Phospholipase A2 Functions in Pseudomonas aeruginosa- Induced Apoptosis
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感染与免疫杂志 2006年第2期
Department of Medical Microbiology, Technische Universitat Munich, 81675 Munich, Germany
Department of Molecular Biology, University of Duisburg-Essen, 45122 Essen, Germany
ABSTRACT
Pseudomonas aeruginosa, a gram-negative, facultative pathogen, causes severe and often even lethal infections in immunocompromised patients, as well as cystic fibrosis patients. We show here that a variety of P. aeruginosa strains activate phospholipase A2 (PLA2), cultured epithelial cells, and fibroblasts, resulting in increased intracellular and extracellular arachidonic acid release. The use of different PLA2 inhibitors revealed that P. aeruginosa-induced arachidonic acid release is mediated by activation of cytosolic PLA2 (cPLA2), whereas iPLA2 or sPLA2 do not seem to be involved in the response to P. aeruginosa. Likewise, the cPLA2-specific inhibitors MAFP and AACOCF3 prevented apoptosis of cultured epithelial cells upon P. aeruginosa infection, whereas inhibitors specific for iPLA2 or sPLA2 were without effect. The physiological significance of these findings is indicated by an inhibition of apoptosis in tracheal epithelial cells upon in vivo infection with P. aeruginosa. The data indicate that arachidonic acid generation by activation of cPLA2 during P. aeruginosa infection plays an important role in the induction of host cell death.
INTRODUCTION
Pseudomonas aeruginosa infections are a severe clinical problem, in particular in immunocompromised individuals and in patients with cystic fibrosis (CF) (53). CF patients are highly susceptible to P. aeruginosa bacteria, leading to chronic lung infection in the majority of these patients. This lung disease is characterized by persistent pulmonary inflammation that finally results in fibrosis and destruction of lung tissue and respiratory failure. At present, P. aeruginosa is the leading cause of mortality in CF patients. Infection of mammalian cells with P. aeruginosa results in the release of cytokines (11, 23, 45), internalization of the bacteria (20, 40, 41), and induction of apoptosis of the infected host cells (5, 19-21, 42). We sought here to identify novel mechanisms of P. aeruginosa-induced apoptosis. It was previously shown that mice unable to respond with host cell apoptosis to pulmonary infection with P. aeruginosa are highly sensitive to the infection, develop sepsis, and die (19). Further, it was demonstrated that P. aeruginosa-induced apoptosis is delayed in cells lacking functional cystic fibrosis conductance regulator (5), whose mutations cause cystic fibrosis. Grassme et al. (19), Jendrossek et al. (25), and Cannon et al. (5) demonstrated an upregulation of the CD95-CD95-ligand system on the surface of infected cells that resulted in apoptosis of P. aeruginosa-infected cells by ligation of the endogenous CD95 receptor. Genetic deficiency of CD95 prevented P. aeruginosa-induced apoptosis (19, 25), suggesting a central role of the CD95-CD95-ligand system for P. aeruginosa-initiated cell death. Further, an involvement of mitochondria (in particular mitochondrial depolarization, synthesis of reactive oxygen intermediates, and release of cytochrome c), an activation of Jun N-terminal kinases (JNK), and a stimulation of caspases were demonstrated to be involved in the induction of apoptosis in mammalian cells upon infection with P. aeruginosa (19, 25, 26).
In the present study we tested a potential role of phospholipases A2 (PLA2) in P. aeruginosa-triggered cell death. PLA2 are lipolytic enzymes that hydrolyze membrane phospholipids and thereby release fatty acids, particularly arachidonic acid from the sn-2 position of glycerophospholipids (13). PLA2 plays an important role in signal transduction, in particular by generation of proinflammatory mediators as prostaglandins and leukotrienes and by membrane remodeling. Several subtypes of mammalian PLA2 have been described that are divided in four main groups according to their function, localization, and calcium dependency. Secretory PLA2 (low molecular mass enzymes belonging to groups I, II, III, V, and X) are cysteine-rich, secreted proteins that require millimolar concentrations of Ca2+ for activity without a preference for a specific fatty acid in the sn-2 position of the phospholipid substrate (13). The second class of PLA2 includes specific acetylhydrolases such as platelet-activating factor. A third class is composed of Ca2+-independent PLA2, e.g., iPLA2 isolated from myocardium (55), CHO cells, and macrophages (1, 28). The group IV cytosolic PLA2 (cPLA2) includes three PLA2 named , , and . cPLA2, an 85-kDa protein, requires micromolar Ca2+ concentrations for activity and has a preference for arachidonic acid (8). The recently described cPLA2, a 110-kDa protein, shows 30% sequence identity with cPLA2 and also depends on Ca2+ but is less selective for cleavage at the sn-2 position than cPLA2 (39, 49). cPLA2 with a molecular mass of 61 kDa and 29% sequence identity with cPLA2 is Ca2+ independent but distinguishable from iPLA2 by its preference for arachidonic acid at the sn-2 position (3). Previous studies implied PLA2 in the host response to such diverse pathogens as Staphylococcus aureus, Escherichia coli, Aeromonas hydrophila, Pasteurella haemolytica, and Bacillus anthracis (6, 9, 16, 18, 24, 44, 54). PLA2 seem to have a dual function in the infection of mammalian cells by pathogens, since many bacterial toxins seem to kill mammalian cells via PLA2 (4, 12, 16, 18, 27), whereas expression of PLA2 was shown to protect mice from acute infections at least with S. aureus, P. aeruginosa, and E. coli (15, 33). Thus, the exact function of PLA2 in infectious processes requires definition.
