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(Journal of Leukocyte Biology. 2002;71:54-64.)
© 2002 by Society for Leukocyte Biology

Expression and functional characterization of CFTR in mast cells

M. Kulka, M. Gilchrist, M. Duszyk and A. D. Befus

Pulmonary Research Group, University of Alberta, Edmonton, Canada

Correspondence: Dr. Dean Befus, Pulmonary Research Group, University of Alberta, Edmonton, Canada T6G 2S2. dean.befus@ualberta.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cell activation requires Cl- flux, which maintains the driving force for entry of extracellular calcium and initiates release of mediators such as histamine. However, chloride channel expression in mast cells has been poorly understood. For the first time, reverse transcriptase-polymerase chain reaction shows that rat-cultured mast cells (RCMC) and peritoneal mast cells (PMC) contain mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR), an important chloride channel. Immunostaining with an anti-CFTR antibody indicates expression of CFTR in PMC and RCMC. Mast cell CFTR is a functional Cl- channel because it is capable of mediating Cl- flux in response to elevated cAMP. An inhibitor of CFTR-dependent Cl- flux, diphenylamine-2-carboxylate down-regulates mast cell mediator release. These results show that rat mast cells express a functional CFTR, which might be important in mediator release.

Key Words: chloride channels • DIDS • DPC • glibenclamide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The product of the cystic fibrosis (CF) gene is the cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a adenosine cyclic 3',5'-phosphate (cAMP)-regulated Cl- channel in the apical membrane of secretory epithelial cells [1 ]. Although CFTR expression has been generally considered to be epithelial cell-specific, evidence for CFTR expression and/or function has also been observed in other cell types, including lymphocytes, Sertoli cells, heart muscle cells, tracheal submucosal gland cells, and hypothalmic neurons [2 3 4 5 6 ]. Although Cl- transport has been generally implicated in the modulation of membrane potential in several cell types, lymphocyte activation, CD8+ T-cell-mediated cytotoxicity, and volume regulation, the physiological relevance of CFTR expression in nonepithelial cells is poorly understood [3 , 7 , 8 ].

Cl- transport has also been implicated in mast cell (MC) activation and degranulation [9 , 10 ]. Cations such as K+ and Ca2+ play a key role in many MC functions [11 ], and recent patch clamp and pharmacological studies have shown that Cl- conductance is an important component of MC activation and secretion [12 13 14 ]. MCs are important effector cells in inflammatory diseases such as asthma and allergy. Following antigen-mediated clustering of immunoglobulin (Ig)E bound to the high-affinity Fc{varepsilon} receptor (Fc{varepsilon}RI), MCs release mediators such as histamine and arachidonic acid metabolites that cause immediate bronchial smooth muscle constriction, bronchial edema, and mucus hypersecretion [15 16 17 18 ]. Ion transport studies suggest that MC mediator release is a multiphasic process. Following activation, the influx of Ca2+ from intracellular and extracellular sources leads to the activation of Ca2+-dependent enzymes and G proteins, initiating the fusion of the granule membrane with the plasma membrane [13 , 19 20 21 22 23 ]. This Ca2+ flux also activates Cl- channels, which balance whole-cell current and allow a sufficient increase in intracellular Ca2+ to initiate Ca2+-dependent events [12 , 24 ]. Experiments using pharmacological blockers provide evidence that MC exocytosis (which ultimately results in release of histamine and other mediators) is dependent on channels that are Cl--selective [12 ]. In fact, drugs that prevent MC secretion such as furosemide and cromolyn block Cl- channel activity [25 ].

The identity of Cl- channels expressed in MCs is poorly known. However, cAMP-activated Cl- conductances have been measured in rat MCs, suggesting the possible expression of CFTR [24 ]. In epithelial cells, CFTR is one of the major Cl- channels controlling Cl- flux across the apical membrane. We postulate that MCs express CFTR mRNA and protein and that the function of CFTR is important for MC activation and subsequent mediator release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and reagents
The pharmacological reagents used in these experiments were: DIDS (Sigma Chemical Co., St. Louis, MO; D-3514), dipalmitoyl phosphatidylcholine (DPC; ICN, Costa Mesa, CA; 193703), 8-bromo-cAMP (Sigma Chemical Co.), 8-(4-chlorophenylthio)-cAMP (CPT-cAMP; Sigma Chemical Co.), and forskolin (Sigma Chemical Co.). DIDS blocks chloride channels and chloride transporters by modifying amino groups but does not affect CFTR chloride transport [26 , 27 ]. DPC blocks chloride flux via CFTR and the Cl-/HCO3 cotransporter by binding to the channel pore [28 ].

Rats and peritoneal mast cells (PMC) isolation
Male Sprague Dawley rats (300–350 g; Charles River, St. Constant, Quebec, Canada) were housed in a pathogen-free, viral antibody-free facility. For MC sensitization, rats were infected with 3000 Nippostrongylus brasiliensis, as previously described [29 ]. PMC were isolated from sensitized rats >= 4 weeks after infection. Rats were sacrificed by cervical dislocation under anesthesia, and PMC were isolated by the following procedure: Ice-cold Hepes Tyrode’s buffer (HTB; 20 mL) was injected into the peritoneal cavity and massaged gently for 30 s; the peritoneum was opened, and the buffer was collected with a transfer pipette and kept on ice or at 4°C for subsequent procedures. Following centrifugation at 200 g, the cell pellet was resuspended in 5 mL fresh HTB and layered on top of a 30%/80% Percoll gradient. The gradient was centrifuged at 500 g for 20 min, and the highly enriched MCs were collected from the pellet [30 ]. PMC were >98% pure and >96% viable.

