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Am. J. Respir. Cell Mol. Biol., Volume 26, Number 5, May 2002 617-626

Early Mitochondrial Dysfunction, Superoxide Anion Production, and DNA Degradation Are Associated with Non-Apoptotic Death of Human Airway Epithelial Cells Induced by Pseudomonas aeruginosa Exotoxin A

Maria-Cristina Plotkowski, Helvécio C. C. Póvoa, Jean-Marie Zahm, Gérard Lizard, Geraldo M. B. Pereira, Jean-Marie Tournier, and Edith Puchelle

Department of Microbiology and Immunology and Laboratory of Electron Microscopy, UERJ, Rio de Janeiro, Brazil; INSERM UMRS U514, Reims, France; INSERM U498, Dijon, France; and Laboratory of Immunopathology, UERJ, Rio de Janeiro, Brazil

    Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

It has been shown that bacterial exoproducts may induce airway epithelium injury. During the epithelial repair process, the respiratory epithelial cells no more establish tight junctional intercellular complexes and may be particularly susceptible to bacterial virulence factors. In this study, we analyzed the effect of Pseudomonas aeruginosa exotoxin A (ETA) at different periods of time and concentrations on 16 HBE 14o- human bronchial epithelial cells in culture conditions inducing a phenotype of repairing cells. ETA treatment for 24 and 48 h led to the killing of 40.0 ± 5.7% and 79.0 ± 1.4% of the cells, respectively, as determined by the dimethylthiazole 2,5 diphenyl tetrazolium bromide assay. At 1,000 ng/ml, ETA led to the killing of 25.2 ± 6.6, 59.4 ± 5.9, and 82.3 ± 3.7% of the cells, after treatment periods of 7, 24, and 48 h, respectively. Cell death could not be inhibited by z-VAD-fmk, a broad spectrum caspase inhibitor. By transmission electron microscopy, ultrastructural characteristics described in apoptosis were not detected in ETA-treated cells. Instead, the mitochondria of cells treated for 24 and 48 h with ETA at 100 and 1,000 ng/ml were highly condensed. Human nasal polyp epithelial cells in primary culture exposed to ETA at 1,000 ng/ml did not exhibit characteristic features of apoptotic cells either. Cytofluorometric analysis of ETA-treated 16 HBE 14o- cells labeled with DiOC6(3) and hydroethidine showed a time- and dose-dependent reduction of the mitochondrial transmembrane potential, detected 7 h after ETA treatment, and an increase in superoxide production, detected at 24 h, respectively. By a photometric assay, DNA degradation was also detected 7 h after cell treatment with ETA at 100 and 1,000 ng/ml. Taken together, our results show that ETA-induced death of epithelial respiratory cells was preceded by early mitochondrial dysfunction and superoxide anion production, but was not followed by the classically described apoptotic pathways.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Pseudomonas aeruginosa is a major respiratory pathogen of immunocompromised individuals and patients with cystic fibrosis (CF). Both inflammatory mediators and bacterial virulence factors likely contribute to injury of infected airway epithelia, to the shedding of surface epithelial cells, and to the persistence of P. aeruginosa infection (1).

The virulence of P. aeruginosa is multifactorial and includes a wide variety of cell-associated and extracellular proteins. Exotoxin A (ETA), reported to be the most toxic exoproduct of P. aeruginosa, is synthesized by most bacterial clinical strains (2) under the regulatory control of the lasR-lasI quorum-sensing system (3). The toxin enters into host cells via receptor-mediated endocytosis, is cleaved in the endocytic vesicles, and translocated into the cytoplasm, where it catalyzes the transfer of the ADP-ribosyl moiety of NAD+ to elongation factor 2 (EF-2). Inactivation of EF-2 leads to inhibition of protein synthesis and to host cell death (4).

In vivo studies have reported that ETA presents considerable target specificity, the inhibition of protein synthesis being greatest and earliest in the liver of inoculated mice (5). Moreover, in vitro studies have shown that cultured cell lines exhibit differential levels of sensitivity to the toxin also (6, 7). Because receptor-mediated endocytosis is a prerequisite for ETA action, variation in cell susceptibility likely depends on the availability of appropriate receptors for the toxin on cell surfaces.

ETA has been detected in respiratory secretions from patients with CF (8), but the role of the toxin in the induction of airway epithelial injury has not yet been clearly established. Indeed, treatment of hamster tracheal explant with high concentrations of purified toxin caused only a mild disorganization of the epithelium (9), whereas studies performed with type II pneumocytes and with different epithelial cell types showed that receptors for this toxin were limited to basolateral domains of cell membranes (10, 11).