In the present study, we investigated the role of PLA2 for the in vitro and in vivo infection of cultured epithelial cells or fibroblasts, respectively, as well as tracheal epithelial cells with P. aeruginosa. We demonstrate an activation of PLA2 by a variety of P. aeruginosa strains and reveal by the use of a panel of PLA2 inhibitors a significant role of cPLA2 for the induction of host cell apoptosis by P. aeruginosa both in vitro and in vivo.
MATERIALS AND METHODS
Materials and cell culture. The human conjunctiva epithelial cell line Chang (ATCC CCL 20.2) was cultured in RPMI 1640 (Gibco-BRL/Life Technologies) supplemented with 5% fetal calf serum (FCS) at 37°C as monolayers in tissue culture flasks in 5% CO2 atmosphere. The human lung fibroblast cell line WI-38 was maintained in minimal essential medium supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1% nonessential amino acids, and 1% penicillin-streptomycin. Infections were performed in RPMI 1640 supplemented with 10 mM HEPES (pH 7.2) to avoid interactions of serum proteins with the bacteria. Prior to infections, FCS and antibiotics were omitted from the culture media. The PLA2 inhibitors MAFP, BEL, 12-Episcalaradial, and AACOCF3 were purchased from Biomol.
Bacterial strains. Three clinical isolates and two laboratory strains of P. aeruginosa were used. The isolate 762 was originally obtained from an urinary tract infection, strain 696 was isolated from the sputum of a hospitalized patient, and strain 769 was from a patient with urosepsis (18). The laboratory strains used were P. aeruginosa ATCC 27853 and PAO-1. Furthermore, we infected cells with a previously described (26) P. aeruginosa strain deficient for the type III secretion system (P. aeruginosa PAK pcrD::Smr, kindly provided by J. Heesemann), S. aureus strain ATCC 8325, and a nonpathogenic E. coli isolate.
Infection experiments. Bacteria originating from glycerol stock cultures were plated overnight on tryptic soy agar plates at 37°C, resuspended in tryptic soy broth (TSB) at an optical density at 550 nm of 0.25, shaken at 120 rpm for 1 h at 37°C, and harvested during logarithmic growth phase by pelleting and resuspension in fresh TSB. Prior to infection, cells were washed twice in RPMI 1640 (Chang cells) or minimal essential medium (WI-38 cells) and maintained in the same medium during infection. Infection was performed by inoculating subconfluent cell layers at a host cell/bacterium ratio of 1:1,000, 1:100, 1:50, or 1:10. Synchronous infection conditions and an enhanced bacterium-host cell interaction were achieved by a 2-min centrifugation (35 x g) of the bacteria onto the cells. The end of the centrifugation step was defined as the starting point of all infections.
Arachidonic acid release assays. Chang cells (0.4 x 105 per well) or WI-38 cells (0.3 x 105 per well) were labeled for 18 to 20 h with 0.05 μCi of [3H]arachidonic acid [5,6,8,9,11,12,14,15-3H(N)] (0.1 mCi/ml stock; New England Nuclear)/ml. Prior to infection, cells ware washed three times with a buffer containing 132 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, and 0.8 mM MgSO4 (H/S) supplemented with 1% bovine serum albumin and 10 mM glucose and incubated in the same buffer during infection. Infection was performed as described above at the indicated multiplicity of infection (MOI).
Cells were preincubated with the PLA2 inhibitors MAFP, 12-episcalaradial, and bromoenol lactone (BEL) at the indicated concentrations for 15 min prior to infection. The slow-acting inhibitor AACOCF3 was added to the cells 3 h prior to infection.
Extracellular arachidonic acid release. To determine the release of arachidonic acid into the extracellular space, supernatants were collected after the indicated time of infection and centrifuged at 2,000 rpm for 5 min in a microcentrifuge to remove any cells that may have detached. The supernatants were quantified by liquid scintillation counting. The remaining cell-bound radioactivity was determined by adding 0.5 ml of 0.1% Triton X-100 in H2O to the cells, and lysed cells were removed from the plates and subjected to liquid scintillation counting. Extracellular arachidonic acid release was calculated as the percentage of radioactivity released into the supernatant of the total radioactivity (the latter being the sum of released and cell-bound radioactivity). Since only free arachidonic acid is released into the supernatant, the measurement of radioactivity in the supernatant is an accurate measurement for the formation of extracellular arachidonic acid.