Cell culture
The rat MC line, RCMC 1.11.2 (kindly provided by A. Froese, Winnipeg, Manitoba), was initially established from Wistar-ICI rats [31 ] and cultured in a humidified atmosphere of 5% CO2 in air at 37°C. Rat-cultured mast cells (RCMC) were placed in RPMI 1640 medium containing 5% fetal bovine serum (FBS; Gibco-BRL, Grand Island, NY), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 mM Hepes. The human lung carcinoma epithelial A549 and human lung adenocarcinoma Calu-3 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin, and were incubated in the same conditions as above.

Antigen (Ag) stimulation and ß-hexosaminidase (ß-hex) assay
PMC were pretreated with ion channel inhibitors for 5 min or untreated. The ion channel inhibitors used were DIDS (20 µM or 80 µM) and DPC (0.5 mM or 1.0 mM) in HTB. DIDS is an irreversible inhibitor of anion channels, including chloride channels [26 , 27 ]. DPC is a reversible inhibitor of CFTR and a bicarbonate exchanger. PMC were resuspended in several different buffers: complete buffer [containing 140 mM NaCl, 4.3 mM Na2PO4·2H2O, 1.2 mM K2PO4, 1.2 mM MgCl2, 5 mM KCl, 0.5 mM CaCl2, 1 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 4 mM glucose, 0.1% bovine serum albumin (BSA)]; chloride-reduced buffer (11.4 mM NaCl, 127 mM NaGluconate, 4.3 mM Na2PO4·2H2O, 1.2 mM K2PO4, 1.2 mM MgCl2, 5 mM KCl, 0.5 mM CaCl2, 1 mM EGTA, 4 mM glucose, 0.1% BSA); bromide-enhanced buffer (11.4 mM NaCl, 127 mM NaBr, 4.3 mM Na2PO4·2H2O, 1.2 mM K2PO4, 1.2 mM MgCl2, 5 mM KCl, 0.5 mM CaCl2, 1 mM EGTA, 4 mM glucose, 0.1% BSA); and iodide-enhanced buffer (11.4 mM NaCl, 127 mM NaI, 4.3 mM Na2PO4·2H2O, 1.2 mM K2PO4, 1.2 mM MgCl2, 5 mM KCl, 0.5 mM CaCl2, 1 mM EGTA, 4 mM glucose, 0.1% BSA). The PMC were stimulated with 48/80 or 10 worm equivalents (WE) of N. brasiliensis worm antigen for 10 min at 37°C [32 ]. PMC were pelleted at 200 g, and the supernatant was removed. Pellet and supernatant samples were assayed for ß-hex by hydrolysis of the fluorescent substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide (Sigma Chemical Co.; M-2133). One unit of enzyme cleaves 1 µmol substrate/h at 37°C [30 ]. Pellet and supernatant samples (50 µL) were added in duplicate wells in a microtitre plate followed by 1 mM substrate (50 µL), and the mixture was incubated at 37°C for 2 h. The reaction was terminated by the addition of 100 µL 0.2 M Tris base, and the optical density (OD) was read at 450 nm (excitation 356 nm). The OD450nm for blank wells, containing only substrate and Tris base, was automatically subtracted from the sample OD450nm, and the percentage ß-hex release was calculated by the formula: OD450nm of substrate samples/(OD450nm of pellet samples + OD450nm of supernatant samples) x 100 = % release. The spontaneous release (in HTB) was subtracted from all samples to give % specific release.

Reverse transcriptase-polymerase chain reaction (RT-PCR), cloning, and sequence analysis
RNA was extracted from PMC and RCMC using the modified Chomczynski and Sacchi method [33 ]. Briefly, 106–107 cells were homogenized with solution D (4 M guanidinium thiocyanate, 0.5% sodium n-laurylsarcosine, 1 M sodium citrate, and 0.1 mM 2-mercaptoethanol). To the homogenate, 2 M NaOAc, water-saturated phenol, and chloroform-isoamyl alcohol were added. The aqueous phase was removed and treated with solution D once again. The RNA was precipitated with absolute ethanol and washed with 70% ethanol and air-dried. The RNA obtained from PMC was treated with 1 U/mL heparinase for 2 h at room temperature to remove contaminating heparin, because high heparin concentrations markedly decrease the efficiency of the RT-PCR procedure [34 ].

Genomic DNA was digested by incubating 10 µg total RNA with 5 U DNAse (amplification grade; Gibco-BRL), 10x DNAse buffer (Gibco-BRL), 10 U RNAse inhibitor (Gibco-BRL), and RNAse-free H2O for 15 min at room temperature. After 15 min incubation, 25 mM ethylenediaminetetraacetate (EDTA) was added, and the sample was heated at 65°C for 20 min to inactivate the enzyme.

RNA was incubated with 1 µL oligo dT (500 µg/mL) at 70°C for 10 min in a thermocycler. An RT master mix was added to the RNA. The master mix contained 4 µL 5x First Strand Buffer (Gibco-BRL), 2 µL 0.1 M dithiothreitol (DTT), 1 µL mixed dNTP stock at 10 mM , 1 µL Sigma sterile water, and 1 µL (200 U) M-multilamellar vesicles (MLV) RT enzyme (Gibco-BRL). This mixture was incubated at 37°C for 1 h and then 70°C for 10 min.