In intact epithelia, junctional complexes between adjacent cells provide high-resistance intercellular seals. Intercellular tight junctions also separate apical and basolateral domains of cell membranes, assisting in the maintenance of epithelial cell polarity (12). During the repair of epithelial wounds, intercellular junctions are disrupted not only in the wounded area but also between uninjured cells at some distance from the area of trauma (13, 14), and apical membranes of airway cells exhibit molecules usually restricted to basolateral membranes (15).

Several pathogens, including some bacteria, viruses, and parasites, are able to trigger apoptosis of mammalian cells (16). Depending on the pathogen, apoptosis may be detrimental (17) or beneficial (18) to the survival of host organisms. Although P. aeruginosa infection can induce the apoptotic death of different mammalian cell types (19), both in vivo (22) and in vitro (23) studies have shown that the normal airway epithelium is highly resistant to P. aeruginosa-induced apoptosis. In contrast, cells that do not form intercellular tight junctions, or polarized cells from confluent monolayers treated with Ca2+ chelators to disrupt tight junctions, were shown to be susceptible to apoptosis induced by P. aeruginosa infection (23). The ability of P. aeruginosa to trigger apoptosis in airway cells was shown to depend also on bacterial virulence factors (23).

P. aeruginosa ETA has been reported to induce apoptosis of different mammalian cell types (24), but the effects of the toxin on the induction of apoptosis in airway cells have not been investigated so far.

In this report, we investigated the effect of purified ETA on the induction of apoptosis of epithelial airway cells of the 16 14o- HBE line in culture conditions inducing a phenotype of nonpolarized repairing airway epithelial cells (14, 27). Cells were shown to be highly susceptible to the toxin in the range of the concentrations detected in respiratory secretions from P. aeruginosa-infected patients (8). However, ETA induced a range of biochemical and morphologic changes in airway cells which are not characteristic of apoptosis. Human airway epithelial cells dissociated from nasal polyps grown in primary culture and exposed to ETA did not exhibit characteristic features of apoptotic cells either.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reagents

Purified ETA was obtained from Swiss Serum and Vaccine Institute (Berne, Switzerland). All other reagents used were from Sigma Chemical Co (St. Louis, MO), unless otherwise indicated.

Airway Epithelial Cell Culture

A 16 HBE 14o- human bronchial epithelial cell line (28), kindly provided by Dr. Gruenert (University of California at San Francisco, San Francisco, CA), was grown in 199 M culture medium containing 10% fetal calf serum, glutamine, and antibiotics (complete culture medium) on uncoated 24- or 96-well culture dishes. To assess cell culture tightness, confluent monolayers were submitted to immunolabeling of ZO-1 protein by fluorescence microscopy and to ultrastructural localization of lanthanum nitrate in intercellular spaces by transmission electron microscopy, as described previously (27). Confluent cultures were treated with ETA at 10 and 100 ng/ml, a range of concentrations detected in respiratory secretions from P. aeruginosa infected patients (8), and also at 1,000 ng/ml for different periods of time. Control cells were incubated with complete culture medium only.

In a few assays, primary cultures of human nasal polyp epithelial cells (HNPC), obtained and cultured on microplate wells coated with thin collagen I films as previously described (15), were exposed to ETA at 1,000 ng/ml for different periods.

Viability of ETA-Treated Cells

Two complementary approaches were used to investigate the cytotoxic effect of ETA: (i) the dimethylthiazole 2,5 diphenyl tetrazolium bromide (MTT) viability assay (29), and (ii) the cytofluorometric analysis of cell membrane permeability to propidium iodide which enters only into dead cells (30). In the MTT assay, ETA-treated and untreated cells were incubated with MTT at 1 mg/ml in culture medium for 1 h. Supernatants were then removed and cells were treated with isopropanol, to dissolve the formazan crystals formed in viable metabolically active cells. The A570 nm of supernatants from ETA-treated and -untreated cells was determined with an automatic microplate scanning spectrophotometer (BioRad Laboratories, Richmond, CA). The percentage of viability of toxin-treated cultures was calculated by the formula (A570 nm of ETA-treated culture/A570 nm of untreated control culture) × 100. In the analysis of cell permeability to propidium iodide by flow cytometry, ETA-treated and control untreated cells were detached from the microplate wells with a 0.05% trypsin-0.02% ethylenediaminetetraacetic acid solution and pooled with spontaneously detached cells present in cell culture supernatants. After rinsing, cells were resuspended in phosphate-buffered saline (PBS) pH 7.2 containing 1% bovine serum albumin (PBS-BSA) and incubated with propidium iodide at 5 µg/ml final concentration for 5 min at room temperature (19). Samples were transported on ice and analyzed with a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a standard argon-ion laser. The rational for this approach is that nonviable necrotic and late apoptotic cells lose their membrane integrity and are therefore permeable to the fluorescent DNA-intercalating dye (31). At least 10,000 cells were analyzed in each assay. The results were expressed as the percentage of the fluorescence medians of ETA-treated cells, taking the median of fluorescence of untreated cells as 100%.