Intracellular arachidonic acid release. Chang cells were seeded at a density of 3 x 105 cells/well in six-well plates. Labeling with [3H]arachidonic acid, pretreatment with PLA2 inhibitors, and infection were performed as described above. Infections were terminated by removal of the supernatants, washing, and the addition of 1 ml of methanol-15% acetic acid to each well. Cells were scraped off the plates, and wells were washed with 0.5 ml of methanol. Chloroform (0.75 ml) was added, and samples were extracted by incubation for 30 min at room temperature. Next, 0.75 ml each of chloroform and H2O were added, and the phases were separated by low-speed centrifugation for 10 min. The lower phase containing the fatty acids was removed, dried, solubilized in 30 μl of petrol ether-diethyl ether (96:1), spotted onto a silica gel 60 plate, and separated by thin-layer chromatography (TLC) in a solvent system consisting of ethyl acetate-glacial acetic acid-2,2,4-trimethyl pentane-H2O (45:10:25:50, by volume). An arachidonic acid standard was run on each plate to confirm migration of free arachidonic acid. After separation, TLC plates were air dried and a Biomax MS film was exposed at –80°C for several days. To quantify free arachidonic acid, spots representing arachidonic acid were scraped off the plates and subjected to liquid scintillation counting. The TLC analysis permits separation of free arachidonic acid from arachidonic acid incorporated into lipids and, therefore, the analysis of free intracellular arachidonic acid.
Detection and quantification of apoptosis. Apoptosis was quantified by fluorescein isothiocyanate (FITC)-annexin V staining and confirmed by trypan blue staining. To this end, cells were infected as described above, treated with trypsin, washed twice in 10 mM HEPES (pH 7.4), 140 mM NaCl2, and 5 mM CaCl2 and resuspended in the same buffer supplemented with FITC-annexin V (dilution 1:50; Roche Biochemicals, Mannheim, Germany). After incubation for 15 min cells were subjected to fluorescence-activated cell sorter (FACS) analysis using a FACSCalibur flow cytometer using the CELLQuest software (Becton Dickinson, Mountain View, Calif.).
In vivo infections. To perform infection of tracheal epithelial cells in vivo, C57BL/6 wild-type mice were anesthetized by intraperitoneal injection of avertin, the trachea was carefully isolated, and the distal part was catheterized and ligated immediately proximal of the catheter. We then injected MAFP, AACOCF3, or buffer into the proximal part of the trachea. After an incubation period of 15 min for MAFP, we injected 108 CFU of P. aeruginosa 762 into the proximal trachea. Mice were killed after 45 min, and the tracheas were rapidly excised and fixed for 30 min in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.2). Epithelial cells were isolated by disintegration of the trachea by scratching with a 27-gauge needle and a 5-min digestion in trypsin. Cells were washed and stained with FITC-annexin V to detect apoptosis. Apoptosis of epithelial cells in the trachea was also determined by TUNEL staining. To this end, the trachea was excised and fixed in 4% PFA in PBS (pH 7.2) for 36 h. The tissues were embedded in paraffin, and sections were deparaffinized and treated for 5 min in 0.1 M citrate (pH 6.0) with 350-W microwave irradiation for 5 min. The sections were then stained for 30 min at 37°C with FITC-coupled dUTP in the presence of terminal deoxynucleotidyl transferase. The samples were washed, incubated at 70°C for 10 min to reduce unspecific binding, and stained with alkaline phosphatase-coupled anti-FITC-antibodies. The slides were finally developed using Fast Red Tablets (Roche) as a substrate resulting in the formation of a red dye.
RESULTS
P. aeruginosa induces extra- and intracellular arachidonic acid release upon infection of epithelial cells. To gain insight into the interactions of P. aeruginosa with epithelial cells, we investigated the role of PLA2 upon bacterial infection. To this end, we measured P. aeruginosa-induced PLA2 activity by quantification of the extracellular and intracellular arachidonic acid release from two different cell lines, i.e., Chang conjunctiva epithelial cells and WI-38, a lung fibroblast cell line. Intracellular arachidonic acid release was determined by prelabeling cells with [3H]arachidonic acid, extraction of fatty acids, and subsequent separation by TLC. Extracellular arachidonic acid release was determined by labeling cells with [3H]arachidonic acid and quantification of released radioactivity in the supernatant by liquid scintillation counting. The latter data were related to the total radioactivity recovered from the samples. The results revealed a marked increase of free, intra- and extracellular arachidonic acid after bacterial infection with the P. aeruginosa strains 696, 762, 769, ATCC 27853, and PAO-1 (Fig. 1 and Fig. 2).
The release of arachidonic acid occurred very rapidly after infection with P. aeruginosa (Fig. 1) and was dependent on the MOI used in the infection experiments (Fig. 2). However, even at the lowest MOI of 10 bacteria/cell, we were still able to detect some release of arachidonic acid (Fig. 2). A type III mutant of P. aeruginosa did not induce a release of intra- or extracellular arachidonic acid (Fig. 2A).
The release of arachidonic acid was not restricted to P. aeruginosa and was also observed upon infection with a different pathogen, i.e., S. aureus (Fig. 2A). In contrast, nonpathogenic E. coli were unable to trigger a significant release of arachidonic acid (Fig. 2A).