The polymerase chain reaction (PCR) master mix contained 2 µL PCR buffer (Gibco-BRL), 0.4 µL 10 mM dNTP mix, 0.3 µL antisense CFTR primer (5' GGT GTC CTA TTC ACC TCA AGT TCT CTG 3'), which binds bp 901–927 of the Rattus norvegicus CFTR gene (NCBI accession #X95927.1), 0.3 µL sense CFTR primer (5' CTC TGT AGA CCA TAC TGG CCT TGA AC 3'), which binds bp 618–643 of the R. norvegicus CFTR gene, 0.6 µL MgCl2 (50 mM; Gibco-BRL), 13.9 µL sterile Sigma water, 2 µL cDNA, and 0.5 µL (2.5 U) Taq DNA polymerase. The mixture was amplified at an annealing temperature of 56°C for 25–45 cycles and found 35 cycles to give the best results. The product was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. The conditions of use for the primers specific for CFTR were optimized using rat lung RNA.

Three 309 bp CFTR PCR products (from separate RNA isolations) were cloned into the pCR 2.1® (Invitrogen, Carlsbad, CA) vector using T4 DNA ligase and then used to transform "super competent" Escherichia coli cells. The E. coli cells were grown for 1 day, the DNA was isolated using a Promega (Madison, WI) isolation kit, and the plasmid was sequenced on the ABI PRISM sequencer model 2.1.1 and analyzed by BLAST [35 ]. All three sequences obtained were 100% homologous to region 618–927 of the rat CFTR gene.

Flow cytometry
Single-cell suspensions of 105 cells were fixed with 5% formalin for 5 min. The fixation reaction was stopped by adding phosphate-buffered saline (PBS)/1% BSA. Whenever antibodies that recognize an intracellular portion of the CFTR protein (C terminus or R domain) were used, cells were permeabilized and blocked with PBS/0.1% saponin/5% dried milk for 24 h at 4°C. Otherwise, cells were blocked with PBS/5% dried milk for 24 h at 4°C. Cells were then incubated with primary monoclonal antibody [(mAb); mouse anti-CFTR C terminus, Genzyme (Cambridge, MA), 2503-01; mouse monoclonal anti-CFTR extracellular domain, Affinity Bioreagents (Golden, CO), MA1-935; or mouse monoclonal anti-CFTR R domain, Genzyme, 1660-01] for 1 h in PBS/0.1% saponin/5% dried milk or PBS/5% dried milk. Cells were washed twice with PBS/0.1%/5% dried milk or PBS/5% dried milk and incubated with the appropriate secondary antibody [rabbit anti-mouse IgG·fluorescein isothiocyanate (FITC), Serotec (Oxford, U.K.), STAR 38; or goat anti-mouse IgM·FITC, Biosource (Camarillo, CA), AM14708] for an additional hour. The isotype-control antibodies used were purified mouse IgG1 {kappa}-isotype standard anti-trinitrophenol (TNP; Serotec, catalogue #03001D) or mouse IgM isotype control anti-TNP (PharMingen, San Diego, CA, #03081D). Five separate samples of each cell type were analyzed with each antibody.

Western immunoblotting and immunocytochemistry
RCMC, PMC, or Calu-3 cells were isolated and washed with PBS, and 1 x 106 cells were lysed with 2x sample buffer [0.5 mL 1 M Tris-Cl, 1 mL DTT, 2 mL 10% sodium dodecyl sulfate (SDS), 1 mL glycerol, 0.5 mL 0.12 bromophenol blue] and supplemented with 2% ß-mercaptoethanol.

Whole-cell extracts (30–40 µg) were separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in TBS-Tween for 24 h at room temperature and then stained with primary antibodies, anti-CFTR (R domain, Genzyme, 1660-01), and isotype control-purified mouse IgG1 {kappa}-isotype standard (anti-TNP; Serotec, 03001D) for 1 h at room temperature. The membranes were washed with TBS-Tween 3x and then stained with the secondary antibody for 1 h. The secondary antibody was sheep anti-mouse IgG conjugated to horseradish peroxidase (HRP; Amersham Life Science, Arlington Heights, IL, catalogue #NA 9310).

The nitrocellulose membranes were developed with chemiluminescence reagent (NEN Life Technologies, Boston, MA, catalogue #NEL 101) for 1 min and placed into an autoradiography cassette containing high-performance chemiluminescence film (Amersham Life Science, catalogue #RPN2103H). The film was exposed for 30 min.

Immunocytochemistry was performed on cytospins of RCMC cells according to instructions of commercial kit (Vectastain ABC kit PK-6200) as follows: Aliquots (50 µL) of RCMC and PMC (1x106 cells/mL) were cytospun onto slides. Slides were air-dried and then stained with primary mAb (extracellular domain-specific) and isotype (mouse IgM) in PBS for 45 min in a humidified atmosphere at room temperature. Slides were washed with PBS for 5 min. Biotinylated antibody solution from a commercial kit was added for 30 min. Slides were washed for 5 min in PBS. Developing reagent was added for 30 min and washed for 5 min. Chromagen was added for 5 min until color developed. Slides were washed and fixed with ethanol and xylene and were mounted with permount. Images were photographed on a phase-contrast microscope at 625x magnification.

36Cl efflux assay
To determine Cl- secretion attributable to CFTR, a 36Cl efflux assay was used [36 ]. This assay is based on the principle that CFTR Cl- secretion is activated by cyclic AMP. This assay requires repeated washing of cells and measurement of 36Cl present in the medium before and after addition of a cAMP analogue.