Mitochondrial Transmembrane Potential and Superoxide Anion Production

Control untreated and ETA-treated cells were trypsinized from the microplate wells and pooled with spontaneously detached cells present in cell culture supernatants. After rinsing, cells were resuspended in PBS-BSA and incubated for 15 min at 37°C with 40 nM 3, 3' dihexyloxacarbocyanine iodide (DiOC6[3]; Molecular Probes, Eugene, OR) and with 2 µM dihydroethidine (HE). Samples were transported on ice and analyzed within 1 h with a FACScalibur flow cytometer. Assays were repeated three times and in each case at least 10,000 cells were analyzed. DiOC6(3) is a lipophilic cationic fluorochrome that accumulates in the mitochondrial matrix proportionally to their transmembrane potential (32), whereas HE is a nonfluorescent reduced probe that enters into eucaryotic cells and is converted to the fluorescent DNA- intercalating ethidium molecule through superoxide anion oxidation (33).

In a few assays, 16HBE 14o- cells were incubated with ETA at 1,000 ng/ml for 24 h. Thereafter, cells were stained for 2 h with DiOC6(3) and for 30 min with Hoechst 33258 (100 µg/ml), and the culture dishes were placed on the stage of an inverted microscope (Nikon TE300; Nikon, Tokyo, Japan) equipped with a cooled CCD camera (Micromax; Roper Scientific, Tucson, Arizona) connected to a PC computer (DELL Optiplex GX1; Dell, Austin, TX) and driven by the Metafluor software (Universal Imaging Corporation, West Chester, PA). Fluorescent images of cells stained by DiOC6(3) and Hoechst 33258 were successively recorded at 488 nm excitation/520 nm emission and 360 nm excitation/420 nm emission, respectively. Five different areas were recorded in untreated and in ETA-treated culture dishes at a ×40 magnification. The fluorescence intensity corresponding to the DiOC6(3) staining was measured in each image. As positive control, 16HBE 14o- cells were exposed for 15 s to UV radiation and fluorescent images of the mitochondrial membrane potential and nuclear DNA staining were recorded 24 h after the UV exposition, as described above. In other few assays, HNPC in primary culture were incubated simultaneously with DiOC6(3), Hoechst 33258 and ETA at 1,000 ng/ml and cell fluorescent images were successively recorded for up 72 h.

Transmission Electron Microscopy

ETA-treated and -untreated cells were fixed for 1 h at 4°C in a solution containing 2% glutaraldehyde, 4% paraformaldehyde, and 5 mM CaCl2 in 0.1M cacodylate buffer pH 7.2. After washing, cells were post-fixed for 1 h at room temperature with a solution containing 1% OsO4, 5 mM CaCl2, and 0.8% potassium ferrocyanide in cacodylate buffer, dehydrated through graded ethanol series and embedded in Epon. Ultrathin sections were observed with a 906 Zeiss transmission electron microscope. In morphometric studies, an image analyzer (SIS-Auto Image Processing System, Münster, Germany) connected to the microscope was used to draw the outlines of mitochondria from untreated and ETA-treated cells. The areas of an equal number of mitochondrial longitudinal and transverse sections were determined in untreated and in toxin-treated cells. A mitochondrial section was arbitrarily considered to be longitudinal when the relationship between the major axis versus the minor axis was >=  1.5, and transverse when this relationship was < 1.5.

DNA Fragmentation Analysis

Two different assays were used to detect and characterize DNA fragmentation : (i) conventional agarose gel electrophoresis was performed according to a method described by Herrmann and coworkers (34); (ii) photometric determination of histone-associated DNA fragments was performed with the sensitive Cell Death Detection Enzyme-Linked Immunosorbent Assay (Roche Diagnostic Corp., Mannheim, Germany), according to the manufacturer's instructions.

