Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-gamma

Luis J. V. Galietta1, Chiara Folli1, Carla Marchetti2, Luca Romano3, Daniela Carpani4, Massimo Conese4, and Olga Zegarra-Moran1

1 Laboratorio di Genetica Molecolare and 3 Clinica Pediatrica, Istituto Giannina Gaslini, 16148 Genoa; 2 Istituto di Cibernetica e Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa; and 4 Telethon Institute for Gene Therapy, Ospedale San Raffaele, 20132 Milan, Italy


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human bronchial epithelial cells were treated in vitro with interferon-gamma or tumor necrosis factor-alpha to assess their effect on transepithelial ion transport. Short-circuit current measurements revealed that Na+ absorption was markedly inhibited by interferon-gamma (10-1,000 U/ml). The cystic fibrosis transmembrane conductance regulator was also downregulated by interferon-gamma as evident at the protein level and by the decrease in the cAMP-dependent current. On the other hand, interferon-gamma caused an increase of the current elicited by apical UTP application, which is due to the activity of Ca2+-dependent Cl- channels. Tumor necrosis factor-alpha caused few changes in ion transport. Transepithelial fluid transport was measured in normal and cystic fibrosis cells. At rest, both types of cells showed an amiloride-sensitive fluid absorption that was inhibited by interferon-gamma but not by tumor necrosis factor-alpha . Our results show that interferon-gamma alters the transepithelial ion transport of cultured bronchial cells. This effect may change the ion composition and/or volume of periciliary fluid.

airway surface fluid; sodium absorption; cystic fibrosis; airway epithelium; chloride transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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THE THIN LAYER OF FLUID that covers the surface of the airways plays an important protective role. Inhaled particles and bacteria are trapped in the mucous layer covering the airway surface fluid (ASF) and are propelled to the oropharynx by the beating of cilia. Bacteria in the ASF are also neutralized by substances like lysozyme, lactoferrin, and the recently discovered defensins (4, 17, 37). The volume and ion composition of the ASF are the result of the balance between absorption and secretion of ions and water. Transepithelial ion transport is based on the activity of specific ion channels and transporters. Water movement across airway epithelium may be driven by osmotic forces as well as by yet to be determined humoral or intracellular mechanisms. Absorption is mainly controlled by the activity of the amiloride-sensitive epithelial Na+ channel (ENaC) (10). Indeed, Na+ absorption, which is followed by Cl- through paracellular or transcellular pathways, drives water from the apical to the basolateral side. The importance of this process is confirmed by the finding that mice with nonfunctional Na+ channels die at birth because they are unable to remove water from the airways (22). Conversely, secretion on the surface epithelium or in the lumen of submucosal glands is due to Cl- exit through ion channels placed on the apical membrane. In cystic fibrosis (CF), a cAMP-regulated anion channel termed the CF transmembrane conductance regulator (CFTR) is mutated, and, therefore, Cl- transport is impaired (41). CF is also characterized by increased activity of the ENaC. These alterations may produce dehydration of mucus secretions in the airways, thus causing airway colonization by pathogenic bacteria (28). An alternative hypothesis proposes that the altered ion concentration in the ASF of CF patients might impair the bactericidal activity of defensins (44).

Under inflammatory conditions, the airway epithelium is exposed to a series of cytokines and soluble mediators that are secreted by lymphocytes, macrophages, and epithelial cells themselves (26, 37). These mediators, which include interferon-gamma (IFN-gamma ) and tumor necrosis factor-alpha (TNF-alpha ), are involved in a complex cascade of events such as expression of inducible nitric oxide synthase (iNOS) (2), induction of intercellular cell adhesion molecule-1 (24), secretion of interleukin-8 (6), and expression of cyclooxygenase (40). It has been shown in intestinal epithelial cells that IFN-gamma and TNF-alpha are able to downregulate CFTR expression (7, 31). These results led us to hypothesize that proinflammatory conditions are also able to affect ion transport in the airway epithelium. Accordingly, we have investigated the effect of IFN-gamma and TNF-alpha on the activity of Na+ and Cl- channels in bronchial epithelial cells. We have also investigated the effect of these stimuli in CF cells in which the transepithelial ion transport is already compromised. Our results show that IFN-gamma , but not TNF-alpha , causes profound changes in ion transport in both normal and CF cells, resulting in an altered fluid transport.