Arachidonic acid release induced by P. aeruginosa infection is diminished by cPLA2 inhibitors but not by sPLA2 or iPLA2 inhibitors. Next, we aimed to further characterize the PLA2 subclass involved in P. aeruginosa-induced arachidonic acid release. To this end, we investigated the influence of various PLA2 inhibitors on arachidonic acid release into the supernatant and on cell-bound arachidonic acid release. Two inhibitors (MAFP, AACOCF3) specific for both cPLA2 and iPLA2 were used, as well as the sPLA2-specific inhibitor 12-Episcalaradial and the iPLA2-specific inhibitor BEL. The results reveal that MAFP and AACOCF3 significantly reduced intra- and extracellular arachidonic acid release upon infection with P. aeruginosa strains 696, 762, 769, and ATCC 27853 (Fig. 2). A dose-response analysis revealed that doses of 1 μM MAFP or 2 μM AACOCF3, respectively, were already sufficient to reduce arachidonic acid release (Fig. 3A and B). In contrast, neither the iPLA2 inhibitor BEL nor the sPLA2 inhibitor 12-episcalaradial affected P. aeruginosa-induced arachidonic acid release upon infection of WI-38 or Chang cells (Fig. 3C and D).
Since MAFP specifically blocks cPLA2 and iPLA2, whereas BEL specifically targets iPLA2 and 12-Episcalaradial inhibits type II sPLA2, the data suggest that cPLA2 is activated by P. aeruginosa.
The cPLA2 inhibitors MAFP and AACOCF3 reduce P. aeruginosa-induced apoptosis. To gain insight into the functional significance of arachidonic acid release, we investigated the role of PLA2 for the induction of apoptosis during P. aeruginosa infection. P. aeruginosa-induced apoptosis of epithelial cells has been previously demonstrated (5, 19, 20) and seems to be critical for the defense of the host against acute infections with these bacteria (19). First signs of apoptosis as determined by breakdown of phosphatidylserine asymmetry in the cell membrane were detected within 15 to 30 min after infection with P. aeruginosa 762. MAFP and AACOCF3 dose dependently inhibited P. aeruginosa-induced apoptosis (Fig. 4A and B), whereas pretreatment with 12-Episcalaradial (sPLA2 inhibitor) (Fig. 4C) was without effect on apoptosis induction. Inhibition of apoptosis by MAFP and AACOCF3, respectively, was not restricted to P. aeruginosa strain 762 and also observed for the P. aeruginosa strains 696, 769, ATCC 27853, and PAO-1 (Fig. 4D), as well as the S. aureus strain ATCC 8325 (not shown).
To test the in vivo role of PLA2 activation for P. aeruginosa-induced apoptosis, we used an in vivo infection model. To this end, we applied the PLA2 inhibitor MAFP into the trachea in vivo, followed by local infection with P. aeruginosa strains 762 or ATCC 27853. The results reveal an induction of apoptosis in respiratory epithelial cells in vivo upon infection with P. aeruginosa, which was prevented by inhibition of cPLA2 (Fig. 5A and B).
DISCUSSION
In the present study we investigated the role of PLA2 in P. aeruginosa infection and its significance in P. aeruginosa-induced epithelial cell death. We show that several P. aeruginosa strains, i.e., the clinical isolates P. aeruginosa 762, 696, and 769, as well as the laboratory strains P. aeruginosa ATCC 27853 and PAO-1, activate cPLA2, release arachidonic acid, and employ this pathway to induce apoptosis of host cells. The use of various subtype-specific inhibitors indicates an activation of cPLA2 by P. aeruginosa-mediating the release of arachidonic acid. Our data further show that cPLA2 activation plays a significant role in P. aeruginosa apoptosis in vitro as well as in vivo, since cPLA2 inhibitors reduce P. aeruginosa-induced cell death. A P. aeruginosa mutant that lacked the type III secretion system failed to stimulate PLA2 and to induce apoptosis. This is consistent with previous findings that demonstrated the failure of a P. aeruginosa type III mutant to induce significant apoptosis of epithelial cells (26).