Effect of Caspase Inhibitor on Airway Cell Death

Cells cultured on uncoated microplate wells were treated with 100 µM of the cell-permeable tripeptide z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk; Bachem Biochimie, Voisins-le-Bretonneux, SARL), a broad spectrum inhibitor of most mammalian caspases (35), for 1 h before incubation with different concentrations of ETA in culture medium. In parallel, untreated cells were exposed to the same concentrations of the toxin. After a 24-h incubation period, the viability of zVAD-fmk-treated and -untreated cells was assessed by the MTT assay.

Statistical Analyses

Results are expressed as means ± SEM of data obtained in experiments performed at least in triplicate. Statistical differences were determined using the Student's t test. A P value below 0.05 was considered statistically significant.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Characterization of 16 HBE 14o- Cells Used to Assess the Effects of P. aeruginosa ETA

Confluent monolayers cultured on uncoated supports did not establish intercellular tight junctional complexes because they were permeable to lanthanum nitrate, which was detected delineating adjacent cells (Figure 1). Moreover, only a few cells exhibited a positive pericellular labeling with a monoclonal antibody against ZO-1, a protein characteristic of tight junctional complexes (data not shown).


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  		Figure 1.   Transmission electron micrograph of 16 HBE 14o- cells cultured on uncoated support showing the cell culture permeability to lanthanum nitrate, which penetrated into the intercellular space delineating adjacent cells (arrows). Original magnification: ×15,000.

Effect of ETA on 16 14o- HBE Epithelial Airway Cell Viability

In preliminary assays, the cells were exposed to the toxin for short periods (30, 60, and 120 min), rinsed, and incubated in complete culture medium for up to 24 h. Thereafter, viability of control and ETA-treated cells was determined by the MTT assay. Figure 2A shows that the viability of cells treated with ETA at 100 ng/ml was only slightly decreased (85.4 ± 5.0 and 78.0 ± 8.4% of control cells) after incubation period of 60 and 120 min, respectively. At 1,000 ng/ml, ETA was significantly (P < 0.01) more cytotoxic after an incubation period as short as 30 min. In other assays, cells were exposed continuously to ETA for longer periods. Cell treatment for 7 h with ETA at 10 or 100 ng/ml did not affect the viability of untight airway cells. However, when the treatment period was extended to 24 and 48 h, or when the ETA concentration was augmented to 1,000 ng/ml, cell viability decreased proportionally to the toxin concentration and incubation period, reaching only 18.2 ± 3.7% in cultures exposed to 1,000 ng/ml of the toxin for 48 h (Figure 2B; Table 1). Insofar as the MTT assay does not allow one to distinguish apoptotic from necrotic cells, toxin-treated cells were exposed to propidium iodide that enters only in cells with permeable membranes, as observed in primary necrosis and in late apoptosis (secondary necrosis), but not in early apoptosis (31). As shown in Figure 2C, cells treated with ETA at different concentrations for 7 h, and with ETA at 10 ng/ml for 24 and 48 h, did not incorporate significantly the dye, as shown by their propidium iodide labeling which was similar to or lower than the labeling of control cells. In contrast, cells treated with ETA at 100 and 1,000 ng/ml for 24 and 48 h incorporated significantly more propidium iodide than controls (Figure 2C), suggesting the occurrence of secondary necrosis. Because a few cells treated with ETA at 10 ng/ml for 24 and 48 h, and with the toxin at 1,000 ng/ml for 7 h, were shown to be dead by the MTT assay (Figure 2B), and did not incorporate the propidium iodide, we hypothesized that a few cells conserved a selective permeability of their membranes and that in these cells, death was possibly related to an apoptotic mechanism.


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  		Figure 2.   Effect of ETA at different concentrations for different periods on the viability of untight epithelial airway cells. (A and B) The ability of ETA-treated cells to reduce MTT was compared with the ability of control untreated cells, taken as 100%. Data represent mean values of three experiments performed in triplicate. Bars represent SEM. (C) The median value of the propidium iodide labeling intensity of ETA-treated cells was compared with the median value of control untreated cells, taken as 100%. Data represent mean values of three assays in which at least 10,000 cells were analyzed. Bars represent SEM. *P < 0.05 when A570nm (A and B) and the median fluorescence intensity (C) from controls was compared with ETA-treated cells. Open bars, 10 ng/ml; dotted bars, 100 ng/ml; striped bars, 1,000 ng/ml.

