Chronic lung infection with Pseudomonas aeruginosa constitutes the most severe manifestation of cystic fibrosis, a scenario that results from defects in early clearance of the microbe. Early clearance involves epithelial cell ingestion of bacteria, rapid activation of nuclear factor-κB and cellular desquamation within minutes of P. aeruginosa infection, processes that are deficient in cells with mutant alleles of Cftr. Analyzing the effect of Cftr genotype on the apoptotic response of airway epithelial cells to P. aeruginosa, we found that human bronchial epithelial cells expressing ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) underwent significantly delayed apoptosis compared with cells expressing wild-type (WT) CFTR. Mice with a WT Cftr allele had apoptotic cells in their lungs after P. aeruginosa infections, whereas mice homozygous for the ΔF508 or G551D Cftr alleles showed little apoptosis in response to acute infection. Pseudomonal infection induced expression of CD95 and CD95 ligand, a response that was also delayed in cells homozygous for mutant Cftr alleles. Thus, WT CFTR expression promotes a rapid expression of CD95/CD95 ligand and apoptotic response to P. aeruginosa infection. Prompt apoptosis of infected epithelial cells may be critical for clearance of P. aeruginosa, and CFTR-associated defects in apoptosis may contribute to the pathogenesis of the lung disease in cystic fibrosis.
A successful response to infection by a microbial pathogen requires activation of an effective inflammatory reaction followed by resolution and return to normal tissue activity. A key mechanism of resolution is apoptosis and removal of cells that have responded to the invading pathogen (1, 2). Epithelial cells on tissue and mucosal surfaces are likely the first to encounter an invading microorganism, and after binding and internalization of the microorganism, coupled with cellular activation leading to production of inflammatory mediators, can serve as part of the overall host defense response. Cells with bound and internalized bacteria can remove the bacteria from the mucosal surface by desquamation, a process that likely occurs via apoptosis of the epithelial cells. For example, uropathogenic Escherichia coli induces apoptosis of bladder epithelial cells after infection, resulting in sloughing of the infected cells and excretion of the organisms (3). Streptococcus pyogenes binds to its receptor, CD44, on the surface of pharyngeal epithelial cells (4), becomes internalized, induces apoptosis (5), and, likely, clearance of the organisms within the infected cell. Inocula of these organisms that cause pathogenic infections presumably overwhelm this defense mechanism or take advantage of the denuded epithelium to enter submucosal areas (6). One such organism, Pseudomonas aeruginosa, must invade buried corneal epithelial cells to initiate experimental keratitis (7), whereas internalization of low inocula of the bacteria by lung cells is essential for clearance from the respiratory tract (8). This internalization event is defective in lung epithelial cells lacking functional cystic fibrosis transmembrane conductance regulator (CFTR), which underlies the hypersusceptibility of patients with cystic fibrosis (CF) to chronic infection with this bacterium.
Over 80% of patients with CF will develop chronic P. aeruginosa infection, indicating a key function for the CFTR in innate immunity to lung infection with this pathogen. In addition to its role as an ion channel (9–11), wild-type (WT) CFTR acts as specific receptor for the binding and internalization of P. aeruginosa by respiratory epithelia (8, 12). This ingestion event serves a protective function against P. aeruginosa infection in the normal lung (8, 12). The steps following internalization of the CFTR/P. aeruginosa complex that lead to clearance of the organism are only partially known, and apoptosis could be a key component in both induction of shedding of the infected cell, as well as termination of proinflammatory signaling.
Although several studies have investigated the effect of CFTR on apoptosis of epithelial cells expressing either WT or ΔF508 CFTR (13–15), only one study examined this effect in the time proximal to P. aeruginosa infection (15). These investigators concluded that there were no differences in the apoptotic response of cells expressing either WT or mutant CFTR to infection with P. aeruginosa. In our more detailed investigations of the kinetics of apoptosis induction, however, we found that the apoptotic response to infection was strongly influenced by the CFTR genotype of the infected cells. As the studies reported here demonstrate, cultured cells expressing ΔF508 CFTR make a delayed apoptotic response to P. aeruginosa compared with cells expressing WT CFTR. Moreover, lungs from P. aeruginosa–infected CF mice show no apoptosis 3 h after infection, whereas apoptotic cells are readily detected in similarly infected controls. We found increased expression of both CD95 and CD95 ligand in WT CFTR–expressing cells after pseudomonal infection, with kinetics that mirror the apoptotic response. Thus, our data indicate that expression of WT CFTR by respiratory epithelial cells allows a brisk increase in expression of CD95 and CD95 ligand after infection with P. aeruginosa, resulting in apoptosis of the infected cell. The sluggish apoptotic response of cells expressing mutant CFTR after pseudomonal infection may contribute to the failure of the innate host response to early infection and ultimately to the overall pathogenesis of CF lung disease.
