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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

¹Department of Physiology and ²Department of Microbiology and Immunology, Dartmouth Medical School, Hanover; and ³Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire

Submitted 1 September 2005 ; accepted in final form 15 October 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), F508, leads to the absence of CFTR Cl^– channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of F508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl^– channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of F508-CFTR.

cystic fibrosis

The reason why P. aeruginosa is particularly successful at colonizing and chronically infecting the lungs of patients with CF is not completely understood, but published studies have proposed several mechanisms: 1) CFTR is a receptor for binding and clearing P. aeruginosa from the airway (57); 2) asialoganglioside 1 (asialoGM1) is a receptor for binding P. aeruginosa (34, 39), and increased expression of asialoGM1 in CF airway epithelial cells leads to decreased clearance of P. aeruginosa (8, 64); and 3) P. aeruginosa grows in complex bacterial communities known as biofilms, which are more resistant than planktonic bacteria to antibiotics (17, 30, 66). In addition, genetic advantages of P. aeruginosa, such as a large genome (67) and a high frequency of hypermutability (55), together with a high burden of infecting bacteria, compartmentalization of infection, and ongoing antibiotic selective pressure, may lead to the exceptional ability of the microorganism to adapt to the CF airway.

Rescue of F508-CFTR by reduced temperature or chemical means has revealed that F508-CFTR is partially functional as a Cl^– channel (11, 18, 45, 74). Thus restoration of Cl^– transport by increasing the export of F508-CFTR from the ER, thereby increasing the expression of CFTR in the apical membrane, may reinstate mucociliary clearance and eradicate P. aeruginosa from the CF airway. Studies suggest that the ER retention of F508-CFTR is not complete and that some F508-CFTR is constitutively expressed in the plasma membranes of primary epithelial cells of individuals homozygous for the F508 mutation (6, 10, 36). However, the F508 mutation also reduces the functional and biochemical half-life of CFTR in the plasma membrane (28, 45, 46, 65). Accordingly, correction of CFTR-mediated Cl^– secretion in patients with CF is likely to require combined therapy that includes 1) promotion of F508-CFTR exit from the ER, 2) activation of F508-CFTR in the apical plasma membrane, and 3) increase of the half-life of F508-CFTR in the apical membrane. Such therapy must remain efficacious, at least initially, in the presence of P. aeruginosa in the lungs of patients with CF.

Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion across polarized human airway epithelial cells by inhibiting the endocytic recycling of CFTR and thus the number of wild-type (WT)-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data indicate that therapeutic strategies based on the restoration of Cl^– transport in the CF airway may be compromised by chronic infection with P. aeruginosa.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell lines and cell culture. Human airway epithelial cells (Calu-3) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in MEM containing 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% FBS in a 5% CO₂-95% air incubator at 37°C as described previously (44). To establish polarized monolayers, Calu-3 cells were seeded onto Transwell permeable supports (0.4-µm pore size, 1 x 10⁶/6.5-mm diameter and 2 x 10⁶/24-mm diameter; Corning, Corning, NY) coated with Vitrogen plating medium containing DMEM (JRH Biosciences, Lenexa, KS), human fibronectin (10 µg/ml; Collaborative Biomedical Products, Bedford, MA), 1% Vitrogen 100 (Collagen, Palo Alto, CA), and BSA (10 µg/ml; Sigma-Aldrich, St. Louis, MO) (44). Cells were grown in an air-liquid interface culture at 37°C for 14–21 days. Parental human bronchial epithelial CFBE41o– cells (F508/F508), originally immortalized and characterized by Gruenert and colleagues (7, 12), were stably transduced with either WT-CFTR or F508-CFTR (generous gift from Dr. J. P. Clancy, Dept. of Pediatrics, University of Alabama at Birmingham, Birmingham, AL; Ref. 3). CFBE41o– cells were maintained in MEM supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 10% FBS, and 1 µg/ml blasticidine (WT-CFTR) or 2 µg/ml puromycin (F508-CFTR) in a 5% CO₂-95% air incubator at 37°C. To establish polarized monolayers, 1 x 10⁶ CFBE41o– cells were seeded onto 12-mm Snapwell or 24-mm Transwell permeable supports (0.4-µm-pore size; Corning) and grown in an air-liquid interface culture at 37°C for 6–9 days and at 27°C for 36 h to increase trafficking and expression of F508-CFTR in the apical membrane (14, 19, 22). Madin-Darby canine kidney (MDCK) cells stably expressing green fluorescent protein (GFP)-WT-CFTR or GFP-F508-CFTR fusion protein were established and maintained in culture in a 5% CO₂-95% air incubator at 37°C in MEM complete medium containing 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 10% FBS, and 150 µg/ml G418 as described previously (50, 51). Addition of GFP to the NH₂ terminus of CFTR had no effect on CFTR localization, trafficking, and function as a Cl^– channel or on CFTR degradation (51). MDCK cells were seeded onto Transwell permeable supports (0.2 x 10⁶ cells on 6.5-mm- or 24-mm-diameter supports with 0.4-µm pore size) and grown in culture at 37°C for 7–10 days as polarized monolayers. Sodium butyrate (5 mM; Sigma-Aldrich) was added to the MDCK cell culture medium 16–18 h before experiments to stimulate CFTR expression (52).