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Cell culture. Human bronchial epithelial cells were cultured as previously described (16). Briefly, cells were detached from the bronchi after overnight incubation at 4°C with protease XIV. The cells were obtained from lung resections (non-CF subjects) or lung transplants (CF patients). After detachment, the cells were plated on petri dishes or culture flasks and cultured for three to six passages in a serum-free culture medium made by mixing LHC9 and RPMI 1640 media (1:1). Subsequently, the cells were plated at high density on Transwell or Snapwell permeable supports (Corning Costar) that had diameters of 24 and 12 mm, respectively. Under these conditions, the medium was DMEM-Ham's F-12 medium plus 2% fetal clone II serum (HyClone) and various hormones including 20 nM hydrocortisone (HC) (16). Where indicated, HC was removed. To monitor epithelial differentiation, transepithelial resistance was measured daily with an epithelial volt-ohmmeter (Millipore-ERS, Millipore) with chopsticklike electrodes. After 4-8 days in complete medium, the bronchial monolayers developed a transepithelial resistance in the range 1,000-4,000 Omega  · cm2 depending on the day of culture and the type of support. Cells on Transwell cups usually showed a higher resistance. Removal of HC decreased the resistance by ~30%. After optimal differentiation, the resistance of CF cells was always higher than that of normal cells. The difference was significant (P < 0.01).

Except where indicated, IFN-gamma (10-1,000 U/ml) and TNF-alpha (40 ng/ml) were applied in the culture medium for 48 h before Ussing chamber experiments were performed. IFN-gamma application always caused an increase in the transepithelial resistance. For example, normal cells on day 8 on Snapwell cups developed a resistance of 1,228 ± 95 Omega  · cm2 (n = 32 monolayers) in complete medium and 3,468 ± 140 Omega  · cm2 with IFN-gamma (P < 0.01; n = 26 monolayers).

Ussing chamber experiments. After 6 (Transwell) or 8 days (Snapwell) in culture, the permeable supports were mounted in Ussing chamber-like systems: Trans-24 miniperfusion system (World Precision Instruments) for Transwell cups and Vertical Diffusion Chamber (Corning Costar) for Snapwell inserts. The apical and basolateral chambers were filled with a Krebs bicarbonate (KB) solution that contained (in mM) 126 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose. The solution was kept in both chambers at 37°C and bubbled with 5% CO2-95% air. The transepithelial potential difference was short-circuited with a voltage clamp (558-C5, Department of Bioengineering, University of Iowa, Iowa City, IA) connected to the apical and basolateral chambers via Ag-AgCl electrodes and agar bridges. The potential difference and fluid resistance between potential-sensing electrodes were compensated for.

After the voltage-clamp condition was established, amiloride was applied to the apical solution to measure the fraction of the basal short-circuit current due to ENaC activity. Amiloride-treated epithelia were then stimulated with apical and basolateral 8-(4-chlorophenylthio) (CPT)-cAMP or apical UTP to determine the corresponding effects. In some experiments, UTP was given after CPT-cAMP. The net effect of UTP in the presence of CPT-cAMP was not statistically different from that obtained in its absence. Therefore, the data obtained with UTP in both conditions were pooled. The absolute effects of IFN-gamma and TNF-alpha on transepithelial currents were measured on Transwell supports. The time course and dose response of cytokines and the effect of channel blockers were studied on Snapwell cups. After normalization for the different surfaces, the amiloride-sensitive (IAmil) and the UTP-dependent (IUTP) currents were not quantitatively different in the two types of supports. However, the cAMP-dependent current (IcAMP) was larger in Snapwell cups.

In some experiments, the basolateral membrane was permeabilized with amphotericin B (250 µg/ml). Membrane permeabilization was assessed by two criteria: 1) progressive decrease in the transepithelial potential measured in open-circuit conditions and 2) progressive decrease in epithelial resistance measured in short-circuit conditions by applying short voltage pulses. When permeabilization reached a stable level, we applied a transepithelial potential of +10 mV to provide a driving force for ion transport.

