium bromide staining.

Cell Treatment with the Adenoviral Constructions Ad--Gal and Ad-CFTR

The adenoviral constructions shuttle plasmids Ad--gal and Ad-CFTR were built (10, 23) and kindly provided by Transgene (Strasbourg, France). The vector preparations (10⁹ pfu/ml) were endotoxin-free. Confluent CF-KM4 cell treatment consisted of exposure to 10 multiplicities of infection of Ad--gal or Ad-CFTR by cells for 24 h. Cells with and without treatment were studied for PCR and their electrophysiologic and pharmacologic properties immediately after this 24-h exposure to virus.

Single-Channel Patch-Clamp Recording

Cells were grown on glass coverslips under the conditions described above. Single-channel currents were recorded from cell-attached configuration. Cells were stimulated with 10 µM forskolin and 250 µm IBMX (dissolved in dimethyl sulfoxide to a final concentration of 0.1%). Experiments were performed at room temperature. Results were displayed conventionally with inward currents (outward flow of anions) indicated by downward deflections. In all the figures, dotted lines give the zero-current baselines when the channels were all in the closed state. Positive potentials indicate depolarization. Potentials were expressed as the bath potential minus the patch electrode potential. The pipette solution (pH 7.4) contained 150 mM NaCl, 2 mM MgCl₂, and 10 mM N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES). The bath contained 145 mM NaCl, 4 mM KCl, 2 mM MgCl₂, and 10 mM TES (pH 7.4). Other details appear elsewhere (9). For selectivity experiments, 150 mM NaCl were substituted either by 150 mM choline chloride or by 40 mM NaCl plus 110 mM sodium gluconate in the pipette solution. Data are presented as the means ± SD of n separate experiments.

Pharmacologic Stimulation of CF-KM4

CF-KM4 cells were grown onto 24-well plates, and 8 d after confluence they were rinsed for 1 h with DMEM/F12. After five 1-h washes, cells were exposed to 100 µM in DMEM/F12 of either carbachol, isoproterenol, or ATP. After a 30-min exposure, supernatants were collected for SLPI analysis. The effects of pharmacologic agents were analyzed by determining the SLPI relative secretory rate (SLPI S.R.), that is, SLPI secreted in the assay versus SLPI secreted in control wells in which vehicle solutions alone were added at the same times as the drugs were added to experimental wells. The mean ratio was calculated from quadruplicate assays. Each experiment was performed three times. Mean ± SD is indicated. SLPI was assayed by enzyme-linked immunosorbent assay (24).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of the CF-KM4 Clone

One selection criterion of the CF-KM4 cell line was its homogeneous epithelial morphology. Phase-contrast microscopic photographs of CF-KM4 cells taken during the exponential growth phase and the stationary phase show that their morphology was typically epithelial in nature (Figure 1A). Like CF-HTGS cells, the CF-KM4 cells formed clusters of jointed adherent cells. At confluence, reached 8 d after seeding, the monolayer composed of tightly molded polygonal cells showed a mosaic morphology (Figure 1B). When examined for cytokeratin, all of the CF-KM4 cells were labeled with the anticytokeratin monoclonal antibody showing, as expected for typical epithelial cells, a strong cytoskeleton network inside the cells (Figure 1C). We also examined the expression of the SV40 large T antigen within the CF-KM4 cells using a monoclonal antibody specific for the large T antigen. CF-KM4 cells reacted positively with the antibody by a nuclear staining in 100% of the cells, indicating that the entire population of CF-KM4 cells was transformed (Figure 1D).

View larger version (178K): [in this window] [in a new window]   Figure 1.   Light microscopy of CF-KM4 cells. (A) In the exponential growth phase (here taken at the second day of culture), phase-contrast observations reveal growing CF-KM4 cell clusters. Bar = 30 µm. (B) At confluence (from the ninth day of culture), CF-KM4 cells present a mosaic morphology. Bar = 30 µm. FITC-conjugated anticytokeratin (C) labeling indicative of the epithelial nature of CF-KM4 cells. FITC-conjugated anti-SV40 (D) labeling was detected exclusively in the nuclei of all observed CF-KM4 cells. Bars (for C and D) = 10 µm.