P. aeruginosa cultures. Lysogeny broth was inoculated with P. aeruginosa strain UCBPP-PA14 (PA14), a relatively recent laboratory strain isolated from a burn patient (59), from a glycerol stock and incubated at 37°C using rotation until the bacterial count reached optical density (OD)₆₀₀ of 1.5, corresponding to a bacterial count of 5 x 10⁹ colony-forming units (CFU)/ml (16–18 h of culture). Bacteria were harvested by centrifuging cultures at 4,800 g for 10 min at 4°C. After being washed with PBS at pH 7.4 and 4°C to eliminate products secreted into the extracellular environment, bacteria were resuspended in PBS to a stock concentration of 5 x 10⁹ CFU/ml. In addition, P. aeruginosa strain PA01 (67) was cultured as described above. Heat-killed bacteria were prepared by incubating the PBS-resuspended cultures at 95°C for 10 min.

Bacteria-free P. aeruginosa filtrates. Bacterial cultures grown as described above were centrifuged at 4,800 g for 10 min at 4°C. Supernatants were harvested and filter sterilized at 4°C using a Steriflip 0.2-µm filter apparatus (Millipore, Bedford, MA), which resulted in bacteria-free filtrates. For Ussing chamber experiments, the bacteria-free filtrates were concentrated 10x at 4°C using a Centricon filter device with a 30-kDa molecular mass cutoff (YM30; Millipore) to minimize the volume of lysogeny broth (filtrate vehicle) used during the experiment. In addition, heat-inactivated filtrates were prepared using incubation at 60°C for 45 min.

Ussing chamber measurements. Monolayers grown on 6.5-mm-diameter Transwell or 12-mm-diameter Snapwell permeable supports as described above were mounted in an Ussing-type chamber (Jim's Instruments, Iowa City, IA, or Physiologic Instruments, San Diego, CA) and bathed in solutions at pH 7.4 that were maintained at 37°C and stirred using bubbling with 5% CO₂-95% air. Short-circuit current (I_sc) was measured by clamping the transepithelial voltage across the monolayers to 0 mV using a voltage clamp (model VCC MC6; Physiologic Instruments) as described previously (29, 44, 53). Current output from the clamp was digitized using an analog-to-digital converter (iWorx, Dover, NH). Data collection and analysis were performed using LabScribe version 1.6 software (iWorx).

I_sc was stimulated with 100 µM 8-(4-chlorophenylthiol) (CPT)-cAMP in Calu-3 and MDCK cells or 20 µM forskolin in WT-CFTR CFBE41o– cells added to the apical and basolateral bath solution or with 50 µM genistein in F508-CFTR CFBE41o– cells added only to the apical bath solution. Different activation protocols were used to stimulate I_sc in CFBE41o– cells expressing WT-CFTR and F508-CFTR because of the different activation profiles of WT-CFTR and the temperature-rescued F508-CFTR in these cell lines (3). To determine the effects of P. aeruginosa on the CFTR-mediated Cl^– currents, 5 x 10⁶ or 5 x 10⁷ CFU/ml of the appropriate strain of washed (PBS resuspended) P. aeruginosa was added to the apical side of the monolayers and incubated at 37°C in a CO₂ incubator. After incubation, the baseline and peak stimulated I_sc were measured. Data are expressed as net stimulated I_sc, which was calculated by subtracting the baseline I_sc from the peak stimulated I_sc. To determine the effects of the bacteria-free PA14 filtrate on CFTR-mediated Cl^– secretion, the monolayers were bathed in sterile bath solution and I_sc was stimulated with CPT-cAMP. The 10x concentrated PA14 filtrate was diluted 1:10 by addition to the apical bath solution, and the change in stimulated I_sc was measured. Data are expressed as the change in stimulated I_sc after addition of filtrate to the apical bath solution. Glibenclamide (200 µM) was added to the apical bath solution to inhibit CFTR-mediated I_sc.