Immunodetection of CFTR. Human bronchial epithelial cells were grown for 6 days on Transwell filters, detached, and lysed in a buffer containing 20 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitors. Equal amounts of protein lysates (1 mg/sample) were immunoprecipitated with anti-CFTR monoclonal antibody MATG 1105 (kindly provided by Dr. A. Pavirani, Transgene, Strasbourg, France) directed against the R domain of CFTR. The antibody (1 µg/sample) was incubated for 1 h at 4°C with the cell lysate and precipitated with protein G Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Immunoprecipitated proteins were then phosphorylated for 1 h at 30°C by the addition of 10 µCi of [gamma -32P]ATP (3,000 Ci/mmol; NEN Life Science, Boston, MA) with 5 U/sample of catalytic subunit of protein kinase A (Sigma) and separated on 6% SDS-PAGE. Controls included HT-29 cells, which have been previously shown to express the CFTR protein (45), and A549 cells, which do not express the endogenous CFTR (32).

Measurements of intracellular Ca2+. Optical measurements of internal Ca2+ were performed as previously described (25). Epithelial monolayers in Snapwell cups were incubated with 5 µM fura 2-AM for 30 min at 37°C in a standard physiological saline containing (in mM) 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, with pH adjusted to 7.4 with NaOH. The cells were then washed several times with the same solution in the absence of the dye at room temperature. The specimen was then mounted on the stage of an inverted microscope (IM35, Zeiss), and the apical side was continually superfused with solutions fed by gravity. The cells were viewed through a ×100 Nikon Fluor objective and illuminated by a xenon lamp (PTI, Brunswick, NJ) equipped with a rotating wheel with six interference filters, four filters centered at 340 nm and two centered at 380 nm. The emissions relative to each series of filters were mediated, and the ratio (R) of the 340- to 380-nm emissions (E340/E380) was calculated. Internal Ca2+ concentration ([Ca2+]i) was calculated according to the function [Ca2+]i = beta KD[(R - Rmin)/(Rmax - R)], where R = E340/E380, Rmin = E340/E380 in 0 Ca2+, Rmax= E340/E380 in Ca2+ saturated solution, beta  = E380(0 Ca2+)/E380(saturated Ca2+), and KD is the dissociation constant of the dye (140 nM) (20). To obtain the parameter values, after each experiment, the cells were incubated in 10 µM 4-bromo calcium ionophore A-23187 for 20-40 min in a 0-Ca2+ bath (0 Ca2+ added plus 1 mM EGTA), then superfused with the saturated Ca2+ solution. At the end of this procedure, 5 mM MnCl2 was added to the bath to quench the fluorescence produced by the dye and determine the autofluorescence values. Background fluorescence determined in this way differed by <5% from the fluorescence measured in similar untreated cultures.

NO and NOS determination. The concentration of nitrite, a stable product of nitric oxide (NO), in the medium of cell cultures was determined with the Griess reaction (18). The NO synthase (NOS) enzymatic activity in cellular extracts was assessed by measuring the conversion of L-arginine to L-citrulline with a commercial kit (Stratagene, La Jolla, CA). CaCl2 was included in the reaction buffer to allow detection of both constitutive NOS and iNOS. Enzymatic activity is expressed as picomoles of arginine converted in 1 h. Activity was normalized for the amount of protein in the cellular extract.

Transepithelial fluid transport. The cells were plated on Transwell cups and cultured with complete serum-containing medium as explained in Cell culture. After 5 days, the apical medium was removed, and the apical side was washed twice with KB solution and then with 1 ml of KB solution containing [3H]inulin (1 µCi/ml). This solution was discarded and replaced with another 220 µl of the same radioactive solution. A 20-µl aliquot was collected immediately from the apical side to determine the radioactivity at time 0. The apical solution was then covered with 1 ml of mineral oil to prevent evaporation. In part of the experiments, amiloride (10 µM) was also included in the inulin-containing solution. The cells were kept in the incubator for 24 h with the complete culture medium on the basolateral side. After this time, the apical aqueous fluid was removed, and the radioactivity was determined. Inulin is poorly permeable through tight junctions. In fact, <3% of apical inulin passed to the basolateral side in 24 h. Thus we assumed that changes in inulin concentration should reflect the transport of fluid across the epithelial monolayer. We also directly measured the volume of the aqueous fluid recovered from the apical side. These values were consistent with those calculated from the inulin concentration.

Statistics. Data are presented as representative traces or as arithmetic means ± SE. Unpaired groups of data were compared with Student's t-test to assess significance.