From the 12 clones we originally obtained (named CF-KM1 to CF-KM12) we selected the transformed CF-KM4 cell line presenting growth and epithelial characteristics, as deduced from the abovementioned features, similar to those of the genuine CF-HTG cells. The isolation and culture of CF-HTG cells have already been described in detail in a previous work (7), and we demonstrated the relatively short lifespan of the culture of these cells. Indeed, the effective passaging of these CF-HTG cells more than five or six times was, we found, impossible. In contrast, to date, we have been able to passage the CF-KM4 cells more than 20 times without any modification to their growth or secretory characteristics. All of the results we present here were obtained during the tenth passage and are completely identical to those of the fourth and also the seventeenth; and as observed for the non-CF-HTG cell line MM-39 (18), CF-KM4 cells did not undergo a "crisis period" between passages 10 and 20.

CFTR Transgene after Ad-CFTR Transfection

To visualize adenovirus-mediated CFTR gene transfer in the CF-KM4 cell line, we looked for the presence of the CFTR transgene by PCR analysis using primers spanning the F508 mutation of the gene. Used as controls, DNA from the normal HTG cell line MM-39 as well as DNA extracted from a person heterozygous for the F508 mutation were also amplified and run into the polyacrylamide gel. The ethidium bromide-stained PAGE of the different PCR products is shown in Figure 2. The 97-base pair (bp) and 94-bp bands correspond to the normal and F508 PCR products, respectively. The additional bands are heteroduplex forms due to competitive hybridization of the mutant and the normal trends toward the end of the PCR reaction. Whereas in MM-39 cells PCR reaction shows the presence of the normal form of CFTR, in CF-KM4 cells only the F508-mutated form is present. After adenovirus-mediated CFTR gene transfer, the 97- and 94-bp bands are observed as well as the heteroduplex forms, indicating that both the normal and the F508-mutated genes are present in the transfected cells.

View larger version (49K): [in this window] [in a new window]   Figure 2.   PCR of CFTR before and after CFTR gene transfer in CF-KM4 cells. Lane 1: Molecular-weight marker. Lane 2: An individual heterozygous for the F508 mutation is presented for comparison. Note the presence of both nonmutated (97 bp) and mutated (94 bp) amplificates as well as higher-mass heteroduplex forms. Lane 3: MM-39 cells present only the nonmutated CFTR form. Lane 4: The transformed CF-KM4 cells have conserved the F508/F508 mutation. Lane 5: After adenovirus-mediated CFTR gene transfer in CF-KM4 cells, both the original mutated and the transferred normal CFTR genes are observed.

Electrophysiologic Characterization of CF-KM4 Cells before and after Transfection with Ad-CFTR

Primary cultures of HTG cells highly express CFTR (8), which is detectable by its low-conductance chloride channel activity (9). To assess whether transformation with SV-40 may have affected CFTR function, we first performed cell-attached patch-clamp experiments to characterize the cAMP-activated CFTR chloride channel in the transformed non-CF MM-39 cells.

In control patch-clamp experiments with 150 mM NaCl in the pipettethat is, in the absence of agonists of the cAMP pathwayno spontaneous cell-attached channel activity was recorded (n = 5). The addition to the bath of a cAMP cocktail (10 µM forskolin plus 250 µM IBMX) caused the activation of previously silent multiple CFTR-like channels (four out of five experiments). Furthermore, in cells incubated with the cAMP cocktail, spontaneous channel activity was recorded in nine out of 10 experiments (Figure 3A1). Under both conditions, the currents reversed at 0 mV (Figure 3C). Two sets of experiments were then performed to ascertain the chloride selectivity of the channels activated by cAMP agonists. With 150 mM choline chloride in the pipette, similar CFTR-like channel activity was recorded in cAMP-treated MM-39 cells (two out of three experiments). The reversal potential was close to 0 mV (n = 2). With 40 mM NaCl and 110 mM sodium gluconate in the pipette, channel activity that reversed at 28 ± 3 mV (n = 3) was detected in cAMP-treated MM-39 cells (not shown). Together, these experiments demonstrate the chloride selectivity of the channels activated in cAMP-treated MM-39 cells. For comparison, Figures 3B and 3C show a representative cell-attached recording of CFTR channels from a CHO cell stably expressing CFTR, in the presence of the cAMP cocktail in the bath. The current-voltage relationship and unitary conductance cAMP-activated chloride channels in MM-39 cells (9.2 ± 2.1 pS, n = 13) were similar to those observed with CHO cells stably expressing CFTR (7 ± 0.8 pS, n = 4; Figure 3C) and to those already described for the parental HTG cells (9) or other preparations (21). Therefore, we concluded that in MM-39 cells the chloride channels activated on-cell by cAMP can be identified as CFTR.