Intact Calu-3 monolayers were bathed in MEM (–FBS). To measure CFTR-mediated Cl^– currents across the apical membrane in Calu-3 cells, basolateral membranes were permeabilized with nystatin (200 µg/ml), and an apical-to-basolateral Cl^– concentration gradient was established (16, 26). A low-Cl^–, high-Na⁺, high-gluconate basolateral bath solution was used to prevent cell swelling due to the increased basolateral Cl^– permeability under these conditions as described previously (15, 16, 26). The basolateral bath solution contained (in mM) 115 Na⁺-gluconate, 5 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, and 10 glucose. The apical bath solution contained (in mM) 120 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, and 10 mannitol (44). Mannitol was substituted for glucose in the apical bath solution to eliminate the contribution of the Na⁺-glucose cotransporter to I_sc as described previously (16). Successful permeabilization of the basolateral membrane was based on the recording of a current consistent with apical-to-basolateral flow of negative charge (16). CFBE41o– cells were bathed in solutions with apical-to-basolateral Cl^– gradient in the presence of amiloride (100 µM) in the apical bath solution to inhibit Na⁺ absorption through ENaC (29). The apical bath solution contained (in mM) 115 Na⁺-gluconate, 5 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 4 Ca²⁺-gluconate, and 10 mannitol. The basolateral bath solution contained (in mM) 120 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, and 10 glucose. MDCK monolayers were bathed in MEM solution in the presence of amiloride (100 µM) in the apical bath solution to inhibit Na⁺ absorption through ENaC.

Paracellular resistance (R_j) was determined according to Ohm's law (R = V/I) in Calu-3 monolayers under symmetrical high-Cl^– conditions with no glucose in the bath solution as described previously (37). The open-circuit voltage was recorded, and subsequently the change in I_sc in response to a 1-mV voltage pulse was measured after inhibiting the apical conductance with 20 µM thiazolidonone CFTR_inh-172 (48, 70).

Antibodies. Anti-gp114 MAb was a generous gift from Dr. Andre Le Bivic (Université de la Mediterranée, Marseille, France; Refs. 5 and 73). Other antibodies used were anti-human CFTR COOH terminus MAb, clone 24-1 (R&D Systems, Minneapolis, MN); anti-CFTR MAb, clone M3A7 (Upstate Biotechnology, Lake Placid, NY); anti-GFP JL-8 MAb (BD Biosciences, San Jose, CA); anti-transferrin receptor MAb (Zymed, San Francisco, CA); anti-Na⁺-K⁺-ATPase MAb (Upstate Biotechnology, Lake Placid, NY); and goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories, Hercules, CA). All purchased antibodies were used at the concentrations recommended by the respective manufacturers.

Determination of CFTR expression in the apical plasma membrane. To determine the effect of P. aeruginosa on the apical membrane expression of CFTR, polarized epithelial cells grown on 24-mm-diameter Transwell permeable supports were incubated at 37°C with washed bacteria or the cell-free bacterial filtrates added to the apical medium. After being incubated, the apical membrane proteins were selectively biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) (EZ-Link; Pierce Biotechnology, Rockford, IL) and isolated using streptavidin agarose beads, and the apical membrane CFTR was detected using Western blot analysis as described previously (69). Determination of the apical membrane expression of gp114 and the basolateral membrane expression of Na⁺-K⁺-ATPase and the transferrin receptor was performed by biotinylating the appropriate plasma membrane domain.

Determination of CFTR internalization from the apical plasma membrane. To determine whether P. aeruginosa caused internalization of CFTR from the apical membrane, studies were conducted as described previously (69). Briefly, apical membrane proteins in polarized MDCK cells were first biotinylated at 4°C using EZ-Link sulfo-NHS-SS-biotin. Subsequently, polarized cells were incubated with warm (37°C) PA14 filtrate added to the apical medium, and the disulfide bonds on sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by GSH added to the apical solution at 4°C. At this point, only proteins internalized from the apical membrane remained biotinylated.

Endocytic assay. Studies were conducted to determine whether PA14 decreased the apical membrane expression of CFTR by accelerating CFTR endocytosis from the apical membrane. An endocytic assay was performed on polarized MDCK cells grown on Transwell permeable supports as described previously (69). In brief, apical membrane proteins were biotinylated at 4°C using EZ-Link sulfo-NHS-SS-biotin. Subsequently, cells were either lysed or incubated with warm (37°C) vehicle (lysogeny broth) or PA14 filtrate added to the apical medium, and the disulfide bonds on sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by adding GSH to the apical solution at 4°C. Endocytosis of CFTR was calculated as the difference between the amount of biotinylated CFTR before 37°C incubation and after GSH treatment.