    RESULTS
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When mounted in Ussing chambers, human normal bronchial epithelia showed a short-circuit current that was largely inhibited by 10 µM amiloride (Fig. 1A). The mean amplitude of the IAmil, which reflects activity of the ENaC, was 5.05 ± 0.36 µA/cm2 (n = 27 monolayers). After incubation of bronchial cells with 1,000 U/ml of IFN-gamma for 48 h, IAmil decreased to 1.93 ± 0.35 µA/cm2 (P < 0.001; n = 11 monolayers; Fig. 1, B and C). The medium utilized for the culture of polarized bronchial cells contained 20 nM HC (16). Because inflammatory stimuli are often antagonized by glucocorticoids (14, 15, 29), we wanted to assess the effectiveness of IFN-gamma in the absence of this hormone. Removal of HC from the culture medium caused a strong reduction in IAmil (Fig. 1C). This result was not unexpected because ENaC is regulated by glucocorticoids in the lung (11). Under these conditions, treatment with IFN-gamma almost completely abolished the residual IAmil (from 0.95 ± 0.14 to 0.09 ± 0.03 µA/cm2; P < 0.001). Similar results were also obtained with bronchial epithelial cells from a CF patient (Fig. 1D). Indeed, IFN-gamma downregulated IAmil in CF cells in either the presence or absence of HC. The size of IAmil in normal and CF cells was not statistically different. We tested also TNF-alpha (40 ng/ml). This cytokine inhibited IAmil only in normal cells grown in the absence of HC (Fig. 1, C and D).


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Fig. 1.   A and B: effect of amiloride in control and interferon (IFN)-gamma -treated cells, respectively, on short-circuit current (Isc). Data are from 2 representative experiments on normal cells. t, Time. C and D: effect of IFN-gamma and tumor necrosis factor (TNF)-alpha on amiloride-sensitive current (IAmil) in normal and CF cells, respectively. Cells were treated with IFN-gamma (1,000 U/ml) or TNF-alpha (40 ng/ml) in presence (+) and absence (-) of hydrocortisone (HC; 20 nM). Values are means ± SE of amount of Isc blocked by amiloride in 10-29 experiments. Significant inhibition by IFN-gamma or TNF-alpha : ** P < 0.01; *** P < 0.001.

The activity of CFTR was determined by looking at the response to a membrane-permeable cAMP analog. On application of CPT-cAMP (500 µM), the short-circuit current of non-CF cells increased by 3.22 ± 0.25 µA/cm2 (n = 20 monolayers; Fig. 2A). After treatment with IFN-gamma for 48 h, the IcAMP was only 0.96 ± 0.14 µA/cm2 (P < 0.01; n = 5 monolayers; Fig. 2, B and C). Removal of HC did not affect these results. Indeed, IcAMP was unaltered in HC-free medium, and the inhibition caused by IFN-gamma was similar to that found in the presence of the hormone. TNF-alpha did not significantly change IcAMP in either condition (Fig. 2C). The current activated by CPT-cAMP was completely blocked by glibenclamide (500 µM) and was absent in CF cells (data not shown). These results are consistent with the assumption that IcAMP is due to CFTR activity. CFTR protein levels were assessed by immunoprecipitation and in vitro phosphorylation. This assay revealed the presence of three bands of 135, 145, and 165 kDa, which represent different stages of CFTR processing. The band with the highest molecular mass should correspond to the mature protein. Stimulation with IFN-gamma (1,000 U/ml) caused a significant decrease in the CFTR bands (Fig. 3). On the contrary, TNF-alpha caused an increase in CFTR protein.


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Fig. 2.   A and B: current activated by 8-(4-chlorophenylthio) (CPT)-cAMP in control and IFN-gamma -treated cells, respectively. Data are from 2 representative experiments on normal cells. C: inhibition of cAMP-dependent current (IcAMP) in cells treated with IFN-gamma (1,000 U/ml) or TNF-alpha (40 ng/ml). Experiments were done with and without HC. Values are means ± SE of current activated by CPT-cAMP from 4-20 experiments. Significant inhibition: ** P < 0.01; *** P < 0.001.



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Fig. 3.   Immunodetection of cystic fibrosis transmembrane conductance regulator (CFTR). Cell lysates from human bronchial (lanes 1-3), A549 (lane 4), and HT-29 (lane 5) cells were processed for immunoprecipitation and phosphorylation as described in METHODS. Human bronchial cells were untreated (lane 1) or treated with IFN-gamma (1,000 U/ml; lane 2) or TNF-alpha (40 ng/ml; lane 3). Nos. on left, molecular mass of different forms of CFTR. Band intensity was analyzed by densitometry. In untreated cells, bands at 135, 145, and 165 kDa correspond to 77,600, 16,900, and 10,316 densitometric units, respectively. IFN-gamma decreased the higher band to undetectable levels, whereas the other 2 were 50 and 57% of control level. TNF-alpha increased CFTR bands by 907, 449, and 426%, respectively. Similar data were obtained in 2 other experiments.