RCMC Calu-3 and A549 were seeded at 70–95% confluence in a 25 cm2 flask and washed 3x with Ringer’s solution (HCO3-, phosphate-buffered 109 mM NaCl Ringer’s solution supplemented with 28 mM lactate). 36Cl- solution (30 µL; sodium salt from ICN; 1 µCi/µL; 1 mCi=37 kBq) was diluted in 5 mL Ringer’s solution and added to the flask containing the cells. The flask was incubated for 2–3 h at 37°C. All assays were performed at 37°C. At time 0, the flask was washed with Ringer’s solution. A fresh 2 mL aliquot of Ringer’s solution was added immediately to the flask, and the measurement of Cl- efflux rate was started. This process was repeated every 15 sec for 1 min, when Ringer’s solution with forskolin (2.5 µM), 8-bromo-cAMP (250 µM), and CPT-cAMP (250 µM) was added, incubated with the cells for 15 sec, and removed. These compounds were not added again for the remaining 4 min of the efflux run. At the end of the run, 50 mM NaOH was added to lyse the cells and to determine the radioactive counts remaining in the cells. Each sample was diluted in scintillation cocktail, and its radioactivity was measured in a scintillation counter. The fraction of intracellular 36Cl remaining in the cells during each time point was calculated, and the time-dependent rates of 36Cl efflux were calculated as ln(36Clt=1/36Clt=2)/(t1-t2), where 36Cl is the percent intracellular Cl at time t, and t1 and t2 are successive time points [37 ].

Statistical analysis
One-way analysis of variance (ANOVA) was performed on ß-hex and 36Cl efflux data, assuming P < 0.01 unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MCs express message and protein for CFTR
To determine whether MCs expressed CFTR, specific primers were designed for rat CFTR, and RT-PCR was used to screen two MC populations for CFTR expression. cDNA preparations made from RCMC, in vivo-derived PMC, and rat lung total RNA contained CFTR mRNA (Fig. 1 ). The 309 bp PCR product represented the fragment size expected from the published rat cDNA sequence (NCBI accession number X95927.1). To avoid genomic contamination, RNA samples were treated with DNAse. RCMC and PMC total RNA preparations (without RT) did not show any amplification, indicating that the primers amplified CFTR mRNA not contaminating genomic DNA (Fig. 1) . PCR amplification was also performed using primers specific for ß-actin to ensure cDNA quality. RCMC, PMC, and lung cDNA expressed the 560 bp ß-actin product, and as expected, RCMC, PMC, and lung RNA (without RT) did not show the product.



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  		Figure 1. RT-PCR showing CFTR mRNA expression by RCMC, PMC, and rat lung cells. RCMC, PMC, and lung RNA was reverse-transcribed into cDNA. Subsequent cDNA samples were PCR-amplified through 35 cycles using CFTR-specific primers (lane 2–4 from left). PCR amplification was also conducted on RCMC, PMC, and rat lung RNA preparations (without reverse transcription) to control for possible genomic contamination (lanes 5 and 6). RCMC, PMC, and lung cDNAs show the expected band of 309 bp representing CFTR, but RCMC, PMC, and rat lung RNA that has also undergone PCR amplification does not contain the CFTR product. ß-Actin amplification (35 cycles) of the same RCMC, PMC, and lung samples is also shown as a control for RNA quality. RCMC, PMC, and rat lung cDNAs contain the expected 560 bp product (lanes 9–11), and the RNA controls do not (lanes 12 and 13). Lanes 1, 6, 7, and 14 contain a 1 kb DNA ladder for comparison. These results are representative of experiments on 10 separate RNA preparations.

 
To ensure that the CFTR primers were specifically amplifying CFTR cDNA, PCR products from three different RNA preparations were amplified using CFTR primers inserted into pCR vectors and cloned in E. coli. Sequencing showed that the PCR products had 100% homology to region 618–927 of the R. norvegicus CFTR gene (Fig. 2 ).



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  		Figure 2. Comparison of PCR amplification product (PCR; 1–309) with the published sequence of the rat CFTR gene sequence (rCFTR; 618–927). The comparison shows 100% homology to the rat CFTR gene. Three PCR products from independent preparations of RNA were cloned and sequenced by the ABI PRISM sequencer model 2.1.1 and analyzed by BLAST.

 
To determine the presence of CFTR protein in rat MCs, a monoclonal anti-CFTR C terminus, a monoclonal anti-CFTR extracellular domain, and a monoclonal anti-CFTR R domain antibody were used in flow cytometry to screen RCMC and PMC for CFTR expression. To confirm that these antibodies recognized native CFTR protein, Calu-3 submucosal airway epithelial cells, which express large amounts of CFTR protein, were used as a positive control, and A549 lung epithelial cells, which do not express CFTR protein, were used as a negative control. As expected, Calu-3 cells were positive for CFTR protein expression using antibodies recognizing the C terminus and extracellular domain, and A549 cells were negative (Fig. 3 ). RCMC and PMC were also positive for CFTR expression using the C terminus and extracellular domain antibodies, because the anti-CFTR antibodies showed a shift in expression from the isotype control antibodies (Fig. 3) . As much as 60.6% of RCMC and 35.6% and 47.6% of Calu-3 cells show positive staining for CFTR (compared with isotype control; Table 1 ). However, the antibody recognizing the R domain did not show a shift even in the Calu-3 cells (>7% of cells were positive; Table 1 ). Because previous studies have shown that antibody to the R domain can recognize CFTR in Western immunoblotting, we extracted cell lysates from PMC, RCMC, and Calu-3, resolved them by SDS-PAGE, and immunoblotted using this antibody. The R-domain antibody demonstrated that RCMC, PMC, and Calu-3 express a CFTR protein of approximately 170 kDa as expected (Fig. 4 ).



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  		Figure 3. Flow cytometry showing expression of CFTR on RCMC (first row), PMC (second row), Calu-3 epithelial cells (third row), and A549 epithelial cells (fourth row) using the three mAb recognizing three regions of the CFTR protein (C terminus, extracellular domain, and R domain) as labeled above. In each flow diagram, the thin line represents fluorescence values obtained using the appropriate isotype control (see Materials and Methods), and the dark line represents fluorescence values obtained using anti-CFTR antibody indicated. Fluorescence values (FL1-H) are in log units on the x-axis, and the cell counts are on the y-axis. Cells were incubated with monoclonal anti-CFTR (5–10 µg/mL) and isotype antibody (5–10 µg/mL) for 1 h at room temperature.