CFT1 cells are respiratory epithelial cells derived from a ΔF508 homozygous patient, which have been immortalized by transfection with human papilloma virus 18 E6 and E7 genes (16). CFT1-LCFSN cells are CFT1 cells complemented with a full-length WT Cftr introduced via a retrovirus, CFT1-ΔF508 cells are control cells transfected with a third ΔF508 Cftr, and CFT1-LC3 cells are transfected with the gene encoding β-galactosidase. Transfected CFT1 cell lines are grown in Ham's F-12 medium containing 10 μg/ml insulin, 1 mM hydrocortisone, 3.75 μg/ml endothelial cell growth supplement, 25 ng/ml epidermal growth factor, 30 nM triiodo-L-thyronine, 5 μg/ml transferrin, and 10 ng/ml cholera toxin (hormone-supplemented F12 medium). Neomycin at 150 μg/ml was used for culture maintenance, but removed before experiments with microorganisms. IB3 cells are bronchial epithelial cells from a ΔF508/W1282X patient that are adeno–12-SV40 immortalized (17). The S9 line was generated from IB3 cells by transfection with a recombinant adeno-associated viral vector encoding WT CFTR. Both S9 and IB3 cells are grown in LHC-8 Basal Medium (Biofluids, Inc., Rockville, MD) supplemented with 5% fetal bovine serum (FBS) and 1:100 Antibiotic-Antimycotic (contains 10,000 U/ml penicillin G, 10,000 μg/ml streptomycin, and 25 μg/ml amphotericin B; Invitrogen, Grand Island, NY). All culture flasks and plates were coated with a solution containing 35 μg collagen /ml (Vitrogen 100; Cohesion, Palo Alto, CA), 1 μg/ml BSA, and 1 μg/ml fibronectin for both S9 and IB3 lines. Mouse mammary tumor cell line C127 transfected with either WT or ΔF508 Cftr was propagated as described previously (8, 18).
Frozen stocks of P. aeruginosa strain PAO1, an isogenic mutant, PAO1 algc::tet, unable to synthesize the lipopolysaccharide (LPS) outer core, or clinical P. aeruginosa isolates 6294 and 324, were streaked on tryptic soy agar plates and grown overnight at 37°C. P. aeruginosa expressing green fluorescent protein, kindly provided by Dr. George O'Toole (19), was grown on tryptic soy agar plates supplemented with 1 mg/ml carbenicillin. The bacteria were diluted in Ham's F12 medium to an optical density at 650 nm (O.D. 650) of 0.4, which corresponds to 2 × 109 colony-forming units (cfu)/ml of bacteria, and appropriately diluted for use in different assays. Bacteria were in stationary phase growth at the time of inoculation. For experiments with bacteria in log phase growth, 5 ml of Luria Broth was inoculated with PAO1, and grown overnight at 37°C. The broth was diluted to an O.D. 650 of 0.1, grown at 37°C for 1 h, centrifuged, washed with phosphate-buffered saline (PBS) and resuspended to an O.D. 650 of 0.4.
CFT1 cells were grown to confluence in hormone supplemented F12 medium, rinsed with PBS, and incubated at 37°C with 106 cfu/ml P. aeruginosa in antibiotic-free, hormone-supplemented F12 (CF12) for specified times. The initial inoculum was verified by serial dilution and plating. At the end of the incubation period, the medium was changed to CF12 with neomycin and the tissue cultures incubated overnight at 37°C. S9 and IB3 cells were grown to confluence in FBS-supplemented LHC-8 media, rinsed with PBS, and incubated at 37°C with 106 cfu/ml P. aeruginosa in antibiotic-free CF12 for specified times. Use of antibiotic-free FBS-supplemented LHC-8 media resulted in bacterial killing, so CF12 was substituted. The uninfected monolayers of S9 and IB3 cells appeared healthy in CF12 at the end of the overnight incubation. Cells were collected by trypsinization, washed, and stained via one of two different protocols to detect apoptosis: (i) FITC-annexin (ApoAlert Annexin V Apoptosis Kit; Clontech, Palo Alto, CA), or (ii) FITC-M30 antibody (M30 CytoDEATH kit; Roche, Basel, Switzerland). Annexin staining detects phosphatidyl serine flipped to the outer leaflet of the plasma membrane, an early apoptotic event. M30 is a monoclonal antibody to a cytokeratin 18 cleavage product of caspase 6 produced early in apoptosis of epithelial cells. Stained cells were analyzed by flow cytometry on either a Facscalibur Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ) or the MoFlo Cell Sorter (Cytomation, Fort Collins, CO). CellQuest software was used for data analysis (Becton-Dickinson). Cytochalasin was obtained from Sigma (St. Louis, MO); PhiPhiLux from OncoImmunin (Gaithersburg, MO); and doxorubicin, puromycin, staurosporin, paclitaxel, and aphidocolin from Calbiochem (La Jolla, CA) (Apoptosis Inducer Set II, used according to manufacturer's instructions).