At present the signals mediating the activation of cPLA2 upon infection with P. aeruginosa are unknown. We have previously shown that P. aeruginosa triggers an activation of the acid sphingomyelinase and the formation of ceramide (19). Ceramide forms membrane platforms that serve to reorganize receptor molecules involved in internalization of the bacteria, as well as the induction of apoptosis (19). However, it is possible that ceramide does not only triggers the reorganization of receptor molecules in ceramide-enriched membrane domains upon infection but also mediates a stimulation of PLA2 (22, 31). In this context it is interesting that ceramide has been shown to bind to the CaLB domain of PLA2 and facilitates membrane docking of and lipid hydrolysis by PLA2 (22, 31). Thus, ceramide might be involved in the spatial reorganization of PLA2 required for the function of this enzyme. In addition, a recent study demonstrated an involvement of ceramide-kinase in the activation of PLA2 by ceramide (36, 37). Since ceramide-1-phosphate was shown to be a direct activator of PLA2 (36, 37), the metabolism of ceramide to ceramide-1-phosphate after infection with P. aeruginosa may also contribute to PLA2 activation. Further, we have previously shown that infection of human and murine epithelial cells and fibroblasts with P. aeruginosa results in the induction of apoptosis by upregulation of the endogenous CD95 receptor/CD95 ligand system (5, 19). The critical role of the CD95/CD95 ligand system for induction of apoptosis was demonstrated by the use of CD95- and CD95-ligand-deficient cells. These cells were resistant to P. aeruginosa-induced cell death (19, 25). Recent studies from Cannon et al. confirmed the CD95-dependent induction of apoptosis in mammalian cells by P. aeruginosa (5). CD95 might also mediate the activation of PLA2 upon infection with P. aeruginosa, although the activation of PLA2 by CD95 and the role of this enzyme for CD95-triggered death seem to be controversial. For instance, studies of Ulisse et al. (51) and Cifone et al. (7) demonstrated an activation of PLA2 by CD95 in Sertoli and HuT78 cells that was mediated by a signaling pathway involving extracellular regulated kinases. Activation of PLA2 activity was required, at least in part, for the induction of CD95-induced cell death in these cells. On the other hand, it was shown by Luschen et al. (32), Enari et al. (17), de Valck et al. (14), and Atsumi et al. (2) that an activation of PLA2 is not required for CD95-triggered apoptosis and cPLA2 seems to be even cleaved by proteolysis after CD95 stimulation.
Next, it is also possible that P. aeruginosa triggers an activation of PLA2 independent of the acid sphingomyelinase, ceramide, and the CD95 receptor/CD95 ligand system. In this respect, it is interesting that activation of PLA2 by P. aeruginosa required a functional type III secretion system. Although the exact nature of proapoptotic P. aeruginosa factors that are delivered into infected cells and mediate apoptosis is unknown, it might be possible that bacterial factors directly or indirectly activate PLA2.
Finally, P. aeruginosa expresses enzymes that display PLA2 activity and the observed PLA2 activity could be caused by an activation of a bacterial PLA2 (43, 44). In particular, it was shown that P. aeruginosa exotoxin U exhibits a PLA2 activity that is activated by still unknown mammalian cell factors (38, 49). Moreover, the PLA2 activity of P. aeruginosa exotoxin U was inhibited by the cPLA2 inhibitor MAFP and, accordingly, MAFP also prevented exotoxin U-induced cell death (38). However, we used doses of MAFP of 10 μM, while inhibition of P. aeruginosa exotoxin U by MAFP required concentrations of the inhibitor up to 1.35 mM and prolonged incubation of the enzyme with the inhibitor up to 16 h (38). Therefore, it seems to be unlikely that the observed effects of MAFP are due to an inhibition of the bacterial protein, and our data strongly suggest that a cellular PLA2 is activated by P. aeruginosa to mediate the release of arachidonic acid.
P. aeruginosa-induced PLA2 might be involved in apoptosis by the activation of several proapoptotic pathways. It was demonstrated that arachidonic acid, but not products of the lipoxygenase and the cyclooxygenase pathway, induces a depolarization of mitochondria, a typical event of mitochondrial alterations during apoptosis (29, 30, 34, 35, 46, 50, 52). Furthermore, it was suggested that PLA2 induces the formation of proapoptotic reactive oxygen intermediates and an activation of sphingomyelinases with a concomitant release of ceramide and induction of apoptosis (10, 56). Finally, a recent study demonstrated a change of the nuclear architecture by PLA2 in the course of apoptosis (47). However, the molecular details of arachidonic acid-induced apoptosis are still unknown.
The present data suggest PLA2 and arachidonic acid as novel components of the death machinery in the host cell and the pulmonary host response upon P. aeruginosa infection. Our studies show that P. aeruginosa infection activates cPLA2, resulting in arachidonic acid release. Pharmacological inhibition of PLA2 prevents P. aeruginosa-induced apoptosis, suggesting an important role of PLA2 in the biological response of the lung to infections with P. aeruginosa.
ACKNOWLEDGMENTS
The study was supported by DFG grant Gu 335-10/3 and MO 435/17-3, a BMBF program, and the MANAD program to E.G.
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Department of Molecular Biology, University of Duisburg-Essen, 45122 Essen, Germany
ABSTRACT
Pseudomonas aeruginosa, a gram-negative, facultative pathogen, causes severe and often even lethal infections in immunocompromised patients, as well as cystic fibrosis patients. We show here that a variety of P. aeruginosa strains activate phospholipase A2 (PLA2), cultured epithelial cells, and fibroblasts, resulting in increased intracellular and extracellular arachidonic acid release. The use of different PLA2 inhibitors revealed that P. aeruginosa-induced arachidonic acid release is mediated by activation of cytosolic PLA2 (cPLA2), whereas iPLA2 or sPLA2 do not seem to be involved in the response to P. aeruginosa. Likewise, the cPLA2-specific inhibitors MAFP and AACOCF3 prevented apoptosis of cultured epithelial cells upon P. aeruginosa infection, whereas inhibitors specific for iPLA2 or sPLA2 were without effect. The physiological significance of these findings is indicated by an inhibition of apoptosis in tracheal epithelial cells upon in vivo infection with P. aeruginosa. The data indicate that arachidonic acid generation by activation of cPLA2 during P. aeruginosa infection plays an important role in the induction of host cell death.