Mitochondrial Transmembrane Potential (Delta psi m) and Superoxide Anion Generation

Mitochondrial dysfuntion has been shown to be an early and irreversible event of apoptosis (32). To further investigate the effects of ETA on epithelial respiratory cells, control untreated and ETA-treated cells were incubated with the Delta psi m-sensitive fluorochrome DiOC6(3) and with HE, which is oxidized to ethidium by superoxide anion generation. The median values of the labeling intensity of ETA-treated cells were compared with median values of control untreated cells, referred to as 100%. As shown in Figure 3 and Table 1, ETA induced a progressive loss in mitochondrial transmembrane potential and a concomitant increase in the generation of superoxide anion. The first phenomenon frequenty occurs in early steps of apoptosis (32), whereas the second has been detected only in certain apoptotic processes (36).


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  		Figure 3.   Cytofluorometric analysis of the effect of ETA at different concentrations for different periods on the mitochondrial transmembrane potential, assessed by the DiOC6(3) labeling (A) and on the generation of superoxide anions, assessed by the labeling with hydroethidine (HE; B). In A and B, the median values of the labeling intensity of ETA-treated cells was compared with the median values of control untreated cells, taken as 100%. Data represent mean values of three assays in which at least 10,000 cells were analyzed. Bars represent SEM. Open bars, 10 ng/ml; dotted bars, 100 ng/ml; striped bars, 1,000 ng/ml. C and D are representative histograms of DiOC6(3) and HE labeling, respectively, for three different experiments. Open areas, control cells; shaded areas, ETA-treated cells.

In a few assays, 16 HBE 14o- cells cultured on glass coverslips were treated with ETA at 1,000 ng/ml for 24 h, and then exposed to DiOC6(3) and Hoechst 33258 to assess chromatin condensation. Cell observation with an inverted microscope equipped with a CCD camera showed no significant difference in mitochondrial transmembrane potential between ETA-treated and -untreated cells (mean green fluorescence intensity of 40.5 ± 5.8 and 35.5 ± 8.5, respectively; Figures 4C and 4D). Hoechst staining did not reveal any significant difference in nuclear morphology between ETA-treated and -untreated cells. In contrast, cells exposed to UV irradiation, a known inducer of cell apoptosis (37), exhibited typical apoptotic nuclear morphology, characterized by condensed and fragmented chromatin (Figure 4F).


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  		Figure 4.   Phase-contrast and fluorescence micrographs of control untreated (A and C), ETA-treated (B and D) and positive control UV-exposed 16HBE 14o- cells (E) stained by DiOC6(3) and Hoechst 33258, to assess mitochondrial membrane potential and nuclear morphology, respectively. Phase-contrast observation of ETA-treated cultures (B) revealed a dramatic detachment of the cells leading to an important reduction in cell numbers, as compared with control cultures (A). The DiOC6(3) staining of control cells (C) was not different from the staining of ETA-treated cells (D). In contrast, cells exposed to UV radiation (E) exhibited a marked decrease in the DiOC6(3) fluorescence and nuclear chromatine condensation (arrows) typically described in apoptotic cells.

It has been reported that cell lines may be prevented from exhibiting characteristic apoptotic morphology (38). To address the question whether the absence of chromatin condensation and/or fragmentation in ETA-treated 16 HBE14o- cells depended on the cell immortalization process, airway cells in primary culture were treated simultaneously with the toxin at 1,000 ng/ml, with DiOC6(3) and Hoechst 33258 and observed continuously under an inverted microscope. No significant difference in mitochondrial fluorescence or nuclear morphology was detected in HNPC exposed to ETA for as long as 48 h. In contrast, between 48 and 72 h, many cells were seen to round up and to detach from the microplate wells (data not shown).

Transmission Electron Microscopy

To further investigate the mechanism of airway cell death by ETA, ultrastructural characterisitics of apoptosis were investigated by transmission electron microscopy. A few 16 HBE cells treated with ETA at 100 ng/ml for 24 h exhibited perinuclear condensed chromatin typical of an apoptotic process, but they also presented disrupted cytoplasmic plasma membranes, suggestive of secondary necrosis (Figure 5A). These ultrastructural characteristics were not observed in untreated cells (data not shown). Other ultrastructural features of apoptosis, such as nuclear and plasma membrane blebbing, presence of cells with fragmentated and/or condensed nuclei, and presence of apoptotic bodies, were never observed in cells under ETA treatment. It is worthy of note that in many ETA-treated cells presenting unchanged nucleus, some mitochondria were found to be highly deformed and condensed (Figures 5C and 5D), compared with mitochondria from untreated cells (Figure 5B). Mitochondrial condensation was confirmed by morphometric studies: the areas of organelles from cells treated with ETA at 100 ng/ml for 24 or 48 h (0.3 ± 0.02 and 0.2 ± 0.02 µm2, respectively) were significantly lower (P < 0.01) than the areas of organelles from control cells (0.8 ± 0.05 µm2). No significant difference was detected between areas of mitochondria from controls and from cells treated with ETA at 100 ng/ml for 7 h (0.7 ± 0.06 µm2).