Mice were anesthetized with isoflourane and each animal infected with 1 × 108 cfu of strain PAO1 suspended in F12 by application of 10-μl aliquots onto each of the animal's nares as they are cradled at a 45° angle. Control animals were anesthetized and 10-μl aliquots of sterile F12 placed on the their nares. After 4 h, the animals were sacrificed by CO2 asphyxiation, the lungs harvested, and the left lung fixed with formalin, embedded in paraffin, and sectioned. Slides were stained with hematoxylin and eosin (H&E) to confirm infection, and with the FITC-dUTP TUNEL assay plus propidium iodide (PI) as a counterstain to assess apoptosis (In Situ Cell Death Detection kit; Boehringer Mannheim, Indianapolis, IN). The TUNEL assay detects DNA fragmentation, a late apoptotic event. Sections were viewed by fluorescent or confocal microscopy.
Epithelial cell monolayers were incubated with a multiplicity of infection of 10 cfu of P. aeruginosa PAO1 per cell for 3 h, then collected by trypsinization and scraping. To kill extracellular, but not intracellular, bacteria, epithelial cells were suspended in media containing 200 μg/ml gentamicin and incubated at 37°C for 45 min. Apoptosis was assessed using the ApoAlert Annexin V Apoptosis Kit (Clontech). Using the MoFlo cell sorter, the 10,000 cells at the highest and lowest extremes (top and bottom 5%) of fluorescence were collected into separate tubes. Events were gated using PI staining to exclude necrotic cells. The sorted cells were then lysed with 0.05% Triton X 100 and the released intracellular bacteria plated onto Pseudocel agar for enumeration.
CFT1-LCFSN cells were grown on glass-bottom microwell dishes (MatTek Corp., Ashland, MA), inoculated with bacteria for the indicated times and stained for apoptosis with annexin V-PE (Pharmingen, San Diego, CA) with 7AAD counterstain (Molecular Probes, Eugene, OR).
Previous studies in our laboratory have demonstrated that increased uptake of P. aeruginosa into respiratory epithelial cells with WT CFTR was correlated with clearance of the organism from the lung (12). To examine the hypothesis that internalization and desquamation may be linked to apoptosis of bacteria-laden cells, CFT1 cells transfected with either WT Cftr, a third copy of ΔF508 Cftr or a control gene were exposed to P. aeruginosa PAO1 and flow cytometry studies were performed using the monoclonal antibody M30, which stains a neoepitope of cytokeratin 18 produced by epithelial cells after cleavage by caspase 6. Exposure of the different lines of transfected CFT1 cells to PAO1 at a multiplicity of infection (MOI) of 1:1 resulted in apoptosis of an increasing proportion of cells as the duration of exposure to the organism increased (Figure 1). The WT CFTR expressing cells reached a plateau of greater than 90% of the cells apoptotic after 4 h of exposure to PAO1 in log phase growth or 5 h of exposure to PAO1 initially in stationary phase growth (Figures 2A and 2B) . The time course for induction of apoptosis in all three lines after exposure to bacteria in stationary phase growth lagged by ∼ 1 h compared with that obtained in the comparable cell line after exposure to bacteria in log phase growth. Although the cells expressing only mutant CFTR underwent apoptosis after exposure to PAO1, the time course appeared delayed by ∼ 1 h for the ΔF508 cells and 2 h for the LC3 cells, compared with the WT CFTR–expressing LCFSN cells. Thus, the WT CFTR–expressing cells appeared more susceptible to apoptosis induced by exposure to P. aeruginosa.
The delay in apoptosis after P. aeruginosa infection in CFTR-defective cells is not limited to the CFT1 cell line. Similar flow cytometry experiments were performed using the cell line IB3, an adeno–12-SV40 immortalized bronchial epithelial cell line derived from a ΔF508/W1282X patient, and the S9 derivative of the IB3 cells generated by transfection with a recombinant adeno-associated viral vector encoding WT CFTR. Although both S9 and IB3 cells appeared more resistant to apoptosis than the CFT1 cells, the time course for induction of apoptosis in the WT CFTR expressing S9 cells was significantly accelerated by ∼ 3 h compared with the IB3 cells after exposure to PAO1 in stationary phase growth at an MOI of 1:1 (Figure 2C).
The difference in the apoptotic response to infection was not limited to infection with strain PAO1, as a clinical isolate from a patient with CF that was obtained early in the course of infection, strain 324, induced apoptosis in WT CFTR–expressing cells more readily than in cells expressing only ΔF508 allele of Cftr (Figure 3). Indeed, after 4 h of exposure to strain 324, ∼ 90% (median 90, range 60–99) of WT CFTR–expressing cells were apoptotic, whereas only 45% (median 45, range 45–62) of cells expressing three copies of ΔF508 Cftr allele had undergone apoptosis. This ∼ 2-fold difference in proportion of apoptotic cells was also seen in experiments with another clinical strain of P. aeruginosa, 6294 (60% apoptotic WT-CFTR [median 60, range 42–71] versus 35% apoptotic ΔF508 CFTR [median 35, range 18–45]), and is comparable to that obtained with PAO1-exposed epithelial cells (64% apoptotic WT-CFTR cells [median 64, range 51–83] versus 48% apoptotic ΔF508 CFTR cells [median 48, range 16–62]), although fewer cells had undergone apoptosis by 4 h of exposure to bacteria. The difference in apoptosis was not due to differences in the growth rate of the bacteria in contact with the different epithelial cell types, as the numbers of live bacteria found in the medium after 6 h of incubation did not differ among the cell types (data not shown).
