Interferon-γ activates outwardly rectifying chloride channels in the human bronchial epithelial cell line BEAS-2B Ning Gea, Youichi Nakamuraa, Yutaka Nakayab, and Saburo Sonea aThird Department of Internal Medicine, and bDepartment of Nutrition, The University of Tokushima School of Medicine, Tokushima Japan Abstract: The mechanism of increased chloride currents by inflammatory cytokine, interferon-gamma (IFN-γ), was investigated in cultured a human bronchial epithelial cell line (BEAS-2B) using cell-attached and inside-out patch configurations. The channel sensitive to chloride ion was activated by forskolin, an activator of adenylate cyclase, or 100μM dibutyryl 5'-cyclic monophosphate in cell-attached configurations. The conductance of this channel was 40±4 pS in symmetrical 150 mM chloride solution between membrane potentials of 0 to +50 mV, and this channel was blocked by 500μM 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), suggesting that this channel was an outwardly rectifying chloride channel (ORCC). Treatment of 10-1000 U/ml IFN-γ for 3 hours, but not IFN-α, significantly increased channel activities of ORCC, and this activation was observed at least 24 hours after treatment. Erythromycin, a macrolide antibiotic, at a concentration of100μM inhibited the activation of ORCC induced by IFN-γ. The findings of the present study indicate that increased mucus secretion during inflammation might be partly due to activation of chloride permeability by cytokine and erythromycin might improve oversecretion of mucus from bronchial epithelium by blocking ORCC. J. Med. Invest. 48:97-101, 2001 Keywords:chloride channel;epithelial cell;outwardly rectifying chloride channel, cystic fibrosis transmembrane conductance regulator INTRODUCTION Infection and inflammation increases mucus production, but a large amount of sputum may cause air flow limitation and recurrent respiratory infection due to impaired mucociliary transport functions (1). The increased secretion of fluid is partly due to the increased permeability to chloride ions (2). Previous studies showed the presence of at least three types of chloride channels in epithelial cells, i.e. cystic fibrosis transmembrane conductance regulator (CFTR) (3, 4), outwardly rectifying chloride channels (ORCC) (5) and stretch-induced chloride channels (6). Transepithelial chloride fluxes are determined mainly by the activity of chloride channels, the ORCC and the CFTR, located in the apical membrane of airway epithelial cells (7). In cystic fibrosis, the CFTR is genetically defective, and transepithelial electrolyte transport in the airway epithelium becomes inadequate resulting in impaired mucociliary clearance and ultimately respiratory failure (8). Therefore, much attention has been paid to the CFTR. However, there have been few studies on ORCC and its characteristics have not been clarified completely. The present study aimed to clarify the ionic mechanism of increased chloride secretion by the inflammatory cytokine, interferon-gamma (IFN-γ). IFN-γ is a pleiotropic cytokine that has a large number of immunologic and non-immunologic functions, which may play an important role in respiratory pathological conditions. Cytokines are also known to modulate ionic currents (9-11). However, there have been no studies on the effect of cytokines, including IFN-γ on chloride channel activities of bronchial epithelial cells. The purpose of the present study was to evaluate the effects of IFN-γ on the ORCC, which participates in the transepithelial chloride fluxes. The results of the study might explain the mechanism of excess mucus secretion in various pathological conditions in airway tracts. MATERIALS AND METHODS Isolation and culture of human bronchial epithelial cells A human bronchial epithelial cell line (BEAS-2B) transformed with SV40 virus was purchased from the American Type Culture Collection (Rockville, MD). The cells at passages 43-45 were used for experiments. The cells were plated in tissue culture plates with 16 mm diameter wells coated with type 1 collagen gel (Vitrogen 100;Celtrix, Santa Clara, CA), and incubated in LHC9/RPMI-1640 medium with gentamycin at 37°C, under 5% CO2 in air. The medium was changed after 24 hr and then every other day. When the cells reached confluency, they were replated on new dishes with cover slips coated with type 1 collagen gel. Cells grown on the cover slips were used for experiments. At