View larger version (25K): [in this window] [in a new window]   Figure 3.   (A and B) Representative cell-attached patch-clamp recordings at various patch potentials (as indicated), showing the activity of CFTR channels only in MM-39 (A1), CF-KM4 cells transfected with Ad-CFTR (A4), and CHO cells stably expressing CFTR (B). In original CF-KM4 cells (A2) and in CF-KM4 cells transfected with Ad--gal (A3), no channel activity was recorded. Bath: 150 mM NaCl-rich solution containing 10 µM forskolin and 250 µM IBMX. Calibration bars: vertical, 1 pA (A1-A4), 0.5 pA (B); horizontal, 250 ms (A1-A4), 750 ms (B). (C) Current-voltage relationship of cAMP-activated CFTR channels in the cell-attached patch-clamp configuration as indicated.

We thereafter performed cell-attached patch-clamp experiments to characterize the cAMP-activated chloride channel in CF-KM4 and CF-KM4 transfected with either Ad--gal or with Ad-CFTR. As for MM-39 cells, in all control patch-clamp experiments no spontaneous cell- attached channel activity was recorded in the absence of cAMP agonists (n = 9, pooled data). In the presence of the cAMP cocktail, opening of CFTR channels was consistently observed only in the CF-KM4 cells transfected with Ad-CFTR (Figure 3A4) but not in CF-KM4 cells (Figure 3A2) or CF-KM4 cells transfected with Ad--gal (Figure 3A3). The chloride channels activated in CF-KM4 cells transfected with Ad-CFTR have a low unitary conductance of 9.2 ± 1.5 pS (n = 7, Figure 3C). As shown in Figure 3C, the I/V relationship was linear and very similar to that measured using MM39 cells or transfected CHO cells.

Although we observed the activity of outwardly rectifying chloride channel in excised membrane patches in all the cell types tested here, including transfected CHO cells, no other channel activity (except CFTR) was recorded in cell-attached patch-clamp configurations. Especially, these large conductance outwardly rectifying chloride channel (ORCC)-like channels were not observed in cell-attached configuration, even in cAMP-treated cells (not shown). These observations further support our conclusion that CFTR was the only ionic channel activated by the cAMP pathway in MM-39 and transfected cells. These data demonstrate that CFTR gene transfer was successful and that the CFTR protein derived from the transgene had cAMP-dependent chloride channel activities and biophysical properties similar to those from the homologous non-CF cells (i.e., MM-39 cells and HTG cells; [9]) or from the well-established CHO cells stably expressing CFTR ([21] and Figure 3B).

Pharmacologic Stimulation of the CF-KM4 Cells before and after Transfection with Ad-CFTR

We previously showed that the SV40-transformed MM-39 cells respond to muscarinic (carbachol), adrenergic (isoproterenol), and nucleotidic (ATP) agonists by increases in the secretion of the specific secretory marker SLPI (18). The amplitudes of these increases at the optimal concentration of agonists (100 µM) are reported in Figure 4A for comparison. They were, respectively, 75 ± 5, 64 ± 12, and 82 ± 10%. In contrast, CF-KM4 cells failed to be stimulated by the cholinergic (carbachol) or the -adrenergic (isoproterenol) agonists. However, response to ATP was conserved (Figure 4B). The mean effective dose of the action of ATP deduced from the dose-response curves (data not shown) was 3 µM, a value similar to that found in normal HTG cells (25).