Infection of CFT1-LCFSN cells and ΔF508 cells with a similar MOI of the P. aeruginosa strain PAO1 algc::tet, a mutant that lacks the LPS outer core–binding ligand for CFTR, induced significantly less apoptosis in both the WT and ΔF508 CFTR cell lines (Figure 3). Although there was a 2-fold increase in the proportion of apoptotic cells after infection of cells expressing WT CFTR compared with cells expressing ΔF508 CFTR (24% apoptotic WT-CFTR cells [median 24, range 22–33] versus 12% apoptotic ΔF508 CFTR cells [median 12, range 9–28]), the difference was not statistically significant. Thus, in the absence of a P. aeruginosa–CFTR interaction, induction of apoptosis in airway epithelial cells is markedly reduced and the difference in responses between WT and ΔF508 CFTR cells is not observed.
To determine whether P. aeruginosa induced apoptosis in a WT CFTR–dependent manner in vivo, we inoculated mice intranasally with 108 cfu of P. aeruginosa strain PAO1. Four hours after inoculation the lungs were harvested, fixed, sectioned, and stained with a TUNEL assay to detect apoptotic cells. Infected mice demonstrated patchy areas of inflammation in the lung as visualized on H&E stains (data not shown). Equivalent areas examined for TUNEL-positive cells showed a striking absence in mice homozygous for either the ΔF508 or G551D Cftr alleles (Figures 4A and 4C), whereas heterozygous littermates had TUNEL-positive cells both in the lumen of bronchi (Figures 4B and 4D, arrows) or scattered among the columnar epithelial cells lining the bronchi (Figure 4D). Thus, the ability to efficiently undergo apoptosis after infection with P. aeruginosa is reduced in transgenic CF mice.
As we had noted previously that interaction of P. aeruginosa with functional CFTR resulted in internalization and clearance of the organism (8, 20), we asked whether apoptosis is dependent on epithelial cell internalization of the bacteria. To assess the association between internalization and apoptosis, CFT1 cells, as well as cells from the mouse mammary tumor cell line C127, transfected with either WT or ΔF508 Cftr, were incubated with P. aeruginosa, treated with gentamicin to kill extracellular bacteria, stained with FITC-annexin, and sorted into most and least fluorescent 10% of cells. The two cell populations were lysed, and the released bacteria plated and counted. Apoptosis correlated significantly with internalization of bacteria for the WT CFTR–expressing cells, but not for cells expressing only ΔF508 CFTR (Figure 5). Interestingly, the magnitude of the ingestion of P. aeruginosa by the transfected mouse cells was considerably higher than for the human cells. Nonetheless, the relative difference in ingestion of P. aeruginosa by apoptotic cells expressing WT versus ΔF508 CFTR was the same. Among the cells not included in the top and bottom 5% gates set to sort into most likely and least likely to be undergoing apoptosis are the vast majority of cells (90%), a large proportion of which were also undergoing apoptosis. Thus, this assay does not quantify the proportion of the original inoculum that may be cleared via apoptosis of the epithelial cells that ingested the bacteria, but only serves to establish a link between internalization of bacteria and apoptosis of cells expressing WT CFTR. Internalization of the organism by apoptotic WT CFTR–expressing cells was confirmed by confocal microscopy (Figure 6) .
Although internalization was associated with induction of apoptosis, internalization does not appear to be necessary for initiation of apoptosis, as cells treated with cytochalasin, which inhibits internalization of bacteria through depolymerization of the actin cytoskeleton, still undergo apoptosis after infection (Figure 7A). At high doses, cytochalasin treatment can induce apoptosis in uninfected cells. However, the dose used in these experiments was low enough that the increase in apoptosis in treated cells was minimal (Figure 7B). Nonetheless, invasion assays in which cells were treated with cytochalasin at the same low dose used to assess apoptosis, inoculated with bacteria, treated with gentamicin, lysed, and plated, confirmed that invasion was inhibited (data not shown). Thus, internalization of P. aeruginosa by respiratory epithelial cells is not necessary to initiate apoptosis, although apoptosis is still associated with P. aeruginosa interacting with WT CFTR on epithelial cells.
To investigate the mechanism of apoptosis induction by infection with P. aeruginosa, monolayers of WT CFTR–expressing cells were loaded with a dye that fluoresces upon cleavage by activated caspase 3 (PhiPhiLux) before inoculation with the bacteria (Figure 8A). The number of activated caspase 3–positive cells lagged slightly behind that of the caspase 6 (M30)-positive cells (Figure 8B), which is consistent with the observation that these caspases are sequentially activated in apoptosis initiated by detachment of intestinal epithelium from the underlying basement membrane (21).
The CFTR-dependent difference in apoptosis of respiratory epithelial cells appears to be specific for apoptosis induced by interaction of the cells with P. aeruginosa, as no consistent differences were noted in the numbers of apoptotic cells expressing either WT or ΔF508 CFTR produced after incubation with a variety of apoptosis-inducing drugs (Figure 8C). Although with exposure to all of the drugs, except puromycin, a slightly increased percentage of apoptotic cells were seen among those expressing WT CFTR, the amount of apoptosis induced in WT and ΔF508 CFTR was remarkably similar.