the same time, they were confirmed to be entirely epithelial cells by their positive staining by the avidin-biotin complex method (ABC kit ; VECTOR Lab., Burlingame, CA). No cells reacted with monoclonal murine anti-swine vimetin antibody (DAKO-Vimetin ; DAKO. Santa Barbara, CA), which was demonstrated to stain fibroblast IMR-90 cells provided by Tokyo Metropolitan Institute of Gerontology. Electrical recording The cover slips with cultured human bronchial epithelial cells (BEAS-2B) were transferred to an organ bath on an inverted microscope. Membrane currents were recorded in inside-out and cell-attached configurations with a patch clamp amplifier (EPC7 List Medical Electronics, Darmstat, Germany), as described by Hamill et al. (12). Patch electrodes were pulled from capillary glass tubes using a two-stage puller (PP-83, Narishige, Tokyo, Japan), coated with sylgard. The electrical resistance of the patch pipette was 5-7 MΩ for single channel recordings. Experiments were conducted at a temperature of 35-37°C. The patch electrodes were maneuvered to the cellular membrane surface, and, when contact was made, negative pressure was applied until a gigaohm-seal was formed. Measurements were filtered and stored on the hard disk of a personal computer for subsequent analysis. Solutions and chemical In cell-attached and inside-out patches, the pipette contained 150 mM NaCl, 1 mM MgCl2, 0.1 mM Ca2+ and 10 mM 3-(N-morpholino) propane sulfonic acid (MOPS) (Sigma Chemical Co. St. Louis, USA), pH 7.2. In inside-out patches, the bath contained a similar solution except for10-8 M Ca2+. The standard bath solution for the cell-attach patches was modified normal Tyrode's solution of the following composition (in mM);137 NaCl, 2.7KCl, 7.5 MOPS, 1.2 CaCl2, 1 MgCl2, 11.5-glucose, pH 7.2. To create a 50 mM concentration of chloride, equimolar chloride was replaced by Na2SO4, pH 7.2. Dibutyryl 5'-cyclic monophosphate, forskolin, and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) were purchased from Sigma Chemical Co. (St. Louis, USA). Data analysis The data acquisition and analysis of single-channel currents was conducted using pCLMP version 6.0 (Axon Instruments, Foster City, CA). The open probability (NP0) was determined from current amplitude histograms and was calculated using the following equation: NP0 = Σ (n x Pn) where n is the number of channels in the patch and Pn is the integrated channels opening. Values are presented as means ± SE. Paired and unpaired t tests were used to study significance between two groups. P<0.05was considered to be significant. RESULTS Characterization of the chloride channels In cell-attached configurations, un-stimulated epithelial cells did not show any current activities. When5'-cyclic monophosphate (db-cAMP), was added to the bathing solution, channel activities were observed in 8 of 10 cells (Fig. 1b). Similar findings were obtained using 100 μM forskolin (n=6). The open probability was increased from 0.01±0.02 to 0.22±0.10 (n=8, p<0.05). A similar current was observed in inside-out configurations without stimulation and Fig. 1c shows typical currents recorded and the I-V relation from inside-out patches. The slope conductance was 40±4 pS between a membrane potential of -0 and + 50mV with symmetrical 150 mM chloride solution (n=6).The reversal potential was 0 mV. Reducing the chloride concentration to one third by replacement with SO4 - caused the I-V relation to shift by -25 mV, suggesting that this channel was relatively selective to chloride. Similar findings were obtained when KCl and K-aspartate solution were used (not shown). Two types of cAMP sensitive chloride channels are reported, i.e. cystic fibrosis transmembrane conductance regulator (CFTR) and outwardly rectifying chloride channels (ORCC). To identify whether this channel is ORCC or CFTR, we tested the effect of 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), a chloride channel blocker sensitive to ORCC. The channel activities were inhibited by500 μM DIDS, from 0.42±0.08 to 0.02±0.05 (n=6, p<0.05, n=4). The above observations show that epithelial cells express a chloride channel, which is usually infrequent in the cell-attached recording configurations and can be activated by the formation of inside-out patches or when stimulated by cAMP. Effect of interferon-γ on epithelial chloride channels In cell-attached configurations application of IFN-γ (100U/ml) did not