INTRODUCTION
Pseudomonas aeruginosa infections are a severe clinical problem, in particular in immunocompromised individuals and in patients with cystic fibrosis (CF) (53). CF patients are highly susceptible to P. aeruginosa bacteria, leading to chronic lung infection in the majority of these patients. This lung disease is characterized by persistent pulmonary inflammation that finally results in fibrosis and destruction of lung tissue and respiratory failure. At present, P. aeruginosa is the leading cause of mortality in CF patients. Infection of mammalian cells with P. aeruginosa results in the release of cytokines (11, 23, 45), internalization of the bacteria (20, 40, 41), and induction of apoptosis of the infected host cells (5, 19-21, 42). We sought here to identify novel mechanisms of P. aeruginosa-induced apoptosis. It was previously shown that mice unable to respond with host cell apoptosis to pulmonary infection with P. aeruginosa are highly sensitive to the infection, develop sepsis, and die (19). Further, it was demonstrated that P. aeruginosa-induced apoptosis is delayed in cells lacking functional cystic fibrosis conductance regulator (5), whose mutations cause cystic fibrosis. Grassme et al. (19), Jendrossek et al. (25), and Cannon et al. (5) demonstrated an upregulation of the CD95-CD95-ligand system on the surface of infected cells that resulted in apoptosis of P. aeruginosa-infected cells by ligation of the endogenous CD95 receptor. Genetic deficiency of CD95 prevented P. aeruginosa-induced apoptosis (19, 25), suggesting a central role of the CD95-CD95-ligand system for P. aeruginosa-initiated cell death. Further, an involvement of mitochondria (in particular mitochondrial depolarization, synthesis of reactive oxygen intermediates, and release of cytochrome c), an activation of Jun N-terminal kinases (JNK), and a stimulation of caspases were demonstrated to be involved in the induction of apoptosis in mammalian cells upon infection with P. aeruginosa (19, 25, 26).
In the present study we tested a potential role of phospholipases A2 (PLA2) in P. aeruginosa-triggered cell death. PLA2 are lipolytic enzymes that hydrolyze membrane phospholipids and thereby release fatty acids, particularly arachidonic acid from the sn-2 position of glycerophospholipids (13). PLA2 plays an important role in signal transduction, in particular by generation of proinflammatory mediators as prostaglandins and leukotrienes and by membrane remodeling. Several subtypes of mammalian PLA2 have been described that are divided in four main groups according to their function, localization, and calcium dependency. Secretory PLA2 (low molecular mass enzymes belonging to groups I, II, III, V, and X) are cysteine-rich, secreted proteins that require millimolar concentrations of Ca2+ for activity without a preference for a specific fatty acid in the sn-2 position of the phospholipid substrate (13). The second class of PLA2 includes specific acetylhydrolases such as platelet-activating factor. A third class is composed of Ca2+-independent PLA2, e.g., iPLA2 isolated from myocardium (55), CHO cells, and macrophages (1, 28). The group IV cytosolic PLA2 (cPLA2) includes three PLA2 named , , and . cPLA2, an 85-kDa protein, requires micromolar Ca2+ concentrations for activity and has a preference for arachidonic acid (8). The recently described cPLA2, a 110-kDa protein, shows 30% sequence identity with cPLA2 and also depends on Ca2+ but is less selective for cleavage at the sn-2 position than cPLA2 (39, 49). cPLA2 with a molecular mass of 61 kDa and 29% sequence identity with cPLA2 is Ca2+ independent but distinguishable from iPLA2 by its preference for arachidonic acid at the sn-2 position (3). Previous studies implied PLA2 in the host response to such diverse pathogens as Staphylococcus aureus, Escherichia coli, Aeromonas hydrophila, Pasteurella haemolytica, and Bacillus anthracis (6, 9, 16, 18, 24, 44, 54). PLA2 seem to have a dual function in the infection of mammalian cells by pathogens, since many bacterial toxins seem to kill mammalian cells via PLA2 (4, 12, 16, 18, 27), whereas expression of PLA2 was shown to protect mice from acute infections at least with S. aureus, P. aeruginosa, and E. coli (15, 33). Thus, the exact function of PLA2 in infectious processes requires definition.
In the present study, we investigated the role of PLA2 for the in vitro and in vivo infection of cultured epithelial cells or fibroblasts, respectively, as well as tracheal epithelial cells with P. aeruginosa. We demonstrate an activation of PLA2 by a variety of P. aeruginosa strains and reveal by the use of a panel of PLA2 inhibitors a significant role of cPLA2 for the induction of host cell apoptosis by P. aeruginosa both in vitro and in vivo.