Grassmé and colleagues demonstrated that P. aeruginosa infection induces apoptosis of lung epithelial cells through activation of CD95/CD95 ligand, a process that was critical to clearance of the organism (22). We investigated whether the CFTR genotype of the respiratory epithelial cells altered the expression of CD95 or CD95 ligand during the course of infection with P. aeruginosa. Both CD95 and CD95 ligand are constitutively expressed on resting epithelial cells at levels that paralleled the CFTR functionality (Figure 9). The LC3 cell line containing two copies of the ΔF508 Cftr allele expressed the least. The ΔF508 CFTR cells with three of these alleles expressed intermediate amounts, and LCFSN cells with WT CFTR expressed over double the basal amounts of CD95 or CD95 ligand found on the surface of LC3 cells. Following P. aeruginosa infection, the LCFSN cells significantly increased surface expression of both CD95 and CD95 ligand within 3 h. The levels continued to rise as length of exposure to bacteria increased. Similarly, the ΔF508 and LC3 cells show a rise in CD95/CD95 ligand expression beginning at 3 h, although, just as seen with the rate of apoptosis, the rate of increased expression lags by ∼ 30 min for ΔF508 and 1 to 2 h for LC3. This delay in CD95/CD95 ligand expression by the cells expressing only mutant CFTR mirrors the delay in apoptosis of these cells after infection with P. aeruginosa and may contribute to an inappropriate resolution of infection in tissues expressing mutant Cftr alleles.
The role that apoptosis of respiratory epithelial cells plays in the pathogenesis of pulmonary infection with P. aeruginosa remains controversial. The studies by Grassmé and colleagues would suggest that induction of apoptosis in lung epithelial cells via the CD95/CD95 ligand system is critical for clearance of the organism. They demonstrated extensive apoptosis of bronchial epithelial cells after infection with P. aeruginosa, a response that was absent in mice deficient in either CD95 (gld mice) or CD95 ligand (lpr mice). The CD95 or CD95 ligand–deficient animals uniformly succumbed to pseudomonal sepsis when inoculated intranasally, whereas only 10% of control animals developed sepsis and died with similar inoculation (22). These results have been challenged by Hotchkiss and coworkers (23), who nasally inoculated mice with P. aeruginosa, as did Grassmé and colleagues, and detected no bronchial epithelial apoptosis via any method other than TUNEL. As an equal number of TUNEL-positive bronchial epithelial cells were detected in infected and control animals, they argue that the TUNEL method can give false positive results in cell types with high endonuclease activity. Grassmé and coworkers have countered that bronchial epithelial cells indeed undergo apoptosis, as noted by electron microscopy (23). They further showed that internalization and activation of apoptosis is dependent on the growth phase of the P. aeruginosa, as only organisms in mid-growth phase interact with epithelial cells. As it is well established that P. aeruginosa levels in the lungs of mice inoculated intranasally increase over a short time period (20, 24), it appears reasonable to conclude that log-phase organisms are more representative of the infecting microbes than are stationary phase organisms.
That CFTR may play a role in the initiation of apoptosis of lung epithelial cells during interaction with P. aeruginosa seems even more contested. Maiuri and colleagues examined intestinal and bronchial biopsy specimens of patients with CF, using the TUNEL assay to stain for apoptosis (13). Approximately 46% of villus enterocytes from 14 small intestinal biopsies of patients with CF showed DNA fragmentation, compared with only 3% of control enterocytes. CF bronchial epithelial cells from two patients undergoing lobectomy due to severe P. aeruginosa infection showed more evidence of DNA fragmentation, particularly in the submucosal glands, than did normal control subjects, as 57% of CF cells stained positive with the TUNEL assay versus only 5% of normal cells. However, because these patients were severely and chronically infected with P. aeruginosa, results with their cells would not be comparable to the process under investigation here, in which early apoptotic responses to P. aeruginosa were found to be deficient in cells with mutant CFTR. Clearly, prolonged exposure of epithelial cells to P. aeruginosa leads to apoptosis, but one cannot compare severely infected patients with CF with uninfected control subjects. Even if this group had compared infected patients with CF with infected patients expressing WT CFTR, they might still have concluded that patients with CF exhibit increased apoptosis of respiratory epithelial cells. Vandivier and coworkers have reported that removal of apoptotic cells is delayed in patients with CF and bronchiectasis, due to cleavage of the phosphatidylserine receptors on the surface of apoptotic bodies by neutrophil elastase impairing uptake by phagocytes (25). Thus, in the setting of a polymorphonuclear infiltrate, as is typical of chronic infection in patients with CF, a slower rate of epithelial apoptosis might be masked by the delayed clearance of apoptotic material.