Endocytic recycling assay. Studies were conducted to determine whether PA14 decreased the apical membrane expression of CFTR by inhibiting the recycling of CFTR from endosomes to the apical membrane, according to a method described in detail previously (69). Briefly, apical membrane proteins were biotinylated using EZ-Link sulfo-NHS-SS-biotin at 4°C, and the endocytic vesicles were loaded with biotinylated proteins by incubation with warm vehicle (37°C lysogeny broth). Cells were cooled immediately to 4°C, and the disulfide bonds on sulfo-NHS-SS-biotinylated proteins in the apical membranes were reduced using GSH. Subsequently, cells were either lysed or incubated at 37°C with warm (37°C) vehicle or PA14 filtrate to allow internalized, biotinylated proteins to recycle to the apical membrane. Cells were then immediately cooled again to 4°C, and the disulfide bonds on sulfo-NHS-SS-biotinylated proteins in the apical membranes were reduced using GSH. Recycling of endocytosed CFTR was calculated as the difference between the amount of biotinylated CFTR after the first and second GSH treatments.

Data analysis and statistics. Statistical analysis of the data was performed using GraphPad Prism version 4.0 for Macintosh (GraphPad, San Diego, CA). Means were compared using a t-test. P < 0.05 was considered significant. Data are expressed as means ± SE.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
P. aeruginosa inhibits CFTR-mediated Cl^– secretion in Calu-3 human airway epithelial cells. Studies were conducted to examine the effects of PA14, a relatively recent laboratory isolate (59), on electrogenic Cl^– secretion across polarized human airway epithelial cells endogenously expressing WT-CFTR. Harvested bacteria were washed with PBS to eliminate products secreted into the extracellular environment and subsequently resuspended in PBS at a stock concentration of 5 x 10⁹ CFU/ml. Polarized (filter grown) Calu-3 cells were incubated at 37°C in a CO₂ incubator in the absence of antibiotics. Washed (PBS resuspended) PA14 bacteria (5 x 10⁷ CFU/ml) were added to the apical side of the monolayers, and I_sc was measured as described in MATERIALS AND METHODS. PA14 reduced the glibenclamide-sensitive, CPT-cAMP-stimulated I_sc after 4–6 h (Fig. 1, A and B). Killing PA14 by incubation at 95°C for 10 min resulted in loss of this activity (Fig. 1, A and C). The effect of PA14 was not observed after exposing the apical side of the monolayers to PA14 for <3 h (data not shown). Additional studies were conducted to determine the effects of another strain of P. aeruginosa on glibenclamide-sensitive, CPT-cAMP-stimulated I_sc. The most thoroughly studied strain of P. aeruginosa, PA01, has been passaged in the laboratory for many years. Thus the gene expression profiles and/or genomic content of PA01 and those of PA14, a relatively recent laboratory isolate, are likely to differ (27, 43, 75). Neither live nor heat-killed PA01 significantly decreased the glibenclamide-sensitive CPT-cAMP-stimulated I_sc after 4–6 h of incubation (Fig. 1, A and D).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Ussing chamber experiments performed to determine the effects of Pseudomonas aeruginosa on CFTR-mediated Cl– secretion across human airway epithelial (Calu-3) cells. Harvested bacteria were washed with PBS to eliminate products secreted into the extracellular environment and subsequently resuspended in PBS to a stock concentration of 5 x 109 colony-forming units (CFU)/ml. Polarized (filter grown) Calu-3 cells were incubated at 37°C in a CO2 incubator in the absence of antibiotics. Washed P. aeruginosa strain UCBPP-PA14 (PA14), a relatively recent laboratory strain isolated from a burn patient (59), or PA01 cells (5 x 107 CFU/ml) were added to the apical side of the monolayers, and the baseline and 8-(4-chlorophenylthiol) (CPT)-cAMP-stimulated short-circuit currents (Isc) were measured as described in MATERIALS AND METHODS. Data are expressed as net stimulated Isc, calculated by subtracting baseline Isc measured before stimulation from peak stimulated Isc. A: summary of experiments demonstrating that PA14 reduced the glibenclamide-sensitive, CPT-cAMP-stimulated Isc across polarized Calu-3 cells after 4–6 h of incubation. Killing PA14 by incubation at 95°C for 10 min resulted in loss of this activity. Neither live nor heat-killed PA01 significantly decreased the glibenclamide-sensitive, CPT-cAMP-stimulated Isc after 4–6 h of incubation. Representative Isc recordings show effects of PA14 (B), heat-killed PA14 (C), or PA01 and heat-killed PA01 (D) on CPT-cAMP-stimulated Isc after 4–6 h of incubation. n = 6–9 experiments/group; *P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Summary of Ussing chamber experiments performed to determine the effects of P. aeruginosa on CFTR-mediated Cl– secretion across the apical plasma membrane (A) and on paracellular resistance (Rj) in Calu-3 cells (B). Polarized cells were incubated with washed (i.e., PBS resuspended) PA14 added to the apical side of the monolayers. A: basolateral membrane was permeabilized with nystatin, and an apical-to-basolateral Cl– gradient was established. Isc was measured, and net stimulated Isc was calculated as described in MATERIALS AND METHODS. PA14 reduced the glibenclamide-sensitive, CPT-cAMP-stimulated Isc across the apical plasma membrane after 4–6 h of incubation. B: Rj was determined according to Ohm's law as described in MATERIALS AND METHODS. PA14 had no effect on Rj in Calu-3 cells. n = 6–9 experiments/group; *P < 0.05.