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  		Figure 5.   Transmission electron micrographs of epithelial airway cells treated with ETA at 100 ng/ ml for 24 h (A, C, and D) and of control untreated cells (B). Note in A the perinuclear chromatin condensation and the disruption of plasma membrane (arrow), suggestive of secondary necrosis. (original magnification: ×7,500). B shows the morphology of mitochondria from a control untreated cell (original magnification: ×35,000). C and D show condensation of the organelle matrix (arrows; original magnification: ×35,000), and the mitochondrial deformation (arrows; original magnification: ×46,000), respectively.

Analysis of DNA Fragmentation Pattern

The biochemical hallmark of apoptosis is the cleavage of chromatin into internucleosomal fragments, resulting in multimers of 180 to 200 bp (31, 38). However, instead of typical laddering pattern, large DNA fragments were observed after agarose gel electrophoresis of DNA extracted from 16 HBE or HNPC cells treated with ETA at 100 and 1,000 ng/ml for 24 h (data not shown). In contrast, the pattern of histone-associated DNA fragments detected by the enzyme-linked immunosorbent assay in ETA-treated 16 HBE 14o- cell supernatants and lysates evokes a mode of DNA fragmentation corresponding to apoptosis (Figures 6A and 6B). In necrosis, cells lose plasma membrane integrity at an early phase of cell death. In contrast, during apoptosis, the integrity of plasma membranes is maintained until the onset of secondary necrosis. Accordingly, in apoptosis, DNA fragments are likely to be detected only in cell lysates, whereas in necrosis, they can be detected both in cell lysates and supernatants. In our study, DNA fragments were detected in cell lysates 7 h after ETA treatment and their concentration increased proportionally with the toxin concentrations and incubation periods (Figure 6A and Table 1). In cell cultures treated for 24 or 48 h with ETA whatever the concentration, higher concentrations of DNA fragments were detected in cell supernatants than in control cell supernatants (Figure 6B), suggesting either the occurrence of an apoptotic process followed by secondary necrosis or a process similar to necrosis. Therefore, the analysis of the mode of DNA fragmentation occurring in the presence of ETA revealed a complex process evoking apoptosis at early times of treatment and necrosis at late times of treatment.


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  		Figure 6.   Photometric detection of DNA fragments in lysates (A) and in supernatants (B) of epithelial airway cell cultures treated with ETA at different concentrations for different periods. Data represent mean values of a typical out of two assays performed in triplicate. Bars represent SD. *P < 0.05 when controls (solid bars) were compared with data from ETA-treated cells. Open bars, 10 ng/ml; dotted bars, 100 ng/ml; striped bars, 1,000 ng/ml.

Effect of Caspase Inhibitor on ETA-Induced Airway Cell Death

Different studies have shown the role of caspases in ETA-induced eukaryotic cell apoptosis (25, 26). Therefore, to further investigate the mechanism of airway cell death, zVAD-fmk, a broad spectrum caspase inhibitor, was added to 16 HBE 14o- cells 1 h before a 24-h incubation period with ETA. As shown in Figure 7, zVAD-fmk had no significant effect on ETA-induced airway cell death, as assessed by the MTT assay.


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  		Figure 7.   Effect of cell treatment with z-VAD-fmk on the viability of ETA-treated 16 HBE 14o- cells, assessed by the MTT test. The ability of ETA-treated cells to reduce MTT was compared with the ability of control untreated cells, taken as 100%. Data represent mean values of a typical assay performed in quadruplicate. Bars represent SD. Striped bars, z-VAD-fmk+; open bars, z-VAD-fmk-.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Infections of the respiratory tract by P. aeruginosa are very frequent in CF and in immunocompromised patients. High proteolytic activity in sputa of these patients, mainly derived from polymorphonuclear leukocytes, has been shown to account for severe pulmonary damage (39). It is less clear, however, whether specific P. aeruginosa extracellular enzymes may contribute to airway damage in these patients.