Airway epithelial cells possess Cl- channels other than CFTR (1, 27) that can be activated by various stimuli such as the application of extracellular UTP or ATP. These compounds act by binding to P2Y2 purinergic receptors and evoking an intracellular Ca2+ increase (27). We asked whether the activity of these channels was also affected by IFN-gamma incubation. Apical application of 100 µM UTP to bronchial cells resulted in a transient stimulation of short-circuit current (Fig. 4A). Incubation with IFN-gamma for 48 h in the absence of HC resulted in an increase in the peak of the IUTP both in non-CF and CF bronchial epithelia (2.3-fold and 5-fold, respectively; Fig. 4). The difference between normal and CF cells was significant (P < 0.001). IFN-gamma also induced a prolongation of the UTP response as shown by the current measured after 15 min of UTP application (Fig. 4, C and D). After 30 min, the current in IFN-gamma -treated epithelia was still elevated (3.05 ± 0.13 µA/cm2; n = 5 monolayers), whereas in untreated cells, it was already decreased to prestimulation levels (data not shown). TNF-alpha significantly increased the IUTP peak only in CF cells (Fig. 4D). The upregulation of the IUTP by IFN-gamma was not modified by HC (data not shown). On the contrary, the small TNF-alpha effect observed in CF epithelia was inhibited by HC.


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Fig. 4.   A and B: typical response to apical UTP in control and IFN-gamma -treated cells, respectively. Data are from 2 representative experiments on CF cells. C and D: upregulation of UTP-dependent current (IUTP) in normal and CF cells, respectively, treated with IFN-gamma (1,000 U/ml) or TNF-alpha (40 ng/ml) in absence of HC. Current was measured at the peak [I(peak)] and 15 min [I(15')] after UTP application. Values are means ± SE from 14-23 experiments. Significant effect: ** P < 0.01; *** P < 0.001. Peak of current activated by UTP after IFN-gamma treatment was higher in CF cells with respect to normal cells (P < 0.01).

IFN-gamma (1,000 U/ml) was applied for 3, 6, 24, and 48 h to determine the time course of IAmil, IcAMP, and IUTP changes. As shown in Fig. 5, the downregulation of ENaC- and CFTR-dependent currents was already observed after 24 h of incubation. At this time, the IUTP began to be more sustained (data not shown). Conversely, the IUTP peak was significantly upregulated only after 48 h (Fig. 5).


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Fig. 5.   Time course of changes in IAmil, IcAMP, and IUTP after 3, 6, 24, and 48 h of treatment with IFN-gamma (1,000 U/ml). Dotted line, time course of untreated cells. Data were normalized to current of untreated cells. Values are means ± SE from 3-5 experiments. IAmil and IcAMP were significantly downregulated at 24 and 48 h, whereas IUTP peak was significantly increased only at 48 h.

Lower concentrations of IFN-gamma were also tested, namely 10 and 100 U/ml. The dose-response relationship shows that IAmil is markedly reduced even at the lowest IFN-gamma concentration tested, whereas IcAMP and IUTP required 100 U/ml to obtain a maximal effect (Fig. 6).


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Fig. 6.   Dose dependence of IFN-gamma effect. Cytokine was applied for 48 h in absence of HC. Data were normalized to current in untreated cells. Values are means ± SE from 3 experiments.

To mimic the UTP effect, we stimulated bronchial cells with the Ca2+ ionophore ionomycin (Fig. 7, A and B). Also in this case, the currents were larger and more sustained in cells exposed to IFN-gamma . For example, in CF cells, the mean currents activated by ionomycin were 1.10 ± 0.37 (n = 3 monolayers) and 6.83 ± 0.31 (n = 3 monolayers) µA/cm2 in control and IFN-gamma -treated cells, respectively (P < 0.01).


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Fig. 7.   IFN-gamma effect on Ca2+-dependent transport. A, C, and E: representative experiments on untreated CF cells. B, D, and F: IFN-gamma -treated CF cells. A and B: response of Isc to apical ionomycin (1 µM). C and D: effect of apical UTP (100 µM) on intracellular Ca2+ concentration ([Ca2+]). E and F: Isc activated by UTP in bronchial cells after permeabilization of basolateral membrane with amphotericin B.