Gottlieb and Dosanjh reported that C127 cells expressing ΔF508 CFTR failed to undergo apoptosis in response to treatment with cyclohexamide or etoposide, whereas isogenic cells expressing the WT CFTR underwent apoptosis with similar treatment (13). The authors proposed that CF cells might show an inability to undergo apoptosis due to an inability to acidify their cytoplasm. Acidification has been observed as an early event in the apoptotic cascade due to the activation of an acid endonuclease required for DNA cleavage (26). Functional CFTR is required for cytoplasmic acidification perhaps acting as an HCO−3 channel itself, or through its interaction with other transporters (27). Thus, defective CFTR leads to defective capacity to undergo cytoplasmic acidification and the inability of cells expressing mutant CFTR to proceed normally through programmed cell death. Because Gottlieb and Dosanjh used cell lines, whereas Maiuri evaluated tissues from patients, it is difficult to compare the results. One explanation offered by Maiuri and colleagues for any potential discrepancy is that CF epithelial cells may undergo increased initiation of apoptosis, but fail to appropriately complete the cell death program. They suggest that the cells may undergo DNA nicking, as seen by the TUNEL assay, but fail to complete the process of DNA degradation by endonucleases. In support of this argument, they noted that the architecture of the intestinal villi was normal despite DNA fragmentation, indicating, perhaps, that the normal process of apoptosis leading to cell shedding at the villus tip was aborted.
Whereas these two groups found differences in apoptosis related to the CFTR genotype of the cells, Rajan and colleagues did not (15). They examined the apoptotic response of several CF and corrected cell lines to infection with P. aeruginosa strain PAO1 and found that apoptosis occurred in cells that lacked tight junctions, but did not occur in infected cells with intact tight junctions. The CFTR phenotype of either the cell lines or the cftr−/− mice did not seem to affect the rates of apoptosis. Furthermore, Gallagher and Gottlieb revisited the findings that they reported earlier (28), and found that transfection of C127-ΔF508 cells with an adenovirus construct containing the WT CFTR did not correct the apparent apoptosis defect noted in their previous study (26). C127 cells or CFT1 cells exposed to UV irradiation underwent apoptosis at the same rates regardless of the CFTR genotype of the cells. They reconciled their previous results with these by postulating that the CFTR-dependent differences in apoptosis noted initially may have been due either to a delay in transport of the apoptosis-inducing drug in the CFTR mutant cells, or an inherent resistance to apoptosis acquired by the C127 ΔF508 cells in the clonal selection process. The authors conclude that CFTR does not play a role in a cell's susceptibility to apoptosis.
Our studies yield the opposite conclusion, namely, that the CFTR genotype of the respiratory epithelial cell significantly affects apoptosis induced by infection with P. aeruginosa. As demonstrated in both in vitro and in vivo models, pseudomonal infection results in apoptosis of a population of WT CFTR expressing respiratory epithelial cells, while cells expressing only defective CFTR demonstrate delayed or no apoptosis in response to infection. The PAO1 algc::tet strain that lacks the LPS outer core ligand that binds CFTR (12) induces significantly less apoptosis than the parental PAO1 strain without inducing a significant difference in apoptosis between WT and ΔF508 CFTR-expressing cells. Clinical strains 324 and 6294 were comparable to strain PAO1 in their apoptosis-inducing effect. Rajan and coworkers also noticed the need for a complete LPS molecule to initiate apoptosis (15). Apoptosis correlates with internalization of P. aeruginosa in WT, but not mutant, CFTR expressing cells. However, as shown previously by several groups (29, 30), internalization of P. aeruginosa is not necessary to initiate apoptosis. The interaction of the organism with CFTR likely initiates mechanisms involved in both processes. For example, we have shown that interaction of P. aeruginosa with the respiratory epithelial cell also increases CD95 and CD95 ligand expression in a CFTR-dependent fashion providing a mechanism for efficient apoptosis. Internalization of the organism has been linked to efficient clearance in a CFTR dependent fashion (8), and can be blocked by incubation with a peptide corresponding to the first extracellular loop of CFTR (12, 31).
The differing results between our studies and the most closely related work of Rajan and colleagues (15) may result from methodological differences that could shed light on the apoptotic process in epithelial cells after pseudomonal infection. For example, Rajan and coworkers looked at apoptosis in a similar in vitro infection model using cultured cells, but used as the apoptosis detection method mitochondrial permeability changes, whereas we measured caspase 6 activation using a surrogate marker–cytokeratin 18 cleavage. They noted no significant differences in the percent apoptotic cells between cells expressing mutant and WT CFTR. Our combined results may indicate an uncoupling of mitochondrial permeability changes and caspase activation in CFTR-defective cells, two processes that may not be linked in P. aeruginosa–induced apoptosis as suggested by others (29). The experiment showing no difference in apoptosis in vivo between WT and transgenic CF null mice was performed in animals infected for 3 d, whereas our differences were found after 4 h. The delayed apoptosis we find in CF cells early following P. aeruginosa infection could be a manifestation of the delayed and inappropriate response by the CF epithelium to early infection. In contrast, the comparable response to apoptosis found 3 d after infection in WT and CF mice likely reflects reactions to a more prolonged exposure, and also represents an infection not encountered normally in non-CF humans. Thus, during prolonged infection the differences in apoptotic responses may not be present.