View larger version (19K): [in this window] [in a new window]   Figure 4.   Responses of CF-KM4 cells to secretagogues before and after CFTR gene transfer. Data are expressed as SLPI S.R. (SLPI secreted in the assays/SLPI secreted in the unstimulated experiment). Each value represents the mean ± SD from quadruplicates. For each experiment, secretions of SLPI by MM-39 cells in response to 100 µM of carbachol (Carb.), isoproterenol (Isop.), or ATP are presented. (A) Responses of MM-39 cells shown for comparison. (B) CF-KM4 cells are not responsive to isoproterenol and carbachol but are responsive to ATP. (C) Adenovirus-mediated -galactosidase gene transfer did not change the response features of CF-KM4 cells to secretagogues. (D) Adenovirus-mediated CFTR gene transfer restored the ability of CF-KM4 cells to respond to carbachol and isoproterenol.

In Figure 4C are the responses to the same agonists of CF-KM4 cells transfected with Ad--gal. Their secretory pattern was found to be similar to that of nontransfected CF-KM4 cells. In contrast, after adenovirus-mediated CFTR gene transfer, a correction of the inability to respond to carbachol and to isoproterenol was observed (Figure 4D). The corrected responses, however, reached higher amplitudes (about 2-fold more) than those of the MM-39 cells. Responses to ATP were not modified.

Of all the agonists tested at high concentrations (1 mM), none significantly altered basal release of LDH from CF-KM4 cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this work we have presented a transformed cell line of CF tracheal gland cells, CF-KM4, that has retained stable and epithelial characteristics similar to the original parental cells. This cell line also constitutively produced SLPI as the tracheal gland serous cells in primary culture (7) and in vivo (26). Furthermore, as is also the case with the parental cells, CF-KM4 cells are not responsive to adrenergic and cholinergic agonists. Consequently, CF-KM4 cells may represent an interesting tool for large-scale study of some pathophysiologic features of CF.

Several cell lines deriving from CF nasal explants or CF primary culture of bronchial cells from the surface epithelium have been developed because of their greater growth potential than primary cultures, giving increased cellular material to study (27). However, it is difficult to acknowledge these studies because the precise phenotype of these cell lines had never been described; that is, they do not appear to be of goblet-cell or ciliated-cell nature (both corresponding to over 90% of the total amount of surface epithelial cells). Therefore, their undetermined nature, or perhaps their insufficient state of differentiation, render them poorly suitable for examining abnormalities in the secretory dysfunction related to CFTR.

To carry out accurate investigations about the physiologic properties of CFTR at the molecular and cellular levels, we therefore focused on developing a CF respiratory cell line from submucosal gland serous cells because (1) they are the cells in which CFTR is expressed at the highest level in the human bronchus (2), and (2) it seemed necessary to establish a cell line with a defined phenotype and with clear characteristics. CF-KM4 cells after transformation have retained similar properties to those of the parental cells, namely, presence of the F508/F508 mutated CFTR genes, an absence of cAMP-dependent chloride channels, and also an absence of a responsiveness to adrenergic and cholinergic agonists by an increase in the secretion of the specific serous-cell marker SLPI. These particular and CF-specific properties might not be a consequence of transformation because introduction of a normal CFTR gene into the CF-KM4 cells corrected the cAMP-dependent chloride channel activity and the defective responsiveness to secretagogues to a level comparable with that observed with the normal homologous cells. Thus, transformation may not have changed the typical CF features of these cells.

Numerous data about CFTR gene transfer concerned pulmonary surface epithelial cells and were focused principally on the correction of the chloride transport defect, a characteristic feature of CF. However, most of this data is still conflicting. Zabner and colleagues have shown that low doses of adenovirus vectors carrying CFTR cDNA can correct defective chloride transport in culture human CF airway epithelial cells (30). On the other hand, inefficiency of gene transfer vector to CF airway epithelia of mice and humans was observed by Grubb and associates (17). It was suggested that this difference may disparately reflect the susceptibility to adenoviral transfection of the epithelial cell types studied in vivo (of columnar phenotype) and in vitro (of basal cell-like phenotype). Again, these discrepancies pinpoint all the care that must be taken when interpreting these results according to the model used.