We measured the effect of apical UTP application (100 µM) on intracellular Ca2+ levels. There was a transient Ca2+ increase that lasted for 2-3 min. The resting levels of Ca2+ and the increase elicited with UTP were not significantly affected by the long-term stimulation with IFN-gamma (Fig. 7, C and D). In normal cells, the net increase in intracellular Ca2+ reached 164 ± 44 (n = 5 monolayers) and 107 ± 28 (n = 4 monolayers) nM for untreated and treated cells, respectively. This difference was not significant. In CF cells, the Ca2+ peak was 277 ± 53 nM (n = 3 monolayers) for control cells and 138 ± 25 nM (n = 3 monolayers) for IFN-gamma -treated cells (P > 0.05).

To remove the contribution of basolateral channels and transporters, we permeabilized the basolateral membrane with amphotericin B (Fig. 7, E and F). Under this condition, apical UTP evoked a peak current of 1.36 ± 0.32 µA/cm2 (n = 3 monolayers) in control cells and 7.14 ± 0.68 µA/cm2 after IFN-gamma incubation (P < 0.01). The response to UTP was more transient in permeabilized cells than in intact epithelia.

We asked whether the production of NO could be one of the mechanisms underlying IFN-gamma effects. Actually, it has been reported that NO is an inhibitor of ENaC in alveolar epithelium (21). To test this hypothesis, we measured nitrite concentration in the supernatant of normal bronchial cells. After 24 and 48 h of stimulation with IFN-gamma in the absence of HC, nitrite levels were very low and not increased with respect to untreated cells. Indeed, at 48 h, nitrite concentration was 1.1 ± 0.3 vs. 2.0 ± 0.6 µM in treated and nontreated normal cells, respectively (P > 0.05). The addition of HC to the culture medium did not change these results. CF cells also showed low nitrite levels. To confirm these data, we measured NOS activity in cellular extracts. The arginine-to-citrulline conversion rate was very low in nontreated normal cells (3.3 ± 2.7 pmol · mg-1 · h-1) and was not significantly changed by IFN-gamma treatment (1.7 ± 0.4 pmol · mg-1 · h-1). Positive controls were murine macrophages (ANA-1) stimulated with 1 µg/ml of LPS for 24 h and rat cerebellar extracts (362.4 ± 9.2 and 329.3 ± 68.3 pmol · mg-1 · h-1, respectively). To further investigate the possible involvement of NO, we treated the cells with N-nitro-L-arginine methyl ester (500 µM), an inhibitor of iNOS, for the entire period of IFN-gamma incubation. Under these conditions, the effect of IFN-gamma on IAmil, IcAMP, and IUTP was unaltered (data not shown). We also treated the bronchial monolayers acutely with NO donors, namely S-nitroso-N-acetylpenicillamine (200 µM) or spermine NONOate (200 µM), on both sides of the epithelium. The size of IAmil, IcAMP, and IUTP was not changed by exposure to NO (data not shown).

To measure fluid transport, a fixed volume of saline solution was applied to the apical side of epithelial cells. After 24 h, we measured the volume of the remaining fluid as explained in METHODS. In control conditions, the apical fluid was reduced by 24-35% (Fig. 8). This corresponds to a fluid absorption of 14.7 ± 1.1 and 10.1 ± 0.9 µl · cm-2 · 24 h-1 in normal and CF cells, respectively. This difference was significant (P < 0.05). Fluid absorption was significantly reduced by apical application of 10 µM amiloride in both types of cells (Fig. 8). However, in CF cells, this effect was less marked. When IFN-gamma was applied for 48 h, fluid absorption was significantly reduced to 5.4 ± 1.7 µl/cm2 in normal cells and 6.4 ± 1.2 µl/cm2 in CF cells (Fig. 8). Conversely, TNF-alpha did not modify fluid transport. We also measured fluid transport in the presence of apical UTP. In untreated epithelia, UTP caused a small and not significant fluid absorption reduction (17 and 19% in normal and CF cells, respectively). This effect was not significantly modified by IFN-gamma treatment (data not shown).