The increased expression of CD95 and CD95 ligand on epithelial cells after infection by P. aeruginosa has been demonstrated by several groups (29, 32). Durieu and coworkers found increased expression of CD95 and CD95 ligand in lung sections obtained from patients with CF colonized with Staphylococcus aureus or P. aeruginosa compared with uninfected control subjects. However, no previous study has highlighted the CFTR dependence of the efficient expression of CD95/CD95 ligand. As CD95/CD95 ligand–dependent apoptosis has also been linked to clearance of P. aeruginosa (22), one might speculate that the sequence of internalization of the bacteria followed by apoptosis of the bacteria laden epithelial cells allows for clearance in a manner analogous to that of uropathogenic E. coli infection of bladder epithelium (3). In CF this process is compromised, perhaps contributing to establishment of chronic P. aeruginosa infection.
An alternate mechanism for aberrant apoptosis of CF epithelial cells after pseudomonal infection is suggested by the studies of Jungas and colleagues (33), who found that cells expressing WT CFTR are more susceptible to apoptosis induced by oxidative stress compared with cells expressing mutant CFTR. The mutant CFTR transports glutathione less rapidly out of the cells allowing protection against oxidative stress due to its direct action as an antioxidant, as well as delayed expression of the pro-apoptotic BCL-2 family member, BAX, which is activated in a glutathione-dependent fashion. This mechanism might be most relevant in the in vivo infection setting, in which neutrophils exert an oxidative stress on the respiratory epithelium while responding to the infection.
Regardless of the underlying mechanism, the decreased internalization and delayed apoptosis of cells expressing only mutant CFTR may contribute to the pathogenesis of pseudomonal infection in the patient with CF by allowing for more prolonged bacterial residence time on the lung mucosa that gives the microbe the opportunity to proliferate and establish infection. Once established, the chronic infection and inflammatory response does lead to ongoing inflammation and apoptosis in the lung, but it is the failure to initiate and resolve P. aeruginosa infection shortly after bacterial exposure within the CF lung that underlies the hypersusceptibility of these patients to P. aeruginosa infection.
This work was supported by National Institutes of Health grants AI22806 and HL58398 to G.B.P. C.L.C was supported by NIH/NIHLB grant HL04277. The authors thank Michelle Lowe for her invaluable assistance at the Confocal and Multi-photon Imaging Core Facility, Brigham and Women's Hospital. They also thank Gloria Meulini and Fadie Coleman for help in breeding the ΔF508 and G551D mice and Ervin Meulini for sectioning the mouse lungs. They thank Vincent Carey for his helpful input regarding the statistical analysis.
|1.||Haslett, C. 1997. Granulocyte apoptosis and inflammatory disease. Br. Med. Bull. 53:669–683.|
|2.||Ishii, Y., K. Hashimoto, A. Nomura, T. Sakamoto, Y. Uchida, M. Ohtsuka, S. Hasegawa, and M. Sagai. 1998. Elimination of neutrophils by apoptosis during the resolution of acute pulmonary inflammation in rats. Lung 176:89–98.|
|3.||Mulvey, M. A., Y. S. Lopez-Boado, C. L. Wilson, R. Roth, W. C. Parks, J. Heuser, and S. J. Hultgren. 1998. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science 282:1494–1497.|
|4.||Cywes, C., I. Stamenkovic, and M. R. Wessels. 2000. CD44 as a receptor for colonization of the pharynx by group A Streptococcus. J. Clin. Invest. 106:995–1002.|
|5.||Tsai, P. J., Y. S. Lin, C. F. Kuo, H. Y. Lei, and J. J. Wu. 1999. Group A Streptococcus induces apoptosis in human epithelial cells. Infect. Immun. 67:4334–4339.|
|6.||Cywes, C., and M. R. Wessels. 2001. Group A Streptococcus tissue invasion by CD44-mediated cell signaling. Nature 414:648–652.|
|7.||Zaidi, T. S., J. Lyczak, M. Preston, and G. B. Pier. 1999. Cystic fibrosis transmembrane conductance regulator-mediated corneal epithelial cell ingestion of Pseudomonas aeruginosa is a key component in the pathogenesis of experimental murine keratitis. Infect. Immun. 67:1481–1492.|
|8.||Pier, G.B., M. Grout, T. S. Zaidi, J. C. Olsen, L. G. Johnson, J. R. Yankaskas, and J. B. Goldberg. 1996. Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271:64–67.|
|9.||Kunzelmann, K. 1999. The cystic fibrosis transmembrane conductance regulator and its function in epithelial transport. Rev. Physiol. Biochem. Pharmacol. 137:1–70.|