MATERIALS AND METHODS
Materials and cell culture. The human conjunctiva epithelial cell line Chang (ATCC CCL 20.2) was cultured in RPMI 1640 (Gibco-BRL/Life Technologies) supplemented with 5% fetal calf serum (FCS) at 37°C as monolayers in tissue culture flasks in 5% CO2 atmosphere. The human lung fibroblast cell line WI-38 was maintained in minimal essential medium supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1% nonessential amino acids, and 1% penicillin-streptomycin. Infections were performed in RPMI 1640 supplemented with 10 mM HEPES (pH 7.2) to avoid interactions of serum proteins with the bacteria. Prior to infections, FCS and antibiotics were omitted from the culture media. The PLA2 inhibitors MAFP, BEL, 12-Episcalaradial, and AACOCF3 were purchased from Biomol.
Bacterial strains. Three clinical isolates and two laboratory strains of P. aeruginosa were used. The isolate 762 was originally obtained from an urinary tract infection, strain 696 was isolated from the sputum of a hospitalized patient, and strain 769 was from a patient with urosepsis (18). The laboratory strains used were P. aeruginosa ATCC 27853 and PAO-1. Furthermore, we infected cells with a previously described (26) P. aeruginosa strain deficient for the type III secretion system (P. aeruginosa PAK pcrD::Smr, kindly provided by J. Heesemann), S. aureus strain ATCC 8325, and a nonpathogenic E. coli isolate.
Infection experiments. Bacteria originating from glycerol stock cultures were plated overnight on tryptic soy agar plates at 37°C, resuspended in tryptic soy broth (TSB) at an optical density at 550 nm of 0.25, shaken at 120 rpm for 1 h at 37°C, and harvested during logarithmic growth phase by pelleting and resuspension in fresh TSB. Prior to infection, cells were washed twice in RPMI 1640 (Chang cells) or minimal essential medium (WI-38 cells) and maintained in the same medium during infection. Infection was performed by inoculating subconfluent cell layers at a host cell/bacterium ratio of 1:1,000, 1:100, 1:50, or 1:10. Synchronous infection conditions and an enhanced bacterium-host cell interaction were achieved by a 2-min centrifugation (35 x g) of the bacteria onto the cells. The end of the centrifugation step was defined as the starting point of all infections.
Arachidonic acid release assays. Chang cells (0.4 x 105 per well) or WI-38 cells (0.3 x 105 per well) were labeled for 18 to 20 h with 0.05 μCi of [3H]arachidonic acid [5,6,8,9,11,12,14,15-3H(N)] (0.1 mCi/ml stock; New England Nuclear)/ml. Prior to infection, cells ware washed three times with a buffer containing 132 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM CaCl2, 0.7 mM MgCl2, and 0.8 mM MgSO4 (H/S) supplemented with 1% bovine serum albumin and 10 mM glucose and incubated in the same buffer during infection. Infection was performed as described above at the indicated multiplicity of infection (MOI).
Cells were preincubated with the PLA2 inhibitors MAFP, 12-episcalaradial, and bromoenol lactone (BEL) at the indicated concentrations for 15 min prior to infection. The slow-acting inhibitor AACOCF3 was added to the cells 3 h prior to infection.
Extracellular arachidonic acid release. To determine the release of arachidonic acid into the extracellular space, supernatants were collected after the indicated time of infection and centrifuged at 2,000 rpm for 5 min in a microcentrifuge to remove any cells that may have detached. The supernatants were quantified by liquid scintillation counting. The remaining cell-bound radioactivity was determined by adding 0.5 ml of 0.1% Triton X-100 in H2O to the cells, and lysed cells were removed from the plates and subjected to liquid scintillation counting. Extracellular arachidonic acid release was calculated as the percentage of radioactivity released into the supernatant of the total radioactivity (the latter being the sum of released and cell-bound radioactivity). Since only free arachidonic acid is released into the supernatant, the measurement of radioactivity in the supernatant is an accurate measurement for the formation of extracellular arachidonic acid.
Intracellular arachidonic acid release. Chang cells were seeded at a density of 3 x 105 cells/well in six-well plates. Labeling with [3H]arachidonic acid, pretreatment with PLA2 inhibitors, and infection were performed as described above. Infections were terminated by removal of the supernatants, washing, and the addition of 1 ml of methanol-15% acetic acid to each well. Cells were scraped off the plates, and wells were washed with 0.5 ml of methanol. Chloroform (0.75 ml) was added, and samples were extracted by incubation for 30 min at room temperature. Next, 0.75 ml each of chloroform and H2O were added, and the phases were separated by low-speed centrifugation for 10 min. The lower phase containing the fatty acids was removed, dried, solubilized in 30 μl of petrol ether-diethyl ether (96:1), spotted onto a silica gel 60 plate, and separated by thin-layer chromatography (TLC) in a solvent system consisting of ethyl acetate-glacial acetic acid-2,2,4-trimethyl pentane-H2O (45:10:25:50, by volume). An arachidonic acid standard was run on each plate to confirm migration of free arachidonic acid. After separation, TLC plates were air dried and a Biomax MS film was exposed at –80°C for several days. To quantify free arachidonic acid, spots representing arachidonic acid were scraped off the plates and subjected to liquid scintillation counting. The TLC analysis permits separation of free arachidonic acid from arachidonic acid incorporated into lipids and, therefore, the analysis of free intracellular arachidonic acid.