P. aeruginosa inhibits transepithelial Cl^– secretion in WT-CFTR- and F508-CFTR-expressing human airway epithelial cells (CFBE41o–). Studies were conducted to confirm that P. aeruginosa decreases transepithelial Cl^– secretion in polarized human airway epithelial cells. To this end, studies were conducted in CFBE41o– cells stably expressing either WT-CFTR or F508-CFTR. To increase trafficking and the apical membrane expression of F508-CFTR, cells were grown for 36 h at 27°C, a temperature that, at least for some cells, increases the expression of F508-CFTR in the plasma membrane (14, 19, 22). To control for any possible effects of reduced temperature on the results, WT-CFTR-expressing cells were also grown at 27°C for 36 h. CFBE41o– cells stably expressing either WT-CFTR or F508-CFTR were incubated at 27°C in a CO₂ incubator in the absence of antibiotics. Washed PA14 bacteria (5 x 10⁷ CFU/ml) were added to the apical side of the monolayers. PA14 inhibited the glibenclamide-sensitive, forskolin-stimulated I_sc in CFBE41o– cells stably expressing WT-CFTR after 4–6 h of incubation (Fig. 3A). The effect was not observed after exposing the apical side of the monolayers to PA14 for <3 h. To determine whether the inhibition of I_sc was reversible, CFBE41o– cells stably expressing WT-CFTR were first incubated with washed PA14 bacteria as described above. After 6 h of incubation, the monolayers were washed and subsequently incubated at 37°C in a CO₂ incubator with sterile medium containing antibiotics. As shown in Fig. 3B, 1 h after washing the bacteria from the apical side of the CFBE41o– monolayers, the glibenclamide-sensitive, forskolin-stimulated I_sc partially recovered to control values. As shown in Fig. 3C, PA14 also inhibited the glibenclamide-sensitive, genistein-stimulated I_sc in CFBE41o– cells stably expressing rescued F508-CFTR after 4–6 h of incubation (Fig. 3C). The effect was not observed after exposing the apical side of the monolayers to PA14 for <3 h. One hour after washing the bacteria from the apical side of the CFBE41o– monolayers, the glibenclamide-sensitive, genistein-stimulated I_sc partially recovered to control values (Fig. 3D).