In CF-KM4 cells, adenovirus-mediated CFTR gene transfer led to correct expression of the transgene as we observed the CFTR-associated chloride channel activity in the transfected cells. The properties of the channel, cAMP dependency and low conductance, were those expected for CFTR and were similar to the ones found in the non-CF homologous cells: the normal HTG cells (9) and the transformed MM-39 cells, the Calu-3 cell line thought to be also glandular serous cells (31), as well as the ones sufficiently described in the literature (21). This transfection was also shown to lead to correction of specific defects of CF cells, namely, an absence of chloride transport and of -adrenergic and cholinergic stimulation of secretion. This potential of correction of CF defects by adenovirus-mediated CFTR gene transfer that we showed in gland cells, together with the study of Pilewski and coworkers, who showed a high efficiency of transfection in tracheal glands using adenovirus-mediated -galactosidase gene transfer in xenografts (32), argue in favor of a possible use of these recombinant adenoviral vectors for gene therapy.

Besides the defect in chloride secretion by CF epithelial cells, CFTR is also thought to be implicated in macromolecule secretion, and there is increasing evidence that a defect in submucosal gland secretion contributes to the airway pathology of CF. The cAMP-dependent secretory process is indeed lost in the cAMP-mediated fluid secretion in CF (33). One of the consequences is that it may then significantly reduce the water content of gland secretions. The resulting change in viscosity would contribute to the accumulation of airway mucus that is a characteristic of this disease. -Adrenergic response to agonists, which increases cAMP level, has been shown to be defective not only for secretion of chloride but also for secretion of macromolecules. McPherson and colleagues (34) have shown a defective -adrenergic response in the secretion of amylase and of glycoconjuguates by CF submandibular acinar cells, and evidence for a direct implication of CFTR on mucin secretion was established when CF-submandibular acinar cells were treated with antibody directed against CFTR (35). We showed previously that, in contrast to the normal cells, primary culture of CF tracheal gland cells was insensitive to neurotransmitters that may act through adrenergic and cholinergic receptors for their secretion of the specific secretory marker SLPI (7). CF-KM4 were also found here to be unresponsive to adrenergic and cholinergic agonists. This defective cholinergic and adrenergic receptor-dependent secretion was corrected after CFTR gene transfer but not after -galactosidase gene transfer. These results are in agreement with those reported by Mergey and associates (36), who stated that gene transfer to CF fetus cell lines restored defective cAMP-dependent secretion not only of chloride ions but also of glycoconjugates. It has been shown that the second messenger systems do not appear to be modified in CF cells, and it was therefore suggested that an as-yet-unidentified system subsequent to second messengers may be involved in the CF defect.

Moreover, one question that consequently remains is why CFTR may interact with the adrenergic and cholinergic receptor activation and not with the purinergic receptor functioning. This difference might indicate either a difference in second-messenger systems involved by this receptor, or of systems distant to second messengers that might be specific for the purinoceptor.

An actual and troublesome side effect of adenovirus-mediated gene transfer is the immune response by the host (37, 38). The cotton-rat model (39) confirmed the role of cellular immunity in the biology of recombinant adenovirus-mediated gene therapy in the lung and suggested that modifications in the design of recombinant adenoviruses will be useful in improving the potential of this technology in gene therapy of CF, such as high levels of transgene expression, more stable expression of the transgene, and less expression of the remaining early and late gene of the adenovirus. When tracheal gland cells were placed in a proinflammatory situation, they were shown to acquire a CF-like phenotype (40). Thus, in the present study, if adenovirus-mediated gene transfer may have induced inflammatory reactions, the corrected CF cells would have shifted to a CF-like phenotype and the gain from the transfer would have been lost. Because a clear responsiveness to secretagogues was observed after gene transfer, it is therefore unlikely that the current constructions we used induced inflammatory-like reactions by the cells. Although this observation is interesting, it remains speculative and additional work must be designed and carried out to clear this crucial point. This appears important to us because HTG cells were recently shown to participate actively in the cytokine network of the lung (41) and may be able to modulate the inflammatory process of the bronchotracheal tree.

In conclusion, this is the first demonstration showing the effectiveness of CF tracheal gland cells to be corrected by adenovirus-mediated CFTR gene transfer both in chloride and also in protein secretion. Because tracheal gland serous cells are a major site of CFTR expression in the human bronchus, the newly established CF-KM4 cell line can be considered an appropriate model to test at the cellular and molecular level the effects of gene transfer. This cell line can therefore also be helpful for designing and testing the newer generations of adenovirus vectors for gene therapy of CF.