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Fig. 8.   Transepithelial fluid transport. Fluid absorption was measured in normal (N) and CF cells under resting conditions (control) or after treatment with IFN-gamma (1,000 U/ml) or TNF-a (40 ng/ml) for 48 h. Effect of apical amiloride (10 µM) is also included for comparison. Values are means ± SE from 5-29 experiments. Significant inhibition caused by treatment with respect to resting fluid absorption: * P < 0.05; ** P < 0.01. Resting fluid transport was significantly smaller (P < 0.05) in CF cells with respect to normal cells.


    DISCUSSION
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IFN-gamma and TNF-alpha are important mediators that modulate the local immune response against infective agents. IFN-gamma is secreted by T lymphocytes and is believed to be involved in the response to viral pulmonary infections (5, 30, 33, 38). IFN-gamma is not normally increased in CF patients (9), but it is presumable that it might increase in these subjects during respiratory viral infections. Local IFN-gamma levels also increase in the lungs after administration of viral and nonviral vectors used in gene therapy studies (34, 39). TNF-alpha is produced by activated macrophages and neutrophils and is elevated in CF (9) and other inflammatory conditions (26, 43).

The binding of IFN-gamma and TNF-alpha to their corresponding receptors elicits a complex cascade of events that is based mainly on the translocation to the cell nucleus of factors like nuclear factor-kappa B for TNF-alpha and signal transducers and activators of transcription for IFN-gamma (3, 8, 35) where they induce the transcription of specific genes. Previous studies from other laboratories (7, 31) showed that IFN-gamma and TNF-alpha downregulate the activity of CFTR in intestinal epithelial cells. We asked whether these cytokines modulate CFTR and possibly other ion transport mechanisms in airway epithelial cells. We have found that IFN-gamma decreases CFTR-related ion transport in bronchial epithelial cells. Immunodetection experiments indicate that this effect is due to the lowering of CFTR expression and not to more indirect mechanisms. We have also observed, for the first time, that IFN-gamma downregulates ENaC-mediated currents and upregulates the response to UTP, i.e., the Ca2+-dependent Cl- transport. This latter effect is a useful indication that IFN-gamma does not merely cause a general reduction in the ion transport ability in epithelial cells, but, rather, it has a more specific effect.

Inflammatory stimuli are often antagonized by glucocorticoids (14, 15, 29). Therefore, we removed HC from the culture medium in part of the experiments. We found that the IFN-gamma effects are largely HC insensitive.

To observe a significant effect on ion transport, IFN-gamma has to be applied for several hours. This relatively slow response suggests that it is based on gene expression changes that could involve the expression of the proteins forming the channels (i.e., ENaC subunits, CFTR, Ca2+-dependent Cl- channels) or the expression of regulatory proteins.

Our findings show for the first time that the Ca2+-dependent Cl- transport can be regulated by extracellular factors. Actually, both UTP- and ionomycin-dependent responses were similarly upregulated by IFN-gamma . This result suggests that IFN-gamma affects a process that is placed downstream of the intracellular Ca2+ elevation, thus excluding mechanisms based on upregulation of the nucleotide receptor or phospholipase C. Moreover, the experiments carried out with the fura 2 probe further demonstrate that IFN-gamma does not increase the amount of Ca2+ mobilized by UTP. We considered the possibility that IFN-gamma might indirectly increase Cl- transport by upregulating the activity of basolateral channels or transporters that provide the driving force for Cl- exit across the apical channels. However, UTP was still able to activate a current that was larger in IFN-gamma -treated cells when the basolateral membrane was permeabilized. This result suggests that IFN-gamma modulates the expression of Ca2+-dependent Cl- channels or the activity of regulatory mechanisms that control channel activity. It is known that Ca2+-dependent Cl- channels are inhibited by feedback mechanisms. This process seems to be due, at least in part, to the intracellular accumulation of D-myo-inositol 3,4,5,6-tetrakisphosphate (42). IFN-gamma could affect this or a similar process. Such a mechanism could be responsible for prolongation of the UTP response in IFN-gamma -treated cells.

In contrast to IFN-gamma , TNF-alpha did not produce dramatic changes in ion transport. This contrasts with immunodetection experiments in which CFTR protein levels were increased by this cytokine. The lack of a clear effect at the functional level could be due to the induction of inhibitory pathways (e.g., phosphatases) that lower CFTR-channel activity. The differences between IFN-gamma and TNF-alpha are not surprising because the two cytokines use different signaling pathways. TNF-alpha acts through the nuclear factor-kappa B transcription factor (3, 35), the activity of which can be antagonized by the glucocorticoid receptor (14, 15, 29). This fact may explain the ability of TNF-alpha to change IAmil and IUTP only in the absence of HC.