 

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  		Table 1. Flow Cytometry Data on RCMC, PMC, CALU-3, and A549 Cells

 


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  		Figure 4. Western blot analysis of Calu-3 (lane 1), PMC (lane 2), and RCMC (lane 3) cell lysates shows CFTR protein expression. Cell pellets were lysed in the presence of 2% ß-mercaptoethanol and SDS and then resolved by 8% acrylamide SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and stained with anti-CFTR (R-domain-specific) mAb for 1 h at room temperature (1/1000 dilution). This experiment is representative of three separate experiments on independently isolated cell pellets.

 
Immunocytochemistry was also performed to determine the localization of CFTR protein expression in RCMC and PMC using the mAb specific for the extracellular domain of CFTR. CFTR protein expression was localized to the periphery of the MC (Fig. 5 A ), and the strongest staining was at the cell membrane. Greater than 98% of the cells observed were positive for CFTR expression. RCMC stained with isotype-control antibody (IgM) showed no staining (Fig. 5B) . PMC stained with anti-CFTR (extracellular domain) mAbs were also positive, although CFTR was not localized to the periphery but rather scattered throughout the cell (Fig. 5C) . More than 90% of the cells observed were positive for CFTR expression. The isotype control for PMC was negative (Fig. 5D) .



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  		Figure 5. Immunocytochemistry of RCMC (A and B) and PMC (C and D) showing expression of CFTR protein. (A) RCMC (at 5x104 cells/mL) cytospins were prepared as described and stained with anti-CFTR antibody (extracellular domain-specific) at 1/100 dilution for 1 h at room temperature. (B) RCMC (at 5x104 cells/mL) cytospins were prepared as described and stained with IgM isotype antibody at 1/100 dilution for 1 h at room temperature. Cells were also counter-stained with hemotoxylin to make nuclei more visible. (C) PMC (at 5x104 cells/mL) cytospins were prepared as described and stained with anti-CFTR antibody (extracellular domain-specific) at 1/100 dilution for 1 h at room temperature. (D) PMC (at 5x104 cells/mL) cytospins were prepared as described and stained with IgM isotype antibody at 1/100 dilution for 1 h at room temperature. Results are typical of two experiments.

 
Elevated cAMP levels induce Cl- secretion in MCs
To assess the Cl- channel function of CFTR in MCs, paired 36Cl- efflux assays with and without a membrane-permeable cAMP agonist mixture (2.5 µM forskolin, 250 µM 8-bromo-cAMP, and 250 µM CPT-cAMP) were performed in RCMC, according to the procedure adapted from Schwiebert et al. [36 ]. Results indicated that similar to Calu-3 cells (which express wild-type CFTR), RCMC show a recognizable Cl- flux when cAMP agonists were added (1 min; Fig. 6 ). The cAMP-dependent Cl- flux is sensitive to DPC (Fig. 6A) , because the addition of DPC to the agonist mixture reduced the Cl- flux by 50%. The A549 epithelial cell line does not express CFTR and does not show an increased Cl- flux after addition of cAMP analogs above basal rates. These findings indicate that the RCMC CFTR protein is a functioning Cl- channel, because the cells display a DPC-sensitive, cAMP-dependent Cl- current.



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  		Figure 6. 36Cl efflux assay showing 36Cl release from RCMC (A), Calu-3 (B), and A549 (C). Cells were grown to confluence and loaded with 36Cl for 2–3 h at 37°C. The unincorporated 36Cl was discarded, and the cells were washed every 15 s to measure the rate of 36Cl release over time. At time 60 s (1 min±5 s), cAMP agonists were added for 15 s. Calu-3 express wild-type CFTR protein, and A549 do not express CFTR (n=7; P<0.01; t0 vs. t60).

 
Release of ß-hex from MCs is ion-selective
CFTR has a characteristic ion permeability (Br>Cl>I), which can be used to distinguish it from other chloride channels. We postulated that if CFTR regulated mediator release from PMC, the ion content of the buffer would also be important in mediator release. The ability of PMC to release granule-associated ß-hex in buffers containing Cl-, Br-, and I- ions was measured (Fig. 7 ) following stimulation with 48/80 (0.75 µg/mL) or antigen (10 WE). Under physiological conditions (140 mM NaCl), PMC stimulated with Ag released 9.9 ± 0.9% of their stored ß-hex (minus spontaneous release 3.5±0.1%). This value was considered to be 100% release in these experiments, and all subsequent values were calculated as a percentage of this (Fig. 7A) . PMC stimulated with Ag in buffer containing NaGluconate (127 mM) or NaI (127 mM) released considerably less ß-hex (42.4±9.7% and 20.7±2.1% of maximum, respectively), whereas PMC in NaBr (127 mM) buffer released almost maximum levels of ß-hex (81.3±8.6%), suggesting that Br- and Cl- ions can facilitate maximum PMC release and that the presence of gluconate or I- ions cannot compensate for their absence.



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  		Figure 7. ß-Hex release of stimulated PMC in different buffers. PMC were placed in different buffers containing specific ions (Cl, Br, Gluconate, or I) and then were stimulated with antigen or 48/80 at 37°C. (A) PMC were stimulated with antigen (10 WE/mL) for 10 min. (B) PMC were stimulated with 48/80 (0.75 µg/mL) for 10 min. Significance values are calculated in relation to antigen or 48/80-stimulated % total release and represent P < 0.01 (n=5 separate experiments).