ETA is a potent inhibitor of protein synthesis produced by most of the clinical isolates of P. aeruginosa. Jaffar and colleagues (8) have shown a positive correlation between the level of this toxin in bronchial secretions and the exacerbation periods in CF. However, insofar as ETA has been reported to bind to alpha 2-macroglobulin receptors (40), located exclusively on basolateral membranes of polarized cells, the mechanism used by this toxin to contribute to epithelial damage in P. aeruginosa-infected patients remained to be determined. The main finding of this report is that ETA induces the death of airway epithelial cells, which are reminiscent of repairing epithelial cells found in remodeling airway epithelium, that do no exhibit intercellular tight junctional complexes (14). Interestingly, it has been recently reported by Rajan and coworkers (23) that the susceptibility of epithelial respiratory cells to P. aeruginosa- induced apoptosis is at least partially dependent on the integrity of tight junctional complexes. Our results not only confirm the susceptibility of airway cells that do not establish tight intercellular junctional complexes to the cytotoxic effect of bacterial exoproducts; they also demonstrate that ETA is a P. aeruginosa virulence factor that may be directly involved in airway cell death.

Studies performed in the early 1990s (24, 41) reported that ETA, as well as other inhibitors of protein synthesis, induced apoptosis of mammalian cells characterized by chromosomal DNA degradation into oligonucleosome-sized fragments, chromatin condensation, and cell nuclei fragmentation. Further studies have confirmed the capability of ETA to induce cell death by apoptosis (25, 26, 42). However, in our experimental conditions, 16 HBE 14o- cells did not exhibit any morphologic changes described in apoptotic cells, as assessed by fluorescence videomicroscopy and by transmission electron microscopy.

In previous studies, it has been reported that cells may undergo apoptosis although lacking some nuclei and cellular morphologic features of apoptotic cells (43, 44), and that cell lines may be prevented from exhibiting apoptotic characteristics through the loss of signal transduction pathways or metabolic components during the immortalization process (38). To ascertain whether the lack of apoptotic characteristics in ETA-treated 16 HBE 14o- cells resulted from the immortalization process, we analyzed the morphologic changes in cells exposed to ionizing radiation, a known inducer of apoptosis (37). In contrast to cells exposed to ETA, UV-treated cells exhibited characteristic nuclei shrinkage and chromatin condensation, easily visualized after nucleous staining with the DNA-binding Hoechst fluorophore. Because different cell types may differ in their susceptibility to apoptotic stimuli, we then investigated the effects of ETA on airway cells in primary culture. For this study, HNPC were grown on a thin film of collagen I, to prevent the establishment of tight junctional complexes (27). After treatment with ETA at 1,000 ng/ml, and with the fluorophores DiOC6(3) and Hoechst, images of the cells were recorded for a 72-h period. Unexpectedly, HNPC did not show nuclear changes or mitochondrial depolarization reported in apoptotic cells, but within 48-72 h they were seen to detach from the microplate wells and to die. Altogether, these results raised doubts about the capability of ETA to induce apoptosis of 16 HBE cells. We therefore investigated additional criteria currently used to define the mechanism of cell death, based on mitochondrial activity, DNA fragmentation pattern, and effect of caspase inhibitor on cell viability.

Mitochondrial changes are known to play key roles in various apoptotic processes (45). In particular, the loss of mitochondrial transmembrane potential has been proposed as a constant, early, and irreversible event of apoptosis (46, 47). However, mitochondrial depolarization has also been described in autophagy and in necrosis (48). In our study, flow cytometric analysis of ETA-treated 16 HBE 14o- labeled with DiOC6(3) showed a time- and dose-dependent depolarization of the mitochondrial membranes. In contrast, when investigated by video microscopy, no significant difference in mitochondrial transmembrane potential was detected between ETA-treated and -untreated cells. The differences between the results obtained with these two approaches may stem from the kind of cells under study. In the FACS assays, both airway cells that had detached spontaneously from the monolayer cultures under the effect of the toxin, and cells detached from the microplate wells by trypsinization, were analyzed. In contrast, in the videomicroscopy assays, only cells remaining adherent to the glass coverslips, which correpond to cells that had to be trypsinized from the microplates in the FACS assay, were analyzed.