activate chloride channels, but when the cells were pretreated with 100 U/ml IFN-γ for more than 3 hours, channel activity was observed 6 in 12 cell-attached patches in the control solution (Fig. 2b) at a pipette potential of +50 mV (membrane potential = resting membrane potential minus 50 mV). The chloride channels are activated 3 hours after application of interferon-γ (from0.01±0.02 to 0.18±0.03, n=6 ; p<0.01), and this activation was observed more than 24 hours after application of IFN-γ. DIDS at a concentration of 500 μM also blocked this channel (Fig. 2c). Fig. 2d summarizes the dose-dependent activation of chloride channels by IFN-γ at 3 hours after application. We also tested the effects of other cytokines on chloride channels. IFN-α did not activate this channel (data not shown). Effect of erythromycin Erythromycin is reported to inhibit chloride secretion across canine tracheal epithelial cells. We studied the effect of erythromycin on single channel current of ORCC. Fig. 3 shows that 100 μM erythromycin inhibited completely channel activities induced by IFN-γ in inside-out patches (n=5). DISCUSSION We have shown that IFN-γ activated chloride channels in time- and dose-dependent manners in cultured human bronchial epithelial cells. The conductance of the channel was 40 pS and the channel activity was inhibited by DIDS, suggesting that this channel was ORCC. In addition, erythromycin could inhibit IFN-γ -induced channel activities. These findings indicated that ORCC might be related to the transepithelial ion flux during inflammation, and that IFN-γ may play an important role in production of mucus during inflammation. Chloride channels are present in the cell membranes of most cells, and a large variety of chloride channels have been documented (13). Chloride channels may play a role in complex cellular regulation including ionic secretion, cell volume control, and endocrine secretions. In the present study, chloride channels were activated spontaneously when the membrane patches were excised especially at depolarized potentials (14), but this was infrequent for the case of cell-attached patches. In agreement with these findings, in the present study, no channel activities were observed in cell-attached recordings in the absence of any reagent in the bath solution, although in contrast, more than half of the inside-out patches showed activities, suggesting the participation of cellular components or mechanisms which keep the channel closed. This channel is also activated by cAMP, suggesting that this channel is CFTR or ORCC. The conductance (40 pS) and sensitivities to DIDS strongly suggest that this channel is ORCC. Cytokines are known to modulate ionic channel currents. Colgan et al. (15) reported that chloride secretion, as measured by the epithelial short circuit current, was diminished as much as 70% by IL-4. Schlichter (16) found that IFN-α can increase the K+ current near the resting potential. In addition, they also found that IFN-α induced nonselective cationic channels. However, cytokine's effects on chloride channels have not yet been studied extensively. The present study firstly demonstrated that IFN-γ activated chloride channels in epithelial cells. IFN-α did not modulate the channel activities, suggesting that this action is specific to IFN-γ. The findings of the study indicated that ORCC was one of the regulators of various stimuli, including IFN-γ, and plays important functions in chloride secretion, although the mechanism of activating the chloride channel by IFN-γ was not examined in the present study. The findings of the present study also indicate that the inhibition of chloride channels might be essential for the treatment of chronic inflammation. The macrolide antibiotics, such as erythromycin, clarithromycin and roxythromycin, have been shown to be effective in the treatment of patients with acute bronchitis and chronic airway diseases including asthma and chronic bronchitis (17-20). The efficacy of antibiotics can be attributed not only to their antimicrobial activities but also to their immunomodulatory action on neutrophils and macrophages. In addition, many patients receiving erythromycin show a reduction in sputum volume and dryness of sputum, the mechanism of which may partly be associated with the direct inhibition of airway secretion by this drug (21). Recently, Ikeda et al. 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