 
Figure 7B shows that in the presence of a physiological concentration of Cl- ions (140 mM), PMC stimulated with 48/80 released a substantial amount of ß-hex (58.4±3.7% of amount stored in cell), which represents maximum release possible in these experiments, and subsequent values were calculated in relation to this maximum release. PMC stimulated with 48/80 in buffer containing NaGluconate (127 mM) or NaI (127 mM) released considerably less ß-hex (30.6±4.5% and 11.9±12.1%, respectively), whereas PMC suspended in NaBr (127 mM) buffer were unaffected, releasing almost the same amount of ß-hex (85.1±2.1%) as cells in physiological NaCl buffer. These data suggest that in the absence of Cl- or Br- ions, there is a significant decrease in PMC mediator release and that the presence of I- ions cannot compensate. Gluconate is a large, negatively charged molecule that has been postulated to compensate for chloride ions in some assays, yet gluconate does not restore PMC ß-hex release. Because ß-hex release is dependent on Br- or Cl- but not on I-, this supports the contention that CFTR-specific chloride ion transport is important in PMC mediator release when stimulated with 48/80 or Ag.

MC mediator release in the presence of the chloride channel inhibitors DIDS, DPC, and glibenclamide
To investigate this postulate, we used pharmacological inhibitors of different ion channels to determine the importance of ion flux and particular channels in ß-hex release by PMC. Whereas RCMC contain few granules and are not activated by conventional MC stimuli such as antigen (unpublished results), PMC are readily activated by Ag and 48/80 (a potent activator of G proteins). PMC were pretreated with the Cl- channel inhibitor DIDS and the CFTR channel inhibitors DPC and glibenclamide for 5 min before activation by 48/80 (0.75 µg/mL) or antigen (10 WE/mL). Thirty minutes after activation, ß-hex release was measured (Figs. 8 9 10 ). DPC, DIDS, and glibenclamide alone had no effect on spontaneous ß-hex release from PMC (unpublished results). DIDS, a chloride-channel inhibitor, dose dependently inhibited antigen-stimulated and 48/80-stimulated PMC ß-hex release (Fig. 8A and 8B) . The lowest concentration of DIDS (10 µM) inhibited 38 ± 13.2% of antigen-stimulated ß-hex release, whereas the highest concentration of DIDS (800 µM) inhibited 138 ± 0.1%, making the IC50 (concentration that inhibits release by 50%) of antigen-stimulated ß-hex release between 20 and 40 µM (Fig. 8A) . The lowest concentration of DIDS, which significantly inhibited 48/80-stimulated ß-hex release, was 40 µM (inhibited release by 39±17.2%; Fig. 8B ). Thus, the IC50 of 48/80-stimulated PMC was between 80 and 100 µM, which is higher than the IC50 of antigen-stimulated PMC. This difference in IC50 between antigen- and 48/80-stimulated PMC may reflect the potencies and differing mechanisms of activation of the two stimuli.



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  		Figure 8. ß-Hex release from stimulated PMC treated with DIDS, a pharmacological inhibitor of chloride channels. PMC were treated with DIDS (chloride-channels inhibitor; 10–800 µM) at 37°C for 5 min in physiological buffer containing 140 mM NaCl. (A) PMC were treated with DIDS for 5 min and stimulated with antigen (10 WE/mL) for 10 min. (B) PMC were treated with DIDS and then stimulated with 48/80 (0.75 µg/mL) for 10 min. Significance values are calculated in relation to antigen or 48/80-stimulated % total release and represent P < 0.01 (n=5 separate experiments).

 


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  		Figure 9. ß-Hex release from stimulated PMC treated with DPC. PMC were treated with DPC (CFTR chloride-channel inhibitor; 0.001–3.0 mM) at 37°C for 5 min in physiological buffer containing 140 mM NaCl. (A) PMC were treated with DPC for 5 min and stimulated with antigen (10 WE/mL) for 10 min. (B) PMC were treated with DPC and then stimulated with 48/80 (0.75 µg/mL) for 10 min. Significance values are calculated in relation to antigen or 48/80-stimulated % total release and represent P < 0.01 (n=5 separate experiments).

 


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  		Figure 10. ß-Hex release from stimulated PMC treated with glibenclamide, a pharmacological inhibitor of CFTR channels. PMC were treated with glibenclamide (CFTR channel inhibitor; 3–300 µM) at 37°C for 5 min in physiological buffer containing 140 mM NaCl. (A) PMC were treated with glibenclamide for 5 min and stimulated with antigen (10 WE/mL) for 10 min. (B) PMC were treated with glibenclamide and then stimulated with 48/80 (0.75 µg/mL; n=6 separate experiments).

 
DPC, a more specific inhibitor of CFTR chloride channels, decreased antigen-stimulated PMC ß-hex release by 31 ± 6.7% (1 µM; Fig. 7A ) but had little effect on 48/80-stimulated release (Fig. 9 B). The IC50 of antigen-stimulated ß-hex release was between 0.1 and 0.5 mM DPC. However 48/80-stimulated ß-hex release was not sensitive to DPC except at the 1 mM and 3 mM concentrations, which showed 14 ± 3.14% and 23 ± 0.1% inhibition, respectively.