In response to mitochondrial dysfuntion, large transmembrane pores are likely to open, a phenomenon known as the mitochondrial permeability transition (MPT). Activation of MPT is associated with the translocation from the mitochondria into the cytosol of numerous mitochondrial proteins such as cytochrome c (49), apoptosis-inducing factor (AIF) (50), and Smac/DIABLO (51). Activation of MPT also allows ions to emerge from mitochondria, and the intake of water into the mitochondrial matrix, resulting in osmotic swelling and rupture of the cristae and of the organelle outer membrane (47, 52). In our study, as well as in others (53, 54), the presence of mitochondrial condensation and the lack of mitochondrial swelling suggest that the activation of MPT played a minor role in ETA-induced cell killing. Interestingly, condensed mitochondria have been described by Laiho and coworkers (55) and by Papapdimitriou and colleagues (56) as a short-term response to cell injury. In these latter studies, however, condensation was followed by a rapid return to the normal configuration, followed by the development of morphologic changes that are normally associated with necrosis. Unlike this transient response to injury, the changes observed in our study were present in cells treated with ETA for as long as 48 h.

In addition to morphologic changes of mitochondria and to the loss of mitochondrial transmembrane potential, we also observed an increase in superoxide anions in ETA-treated cells. As oxygen intermediate anions react with macromolecules, either damaging them directly or setting in motion a chain reaction wherein the free radical is passed from one macromolecule to another, superoxide anion production may induce modifications at various cellular levels. In the present work, the enhanced production of superoxide anions may have produced cell membrane damage revealed by increased permeability to propidium iodide as well as by the irregular degradation of DNA (57).

DNA degradation into oligonucleosome-sized fragments resulting from the activation of endonucleases has been considered the biochemical hallmark of apoptosis. This produces a characteristic laddering pattern of cleavage when DNA from apoptotic cells is subjected to conventional agarose gel electrophoresis. In our study, no typical ladder pattern of fragmentation was detected by agarose gel electrophoresis of DNA extracted from ETA-treated 16 HBE or HNPC cells in primary culture. Cleavage of DNA to 50-kbp fragments, preceeding the appearance of 180- to 200-bp oligonucleosomal fragments, has been detected in several models of epithelial and mesenchymal cell apoptosis (58). In one of these models, although the cells exhibited classic apoptotic morphology, no subsequent internucleosomal cleavage was observed. Because DNA laddering pattern can no longer be considered mandatory in an apoptotic cell death, we next assessed DNA fragmentation in ETA-treated cells by using a photometric determination of histone-associated DNA fragments assay. DNA fragments were detected in the cytoplasm of ETA-treated cells that preserved the integrity of their plasma membranes, a feature described in apoptotic cells.

Finally, in our attempt to define the capability of ETA to induce apoptosis of airway cells, we analyzed the effects of cell treatment with the permeable caspase inhibitor z-VAD-fmk on 16 HBE cell viability. Due to an aspartate residue mimicking the cleavage site and a fmk group forming a covalent inhibitor/enzyme complex, the inhibitor instantly and irreversibly binds to the catalytic site of caspases. In our study, no significant difference was observed between z-VAD-fmk- treated and -untreated cell viability. This result contrasts with those obtained in other studies on the effect of P. aeruginosa ETA on mammalian cells, in which cell apoptosis could be significantly inhibited by the caspase inhibitor (25, 26). Taken together, our observations suggest that ETA induces the death of airway cells that mimic repairing cells found in wounded epithelia by a yet unknown mechanism different from the classically described apoptosis mechanism. We speculate that in infected patients ETA-induced death of airway cells may represent an additional mechanism of epithelial injury favoring the persistence of bacterial infection.

                              
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TABLE 1
Characteristics of untight epithelial respiratory cells treated with ETA at different concentrations for different periods*

    Footnotes

Address correspondence to: Maria-Cristina Plotkowski, Department of Microbiology and Immunology, FCM/ UERJ, Av. 28 de Setembro, 87 fundos, 3° andar. 20 551-030, Rio de Janeiro, Brazil. E-mail: mcplot{at}uerj.br

Abbreviations: cystic fibrosis, CF; elongation factor 2, EF-2; exotoxin A, ETA; dihydroethidine, HE; human nasal polyp epithelial cells, HNPC; mitochondrial permeability transition, MPT; dimethylthiazole 2,5 diphenyl tetrazolium bromide, MTT.
(Received in original form January 3, 2001 and in revised form January 15, 2002)

Acknowledgments: The authors thank Dr. Claude Galabert (CERM, Hôpital Renée Sabran, Giens, France) for providing the exotoxin A, Iris M. P. Alvim for her help with flow cytometric analysis, and Marco Antônio de F. Maciel for his help with transmission electron micrographs. This work was supported by grants from Association Vaincre la Mucoviscidose (France), FAPERJ, and from FINEP/MCT/PRONEX (41 960 881.00; Brazil).
    References
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

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