We have considered the possibility that IFN-gamma might affect ion transport by inducing NO synthesis because iNOS can be elicited by different cytokines and bacterial lipopolysaccharides (2). This hypothesis is based on the recent finding that NO inhibits Na+ absorption in alveolar epithelium (21). Therefore, we assumed that the possible induction of iNOS by IFN-gamma could similarly decrease IAmil in our cells and possibly also affect IcAMP and IUTP. Several lines of evidence seem to rule out this hypothesis. First, the treatment with IFN-gamma does not increase the nitrite concentration in the cell supernatant and does not induce NOS activity in cellular extracts. Second, the NOS inhibitor N-nitro-L-arginine methyl ester does not prevent the effect of IFN-gamma . Finally, cell stimulation with NO donors does not mimic the effect of IFN-gamma .

We compared the effects of IFN-gamma and TNF-alpha in normal and CF cells. This study is interesting insofar as it explores whether stimuli that mimic proinflammatory conditions are able to alter ion transport in CF patients and whether this alteration attenuates or further emphasizes the CF basic defect. We have found that IFN-gamma also downregulates Na+ absorption in CF cells. The effect seems to be quantitatively similar to that observed in normal cells. Regarding CFTR, we have used cells from a Delta F508 homozygote that have virtually no cAMP-dependent Cl--channel activity. However, it is presumable that IFN-gamma might also downregulate CFTR in those patients with milder mutations who have a residual channel activity, thus worsening the basic functional defect. The upregulation of IUTP is higher in CF cells. A higher activity of Ca2+-dependent Cl- channels has been also observed in CF mice (13, 19). A peculiarity of our observations is that ENaC activity was not higher in CF with respect to normal cells as evidenced by other investigators (12). Our findings may suggest that the upregulation of ENaC in CF is influenced by factors other than the simple interaction between ENaC and CFTR. These factors could be variably affected in vitro by cell culture conditions and in vivo by other unknown factors. Actually, it has been shown that the amiloride-sensitive Na+ transport in the tracheae of Delta F508 and CFTR knockout mice is lower than in normal animals (13, 23). Measurements of fluid transport showed that bronchial epithelia have a fluid absorption that is based on the amiloride-sensitive Na+ transport. IFN-gamma treatment significantly decreased fluid absorption in normal and CF cells as expected from the inhibition of Na+ currents in short-circuit conditions. In CF cells, the amount of amiloride-sensitive fluid transport was not elevated with respect to normal cells, confirming data obtained by others (36).

Two models are presently proposed to explain the physiological NaCl absorption and the pathogenesis of CF. In one model, Na+ goes through the ENaC and Cl- follows passively through the paracellular route (28). Because ENaC is hyperactive, dehydration of the apical epithelial surface might impair mucociliary clearance favoring airway infections. The other model postulates that rather than moving through the paracellular pathway, Cl- is transported through the CFTR (44). In CF epithelia, effective NaCl absorption would be impeded, and, consequently, periciliary fluid would have a high salt concentration that inactivates the defensins. The effect of IFN-gamma in vivo would be quite different depending on the model. In the former case, the inhibition of Na+ transport elicited by this cytokine would increase apical volume and, therefore, would facilitate mucociliary clearance. In the latter model, the inhibition of Na+ transport would further increase the apical salt concentration, thus inactivating the defensins and facilitating bacterial infection. Whatever the correct model is, the finding that ion transport in airway epithelia can be modulated by an extracellular mediator has interesting implications. First, it indicates that some inflammatory stimuli may change airway ion transport, leading to possible alterations in mucociliary clearance. Furthermore, assessment of the molecular mechanisms underlying the regulation of ENaC, CFTR, and other Cl- channels could lead to pharmacological tools useful in manipulating transepithelial ion transport. This could be of extreme importance in the treatment of CF and other chronic diseases of the airways.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the North American Cystic Fibrosis Foundation, Telethon Italy Grant E.593, and a grant from the Associazione Lombarda for Cystic Fibrosis.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. J. V. Galietta, Lab. di Genetica Molecolare, Istituto Giannina Gaslini, L.go G. Gaslini 5, 16148 Genoa, Italy (E-mail: galietta{at}unige.it).

Received 6 October 1999; accepted in final form 1 January 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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