Glibenclamide, a sulfonylureal used in the treatment of ischemia, has previously been shown to block epithelial CFTR channels [38 ]. In patch clamp experiments, glibenclamide inhibits CFTR chloride current with an IC50 of 22–38 µM, and 100 µM glibenclamide caused nearly complete inhibition [39 ]. Thus, ß-hex release was measured from PMC treated with glibenclamide (5 min) and activated with 48/80 or Ag (Fig. 10) . Glibenclamide (3, 30, and 300 µM) had no effect on antigen-stimulated PMC ß-hex release (Fig. 10A) . The difference was not significant as tested by ANOVA (P=0.202, and F=1.69). Similarly, 48/80-stimulated ß-hex release was partially inhibited by 3, 30, and 300 µM glibenclamide (22.9±5.5%, 19±7.1%, and 23.9±6.7%, respectively; P=0.02, and F=3.96).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cl- channels regulate cell volume, membrane potential, pH, and osmolarity and can be classified according to their mechanism of activation. Cl- channels are involved in degranulation of rat mucosal MCs by maintaining MC resting membrane potential, counteracting the depolarization of the cell during degranulation, and maintaining the driving force for Ca2+ entry [24 , 40 ]. Because chloride channels in MCs are poorly understood, our aim was to characterize and determine the role of CFTR, a complex chloride channel, in the release of MC mediators.

This study found that CFTR mRNA and protein is expressed in in vivo-derived and cultured rat MCs. PCR shows that PMC and RCMC express mRNA for CFTR and that this amplification product is, in fact, from the rat CFTR gene. Flow cytometry data show that as many as 60% of RCMC and PMC are positive for CFTR, which is similar in intensity to the Calu-3 epithelial cell line. The antibody to the C terminus of CFTR used in flow experiments has been used by others to demonstrate that cAMP induces the recruitment of CFTR from cytoplasmic pools to the apical plasma membrane [39 , 40 ]. The protocol in this study involved permeabilization of the MCs before immunostaining. Thus, the C-terminus antibody is capable of detecting intracellular as well as surface expression of CFTR.

Our data also show that MCs stimulated with membrane-permeable analogues of cAMP can induce Cl- secretion, resulting in a net efflux of 36Cl. These data coincide with previous observations in MCs, which found that internally applied cAMP induces a Cl- current within 10–30 sec of activation [41 , 42 ]. Despite its structural similarity to members of the adenosine 5'-triphosphate (ATP)-binding cassette family, CFTR is a Cl- channel [43 , 44 ] whose activity is under complex regulation by phosphorylation, nitric oxide, and cytoplasmic ATP [3 , 43 , 45 , 46 ]. In all cells in which CFTR is expressed, it is regulated by cAMP-dependent phosphorylation [1 , 47 , 48 ]. CFTR-dependent Cl- current is controlled by cAMP-dependent internal messengers that phosphorylate serine residues on the regulatory (R) domain of the CFTR molecule and cause a conformational change allowing the Cl- channel to be opened [49 ]. Elevated cAMP levels can also induce the activation of other ion channels such as cAMP-sensitive K+ channels capable of hyperpolarizing the membrane and allowing for Cl- flux through open chloride channels. Our data show that elevation of intracellular cAMP in MCs activates a net outward Cl- flux that is partly inhibited by DPC. This supports the hypothesis that MCs express a functional CFTR capable of conducting Cl- ions.

Although our data clearly show that MCs express a CFTR Cl- channel, the role of CFTR in MC function is less clear. Pharmacologic inhibition of chloride channels and ion replacement studies are often the best tools available to study the importance of particular ions in cell functions, and in this study, these methods indicate that chloride and chloride channels are important in MC ß-hex release. PMC stimulated with 48/80 (0.75 µg/mL) in the presence of low Cl- (16 mM) released less ß-hex than in the presence of physiological levels of Cl- (140 mM), suggesting that Cl- ion exchange is important for 48/80-stimulated secretion. A previous study concluded that MC exocytosis was not dependent on activation of Cl- current by 48/80 [50 ]. However, in that study, MCs were activated with a high concentration of 48/80 (10 µg/mL), and the investigators suggested that Cl- current may enhance secretion at suboptimal stimulation [50 ]. We used a lower concentration of 48/80 (0.75 µg/mL) in our study, and at this dosage, our results suggest that PMC mediator release is Cl- ion-dependent.

The CFTR chloride channel displays ion selectivity and permeability such that substitution of Br- or I- for Cl- in the buffer of a patch clamp system can alter the current through the channel. Normally, in the wild-type CFTR channel, the sequence of anion permeability through the channel pore is Br > Cl > I and is strongly regulated by a "selectivity filter" close to the cytoplasmic end of the pore [51 ]. In an experimental system where Br- not I- ions can partially substitute for Cl-, the presence of a functional CFTR channel is suspected. PMC stimulated with 48/80 or antigen in a buffer containing Br- instead of Cl- ions released 85.1% of maximum ß-hex, whereas PMC stimulated in a buffer containing I- ions released 11.9% of maximum ß-hex, suggesting a Br > I selectivity. Based on this selectivity, a CFTR-dependent ion flux is suspected to be present during MC ß-hex release.

The presence of a functional CFTR channel is further characterized by its sensitivity to a variety of pharmacologic inhibitors known to block CFTR function. CFTR Cl- channel activity (as measured by patch clamp) in epithelial cells can be blocked with DPC and glibenclamide but not with the broad anion-exchanger inhibitor DIDS [52 ]. In our study, DPC blocked ß-hex release from PMC stimulated with antigen, but the effect of glibenclamide was more difficult to interpret. Glibenclamide did not block ß-hex release from PMC stimulated with antigen or 48/80, suggesting that ion involvement in degranulation is a complex process. The inability of glibenclamide to block degranulation may reflect that 48/80 and antigen have different mechanisms of activation, and glibenclamide is also an inhibitor of Ca2+-activated Cl- channels and ATP-activated K+ channels, the blockade of which may modify Cl- currents [53 ].

The broad-range inhibitor of several ion channels, DIDS, also reduced the release of ß-hex from 48/80-stimulated PMC, indicating that the role of chloride channels (and other ion channels in general) other than CFTR in mediator release should not be ruled out. In fact, CFTR can regulate ion flux directly through DIDS-sensitive channels, and blocking these channels would affect CFTR’s ability to affect the flow of ions across the plasma membrane [54 ].