View larger version (45K): [in this window] [in a new window]   Fig. 3. Summary of Ussing chamber studies performed to determine the effects of P. aeruginosa on Cl– secretion in CFBE41o– cells stably expressing either wild-type (WT)-CFTR (A and B) or F508-CFTR (C and D). Expression of F508-CFTR in the apical membrane was increased by reducing the temperature in the chambers (27°C) as described in MATERIALS AND METHODS. Cells were incubated with PA14 added to the apical side of the monolayer. A: PA14 decreased the glibenclamide-sensitive, forskolin-stimulated Isc across the CFBE41o– monolayer stably expressing WT-CFTR after 4–6 h of incubation. B: to determine whether the inhibition of stimulated Isc was reversible in the WT-CFTR-expressing CFBR41o– cells, the monolayers were washed after 6 h of incubation with PA14 and subsequently incubated at 37°C with sterile medium containing antibiotics for 1 h (PA14+Wash). The glibenclamide-sensitive, forskolin-stimulated Isc was measured and net stimulated Isc was calculated as described in MATERIALS AND METHODS. C: PA14 also decreased the glibenclamide-sensitive, genistein-stimulated Isc across the CFBE41o– monolayer stably expressing F508-CFTR after 4–6 h of incubation. D: to determine whether inhibition of stimulated Isc was reversible in F508-CFTR-expressing CFBE41o– cells, the monolayers were washed after 6 h of incubation with PA14 and subsequently incubated at 37°C with sterile medium containing antibiotics for 1 h (PA14+Wash). The glibenclamide-sensitive, genistein-stimulated Isc was measured and the net stimulated Isc was calculated as described in MATERIALS AND METHODS. n = 6–9 experiments/group; *P < 0.05.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Summary of Ussing chamber studies performed to determine the effects of P. aeruginosa on Cl– secretion in MDCK cells stably expressing either WT-CFTR or F508-CFTR or parental (nontransfected) cells. Expression of F508-CFTR was induced with sodium butyrate as described in MATERIALS AND METHODS. Cells were incubated with PA14 added to the apical side of the monolayers. PA14 reduced the glibenclamide-sensitive, CPT-cAMP-stimulated Isc across polarized MDCK cells after 4–6 h of incubation. n = 12–18 experiments/group; *P < 0.05.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Western blot analysis experiments were performed to examine the effects of P. aeruginosa on apical membrane expression of CFTR in MDCK cells stably expressing WT-CFTR. Cells were incubated with live PA14 (PA14), live PA01 (PA01), or heat-killed PA14 cells (PA14+Heat) added to the apical side of the monolayers. Live PA14 cells decreased the expression of WT-CFTR in the apical membrane after 4–6 h of incubation. In contrast, neither heat-killed PA14 nor live PA01 cells had a significant effect on the apical membrane expression of CFTR. A: representative Western blot images. P. aeruginosa had no effect on total cellular expression of CFTR. B: summary of data derived from these experiments. C: representative Western blot images demonstrating that PA14 had no effect on expression of gp114 in the apical plasma membrane, expression of Na+-K+-ATPase and transferrin receptor (TfR) in the basolateral membrane, or expression of ezrin in cell lysates. n = 3–6 experiments/group; *P < 0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Western blot experiments were performed to examine the effects of cell-free PA14 filtrate on the apical membrane expression of WT-CFTR in MDCK cells. Polarized MDCK cells were incubated with PA14 filtrate (PA14F) added to the apical side of the monolayers, and apical membrane expression of CFTR was measured using cell surface biotinylation. A: summary of the results of studies performed to determine correlation between apical membrane expression of CFTR and age of PA14 culture used to harvest PA14 filtrate. The small inhibitory effect of the lag and log phase filtrates dramatically increased in the stationary phase filtrates after only 10-min incubation with MDCK cells and was independent of the filtrate's total protein content (data not shown). Representative Western blot images (B) and graph summarizing the results of these studies (C) demonstrating that late stationary phase (16–18 h) PA14 filtrate decreased expression of WT-CFTR in the apical membrane after 10-min incubation. The inhibitory effect was absent in the heat-inactivated PA14 filtrate (PA14F+Heat). n = 3–6 experiments/group; *P < 0.05.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Representative Western blot experiments performed to determine whether cell-free PA14 filtrate affected the apical membrane trafficking of WT-CFTR in polarized MDCK cells. A: after incubation with PA14 filtrate at 37°C, cells were immediately cooled to 4°C and apical membrane proteins were biotinylated. Alternatively, after incubation with PA14 filtrate, apical membranes were washed and incubated with sterile medium at 37°C for 10 min before apical membrane proteins were biotinylated. Disappearance of CFTR from the apical membrane during incubation with PA14 filtrate was followed by partial recovery observed 10 min after washing off the PA14 filtrate. PA14 filtrate had no effect on total cellular expression of CFTR. B: cells were incubated with PA14 filtrate at 37°C for 0, 3, 5, or 10 min and then immediately cooled to 4°C, and subsequently the apical membrane proteins were biotinylated (top). Alternatively, the apical membrane proteins were first biotinylated with EZ-Link sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin), and subsequently PA14 filtrate was added to the apical medium for 0, 3, 5, or 10 min (bottom). After incubation, the disulfide bonds on sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by GSH so that only CFTR internalized from the apical membrane into an intracellular compartment remained biotinylated. P. aeruginosa caused internalization of CFTR from the apical membrane. n = 2 experiments/group.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Ussing chamber experiments were performed to determine the effects of PA14 filtrate on CFTR-mediated, CPT-cAMP-stimulated Cl– secretion across polarized Calu-3 cells. Because the effect of PA14 filtrate on apical membrane expression of CFTR was rapid and partially reversed minutes after the filtrate was washed off, studies were designed to monitor the acute effect of PA14 filtrate on the CPT-cAMP-stimulated Isc as described in MATERIALS AND METHODS. Polarized Calu-3 cells were bathed in a sterile bath solution, and Isc was stimulated using CPT-cAMP. Subsequently, 10x concentrated PA14 filtrate or vehicle (lysogeny broth) was diluted 1:10 by being added to the apical bath solution, and the change in the stimulated Isc was measured as described in MATERIALS AND METHODS. PA14 filtrate reduced the glibenclamide-sensitive CPT-cAMP-stimulated Isc after 10-min incubation. Note that vehicle alone increased the CPT-cAMP-stimulated Isc. Heat-inactivated PA14 filtrate (PA14F+Heat) lost the inhibitory effect and behaved similarly to vehicle, causing an increase in CPT-cAMP-stimulated Isc. n = 3–6 experiments/group; *P < 0.05.