|10.||Schwiebert, E. M., D. J. Benos, M. E. Egan, M. J. Stutts, and W. B. Guggino. 1999. CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79:S145–S166.|
|11.||Ramsey, B. W. 1996. Management of pulmonary disease in patients with cystic fibrosis. N. Engl. J. Med. 335:179–188.|
|12.||Pier, G. B., M. Grout, and T. S. Zaidi. 1997. Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc. Natl. Acad. Sci. USA 94:12088–12093.|
|13.||Maiuri, L., V. Raia, G. De Marco, S. Coletta, G. de Ritis, M. Londei, and S. Auricchio. 1997. DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis? FEBS Lett. 408:225–231.|
|14.||Gottlieb, R. A., and A. Dosanjh. 1996. Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. Proc. Natl. Acad. Sci. USA 93:3587–3591.|
|15.||Rajan, S., G. Cacalano, R. Bryan, A. J. Ratner, C. U. Sontich, A. van Heerckeren, P. Davis, and A. Prince. 2000. Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors. Am. J. Respir. Cell Mol. Biol. 23:304–312.|
|16.||Yankaskas, J. R., J. E. Haizlip, M. Conrad, D. Koval, E. Lazarowski, A. M. Paradiso, C. A. Rinehart, Jr., B. Sarkadi, R. Schlegel, and R. C. Boucher. 1993. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am. J. Physiol. 264:C1219–C1230.|
|17.||Flotte, T. R., R. Solow, R. A. Owens, S. Afione, P. L. Zeitlin, and B. J. Carter. 1992. Gene expression from adeno-associated virus vectors in airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 7:349–356.|
|18.||Marshall, J., S. Fang, L. S. Ostedgaard, C. R. O'Riordan, D. Ferrara, J. F. Amara, H. Hoppe IV, R. K. Scheule, M. J. Welsh, A. E. Smith, and S. H. Cheng. 1994. Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro. J. Biol. Chem. 269:2987–2995.|
|19.||Bloemberg, G. V., G. A. O'Toole, B. J. Lugtenberg, and R. Kolter. 1997. Green fluorescent protein as a marker for Pseudomonas spp. Appl. Environ. Microbiol. 63:4543–4551.|
|20.||Schroeder, T. H., N. Reiniger, G. Meluleni, M. Grout, F. T. Coleman, and G. B. Pier. 2001. Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract. J. Immunol. 166:7410–7418.|
|21.||Grossmann, J., S. Mohr, E. G. Lapentina, C. Fiocchi, and A. D. Levine. 1998. Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells. Am. J. Physiol. 274:G1117–G1124.|
|22.||Grassmé, H., S. Kirschnek, J. Riethmueller, A. Riehle, G. von Kurthy, F. Lang, M. Weller, and E. Gulbins. 2000. CD95/CD95 ligand interactions on epithelial cells in host defense to Pseudomonas aeruginosa. Science 290:527–530.|
|23.||Hotchkiss, R. S., W. M. Dunne, P. E. Swanson, C. G. Davis, K. W. Tinsley, K. C. Chang, T. G. Buchman, and I. E. Karl. 2001. Role of apoptosis in Pseudomonas aeruginosa pneumonia. Science 294:1783|
|24.||Allewelt, M., F. T. Coleman, M. Grout, G. P. Priebe, and G. B. Pier. 2000. Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread. Infect. Immun. 68:3998–4004.|
|25.||Vandivier, R. W., V. A. Fadok, P. R. Hoffmann, D. L. Bratton, C. Penvari, K. K. Brown, J. D. Brain, F. J. Accurso, and P. M. Henson. 2002. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. J. Clin. Invest. 109:661–670.|
|26.||Gottlieb, R. A., J. Nordberg, E. Skowronski, and B. M. Babior. 1996. Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification. Proc. Natl. Acad. Sci. USA 93:654–658.|
|27.||Illek, B., J. R. Yankaskas, and T. E. Machen. 1997. cAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia. Am. J. Physiol. 272:L752–L761.|
|28.||Gallagher, A. M., and R. A. Gottlieb. 2001. Proliferation, not apoptosis, alters epithelial cell migration in small intestine of CFTR null mice. Am. J. Physiol. Gastrointest. Liver Physiol. 281:G681–G687.|
|29.||Jendrossek, V., H. Grassmé, I. Mueller, F. Lang, and E. Gulbins. 2001. Pseudomonas aeruginosa-induced apoptosis involves mitochondria and stress-activated protein kinases. Infect. Immun. 69:2675–2683.|
|30.||Evans, D. J., D. W. Frank, V. Finck-Barbancon, C. Wu, and S. M. Fleiszig. 1998. Pseudomonas aeruginosa invasion and cytotoxicity are independent events, both of which involve protein tyrosine kinase activity. Infect. Immun. 66:1453–1459.|
|31.||Esen, M., H. Grassmé, J. Riethmuller, A. Riehle, K. Fassbender, and E. Gulbins. 2001. Invasion of human epithelial cells by Pseudomonas aeruginosa involves src-like tyrosine kinases p60Src and p59Fyn. Infect. Immun. 69:281–287.|
|32.||Durieu, I., C. Amsellem, C. Paulin, M. T. Chambe, J. Bienvenu, G. Bellon, and Y. Pacheco. 1999. Fas and Fas ligand expression in cystic fibrosis airway epithelium. Thorax 54:1093–1098.|
|33.||Jungas, T., I. Motta, F. Duffieux. P. Fanen, V. Stoven, and D. M. Ojcius. 2002. Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 277:27912–27918.|