Ag-stimulated and 48/80-stimulated MCs appear to be differentially sensitive to the ion inhibitors. Ag activates PMC through the Fc{varepsilon} receptor [55 ], whereas 48/80 is a cation that complexes with the negatively charged sialic acid residues in the cell membrane and nonspecifically activates phospholipase C [56 ]. Thus, the difference in sensitivity to DPC, DIDS, and glibenclamide of antigen- and 48/80-stimulated PMC is a reflection of the different modes of stimuli. Yet, the IC50 of DIDS-treated PMC stimulated with 48/80 is 10 times higher than the IC50 of DIDS-treated PMC stimulated with antigen. Therefore, 48/80 stimulation of PMC might be less dependent on DIDS-sensitive Cl- flux. DPC inhibits antigen-stimulated but not 48/80-stimulated ß-hex, which suggests that Cl- flux via CFTR might be more important at suboptimal stimulation, which occurs via antigen-IgE-Fc{varepsilon} receptor complexes. The role of other DIDS-sensitive channels in ß-hex release is possible. DIDS and DPC can block the Cl-/HCO3- exchanger, which is an alkalinizing mechanism responsible for maintaining internal pH in MCs. DIDS inhibition of the Cl-/HCO3- exchanger would alter intracellular pH and indirectly inhibit CFTR-dependent ion flux [9 , 52 ].

Proposed model for Cl- flux in MCs
MC resting membrane potential is regulated by two types of ion channels: an inwardly rectifying K+ current and an outwardly rectifying Cl- current [57 ]. Upon stimulation by antigen, a small increase in membrane conductance occurs and causes an influx of extracellular Ca2+. Ca2+ entry is driven by a hyperpolarized membrane. However, the resting potential of rat PMC is unstable as measured in the permeabilized patch configuration, and an inward Ca2+ current would depolarize the cell easily. It has been proposed that Cl- channels open and clamp the membrane potential at the electromotive force of chloride ions (ECl) [20 ], allowing for Ca2+ influx driven by a hyperpolarized membrane.

In electrophysiological studies [14 ], an outward membrane current (corresponding to the influx of Cl-) is observed in rat PMC when they are stimulated with substance P or compound 48/80. These studies demonstrate the existence of hyperpolarization resulting from Cl- influx into the cell, which creates a driving force to facilitate the entry of Ca2+ required to initiate histamine secretion. When rat MCs are stimulated with antigen, there is a large increase in the rate of Cl- uptake into the cell consistent with the hypothesis that overall Cl- influx is important for MC exocytosis [24 ]. The putative Cl- channel blocker, DIDS, dose-dependently inhibits the antigen-stimulated histamine secretion but does not inhibit the antigen-stimulated increase of Cl- uptake. These results indicate that initial rises in [Cl]i might be mediated by channels other than DIDS-sensitive Cl- channels, such as a cotransporter or CFTR. The Na/K/2Cl-cotransporter inhibitor, furosemide, abolishes the increased antigen-induced Cl- uptake but does not affect antigen-induced histamine release [58 ]. Our studies show that CFTR is likely the DIDS-insensitive Cl- channel that is important in this first step of MC exocytosis.

One of the major physiological conditions of CF is massive inflammation in the lung. The mechanism of this inflammation is unexplained, although the expression of a nonfunctional CFTR protein is thought to play a role. Traditionally, the CFTR mutation has been thought to be most influential in disrupting epithelial cell function. Pseudomonas aeruginosa is the most common infection found in lungs of CF patients. Aberrant CFTR function correlates with increased levels of apical asialoGM1, asialylated glycolipids that function as P. aeruginosa receptors [59 ]. However, CFTR might also be an important regulator of nonepithelial cell function, particularly in cells involved in immunity. CD4+ T lymphocytes expressing mutant CFTR and stimulated with concanavalin A secrete 45% less interleukin (IL)-10 compared with T lymphocytes from healthy controls [60 ]. Recruitment of neutrophils to sites of infection in CF patients is enhanced yet ineffective at clearing bacterial infection [61 , 62 ]. Opsonic quality of naturally occurring antibodies to P. aeruginosa is decreased markedly in chronically infected CF patients [63 ]. PMC pretreated with P. aeruginosa isolates from patients with CF show a >=47% decrease in release of histamine when stimulated with A23187 [64 ]. This suggests that mast-cell secretion of mediators such as histamine is influenced by bacterial infection and might play a role in the pathogenesis of CF infection. MCs possess important antimicrobial actions that are important in the innate immune response [65 ]. In fact, MCs secrete histamine, ß-hex, and serotonin in response to gram-negative rods [66 , 67 ], a process that may be compromised in patients with CF.

Furthermore, there is evidence that CF cells may be less sensitive to MC mediators. For example, tracheal gland cells isolated from CF patients show a smaller peak in [Ca2+]i in response to histamine than tracheal gland cells from normal subjects [68 ]. Therefore, MC function and ability to clear bacterial infections may be fundamentally altered in CF. This study offers insight into mechanisms regulating MC exocytosis and suggests that epithelial cells may not be the only cell type responsible for the CFTR-defective phenotype seen in many CF patients and CFTR knockout mice.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the Canadian Institutes for Health Research (CIHR), Arthritis Association of Edmonton, and the Walter H. Johns Graduate Fellowship. The authors thank Dr. Angus MacDonald, Dr. Paul Man, and Dr. Phillip Halloran for their support and scientific input during the course of this work.

Received October 13, 2000; revised April 16, 2001; accepted July 9, 2001.


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 DISCUSSION
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