View larger version (26K): [in this window] [in a new window]   Fig. 9. Endocytic assays were performed to determine the mechanism by which PA14 filtrate decreased CFTR expression in the apical membrane. After biotinylating (BT) the apical membrane proteins in polarized MDCK cells stably expressing WT-CFTR at 4°C (EZ-Link sulfo-NHS-SS-biotin), cells were incubated with warm (37°C) PA14 filtrate (PA14F) or vehicle (lysogeny broth). Subsequently, the disulfide bonds on sulfo-NHS-SS-biotinylated proteins remaining in the apical membrane were reduced by adding GSH to the apical solution at 4°C. At this point, the biotinylated proteins resided within the endosomal compartment. As demonstrated by the representative Western blot image shown in A, CFTR endocytosis was linear between 0 and 3 min in the vehicle-treated cells. Thus the data reported are from the 3-min time point. Representative Western blot image shown in B and graph summarizing the results of these studies (C) show that PA14 filtrate did not increase CFTR endocytosis. n = 3 experiments/group.

View larger version (36K): [in this window] [in a new window]   Fig. 10. Endocytic recycling assays were performed to determine the mechanism by which PA14 filtrate decreased expression of CFTR in the apical membrane. The apical membrane proteins in polarized MDCK cells stably expressing WT-CFTR were biotinylated at 4°C. Because the endocytosis of CFTR increased linearly until 3 min (see Fig. 9), the endocytic vesicles were loaded with biotinylated proteins by being incubated with warm vehicle (37°C lysogeny broth) for 3 min. Cells were then cooled to 4°C, and the disulfide bonds on sulfo-NHS-SS-biotinylated proteins in the apical membranes were reduced using GSH. Subsequently, cells were either lysed or incubated with warm (37°C) vehicle or PA14 filtrate to allow internalized, biotinylated proteins to recycle to the apical membrane. Cells were then cooled again to 4°C, and the disulfide bond on sulfo-NHS-SS-biotinylated proteins in the apical membranes was reduced using GSH. Recycling of endocytosed CFTR was calculated as the difference between the amount of biotinylated CFTR after the first and second GSH treatments. As demonstrated by the representative Western blot image shown in A, CFTR recycling was linear between 0 and 3 min in the vehicle-treated cells. Thus data reported are from the 3-min time point. Representative Western blot image shown in B and summary of the results of these studies (C) demonstrate that PA14 filtrate inhibited the endocytic recycling of CFTR. n = 3 experiments/group; *P < 0.05.

	DISCUSSION

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It is generally accepted that the major effect of P. aeruginosa on airway epithelial cells is mediated by an interaction with the basolateral membrane. For example, P. aeruginosa access to the basolateral membrane as a result of epithelial injury and/or incomplete polarization leads to increased bacterial binding, invasion, activation of proinflammatory effectors, and cytotoxicity (21, 35, 41). However, compelling evidence indicates that the interaction of P. aeruginosa with the apical membrane of polarized human airway epithelial cells also activates signaling pathways (1, 33, 34, 57). For example, the addition of P. aeruginosa to the apical surface of polarized human airway epithelial cells elicits specific changes in gene expression (33). Because the inhibition of CFTR-mediated Cl^– secretion was at least partially reversible and was not associated with a change in R_j, it is reasonable to conclude that the effects of P. aeruginosa on CFTR did not result from an injury to the monolayers. Thus, emerging data, including our observations in the present study, indicate that P. aeruginosa affects the human airway epithelial cell function even before an established endobronchial injury. To our knowledge, we have demonstrated for the first time in the present study that P. aeruginosa added to the apical side, which is the side of the airway epithelium initially exposed in vivo to pathogens (2, 58), decreases transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR. However, we cannot rule out the possibility that PA14 affects the biophysical properties of CFTR in addition to its effect on CFTR trafficking.

Studies have demonstrated that P. aeruginosa uses asialoGM1 for binding (34, 39) and CFTR for ingestion by epithelial cells (57). Data reported herein describe another mechanism of interaction between P. aeruginosa and epithelial cells, which, unlike the first two mechanisms, does not require a direct contact of the bacterium with epithelial cells and/or CFTR. Studies have demonstrated that P. aeruginosa also affects amiloride-sensitive Na⁺ transport in polarized epithelial cells. However, it is unlikely that the effects on CFTR-mediated Cl^– transport and amiloride-sensitive Na⁺ transport could be mediated by the same factors, because inhibition of Na⁺ transport is mediated by heat-stable hemolysin (68) and a nonsecreted product (20). In addition, it is unlikely that rhamnolipids could mediate the effect on CFTR, because rhamnolipids inhibit I_sc by affecting Na⁺ rather than Cl^– transport and are secreted by PA01 (25), a strain that has no effect on CFTR-mediated Cl^– transport.

The effect of P. aeruginosa on CFTR-mediated Cl^– secretion across polarized human airway epithelial cells in the present study may be clinically relevant. Strain PA14 as well as clinical isolates of P. aeruginosa (6 isolated from patients with CF and 6 isolated from patients without CF; Swiatecka-Urban A, Su JR, and Stanton BA, unpublished observations) decreased CFTR-mediated Cl^– transport in polarized human airway epithelial cells at concentrations comparable to those in patients with CF (13, 63, 72) and in severely ill patients without CF (56). Thus our study suggests that similar inhibition of Cl^– transport may occur in patients infected with P. aeruginosa. In patients without CF, the decline in CFTR-mediated Cl^– secretion may be transient. In patients with CF, chronic infection with P. aeruginosa may compromise future therapy designed to restore CFTR-mediated Cl^– transport and mucociliary clearance. Because such therapy may not work in patients with established bacterial colonization and/or chronic pulmonary infection, treatment may have to be initiated before the onset of airway bacterial colonization. Alternatively, combined therapy may require the inhibition of the secreted product or products that accelerate internalization of CFTR from the apical membrane as well as the following: 1) promotion of ER exit and plasma membrane expression of F508-CFTR, 2) activation of F508-CFTR in the apical plasma membrane, and 3) increase in the half-life of F508-CFTR in the apical plasma membrane.

In summary, our data provide the first direct evidence that P. aeruginosa inhibits CFTR-mediated Cl^– secretion across polarized human airway epithelial cells expressing either WT-CFTR or rescued F508-CFTR by specifically inhibiting the endocytic recycling of CFTR Cl^– channels. Inhibition of this effect may be necessary to allow pharmacological restoration of CFTR-mediated Cl^– transport in the airways of patients with CF.

	GRANTS

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This study was supported by National Institutes of Health (NIH) Grant P20-RR-018787 (to A. Swiatecka-Urban) and Shwachman Award SWIATE03QO from the Cystic Fibrosis Foundation (to A. Swiatecka-Urban); NIH Grant P20-RR-018787, NIH Grant R01 AI-51360-01, and a grant from the Pew Charitable Trusts (to G. A. O'Toole); and NIH Grant R01 DK-45881, NIH Grant R01 DK-34533, NIH Grant P20-RR-018787, and a Research Development Program grant from the Cystic Fibrosis Foundation (to B. A. Stanton).

	ACKNOWLEDGMENTS

 
We thank Dr. John Wakefield of Tranzyme (Birmingham, AL), who generated the CFBE41o– cells stably expressing either WT-CFTR or F508-CFTR, and Dr. J. P. Clancy of the Dept. of Pediatrics, University of Alabama at Birmingham, who provided stable CFBE41o– cells. We also thank Dr. Andre Le Bivic of l'Université de la Mediterranée (Marseille, France) for providing anti-gp114 antibody.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Swiatecka-Urban, Dept. of Physiology, Dartmouth Medical School, 1 Rope Ferry Road, HB 7701, Hanover, NH 03755 (e-mail: agnieszka.swiatecka-urban{at}dartmouth.edu)

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. Section 1734 solely to indicate this fact.

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