Detection and quantification of apoptosis. Apoptosis was quantified by fluorescein isothiocyanate (FITC)-annexin V staining and confirmed by trypan blue staining. To this end, cells were infected as described above, treated with trypsin, washed twice in 10 mM HEPES (pH 7.4), 140 mM NaCl2, and 5 mM CaCl2 and resuspended in the same buffer supplemented with FITC-annexin V (dilution 1:50; Roche Biochemicals, Mannheim, Germany). After incubation for 15 min cells were subjected to fluorescence-activated cell sorter (FACS) analysis using a FACSCalibur flow cytometer using the CELLQuest software (Becton Dickinson, Mountain View, Calif.).
In vivo infections. To perform infection of tracheal epithelial cells in vivo, C57BL/6 wild-type mice were anesthetized by intraperitoneal injection of avertin, the trachea was carefully isolated, and the distal part was catheterized and ligated immediately proximal of the catheter. We then injected MAFP, AACOCF3, or buffer into the proximal part of the trachea. After an incubation period of 15 min for MAFP, we injected 108 CFU of P. aeruginosa 762 into the proximal trachea. Mice were killed after 45 min, and the tracheas were rapidly excised and fixed for 30 min in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.2). Epithelial cells were isolated by disintegration of the trachea by scratching with a 27-gauge needle and a 5-min digestion in trypsin. Cells were washed and stained with FITC-annexin V to detect apoptosis. Apoptosis of epithelial cells in the trachea was also determined by TUNEL staining. To this end, the trachea was excised and fixed in 4% PFA in PBS (pH 7.2) for 36 h. The tissues were embedded in paraffin, and sections were deparaffinized and treated for 5 min in 0.1 M citrate (pH 6.0) with 350-W microwave irradiation for 5 min. The sections were then stained for 30 min at 37°C with FITC-coupled dUTP in the presence of terminal deoxynucleotidyl transferase. The samples were washed, incubated at 70°C for 10 min to reduce unspecific binding, and stained with alkaline phosphatase-coupled anti-FITC-antibodies. The slides were finally developed using Fast Red Tablets (Roche) as a substrate resulting in the formation of a red dye.
RESULTS
P. aeruginosa induces extra- and intracellular arachidonic acid release upon infection of epithelial cells. To gain insight into the interactions of P. aeruginosa with epithelial cells, we investigated the role of PLA2 upon bacterial infection. To this end, we measured P. aeruginosa-induced PLA2 activity by quantification of the extracellular and intracellular arachidonic acid release from two different cell lines, i.e., Chang conjunctiva epithelial cells and WI-38, a lung fibroblast cell line. Intracellular arachidonic acid release was determined by prelabeling cells with [3H]arachidonic acid, extraction of fatty acids, and subsequent separation by TLC. Extracellular arachidonic acid release was determined by labeling cells with [3H]arachidonic acid and quantification of released radioactivity in the supernatant by liquid scintillation counting. The latter data were related to the total radioactivity recovered from the samples. The results revealed a marked increase of free, intra- and extracellular arachidonic acid after bacterial infection with the P. aeruginosa strains 696, 762, 769, ATCC 27853, and PAO-1 (Fig. 1 and Fig. 2).
The release of arachidonic acid occurred very rapidly after infection with P. aeruginosa (Fig. 1) and was dependent on the MOI used in the infection experiments (Fig. 2). However, even at the lowest MOI of 10 bacteria/cell, we were still able to detect some release of arachidonic acid (Fig. 2). A type III mutant of P. aeruginosa did not induce a release of intra- or extracellular arachidonic acid (Fig. 2A).
The release of arachidonic acid was not restricted to P. aeruginosa and was also observed upon infection with a different pathogen, i.e., S. aureus (Fig. 2A). In contrast, nonpathogenic E. coli were unable to trigger a significant release of arachidonic acid (Fig. 2A).
Arachidonic acid release induced by P. aeruginosa infection is diminished by cPLA2 inhibitors but not by sPLA2 or iPLA2 inhibitors. Next, we aimed to further characterize the PLA2 subclass involved in P. aeruginosa-induced arachidonic acid release. To this end, we investigated the influence of various PLA2 inhibitors on arachidonic acid release into the supernatant and on cell-bound arachidonic acid release. Two inhibitors (MAFP, AACOCF3) specific for both cPLA2 and iPLA2 were used, as well as the sPLA2-specific inhibitor 12-Episcalaradial and the iPLA2-specific inhibitor BEL. The results reveal that MAFP and AACOCF3 significantly reduced intra- and extracellular arachidonic acid release upon infection with P. aeruginosa strains 696, 762, 769, and ATCC 27853 (Fig. 2). A dose-response analysis revealed that doses of 1 μM MAFP or 2 μM AACOCF3, respectively, were already sufficient to reduce arachidonic acid release (Fig. 3A and B). In contrast, neither the iPLA2 inhibitor BEL nor the sPLA2 inhibitor 12-episcalaradial affected P. aeruginosa-induced arachidonic acid release upon infection of WI-38 or Chang cells (Fig. 3C and D).
Since MAFP specifically blocks cPLA2 and iPLA2, whereas BEL specifically targets iPLA2 and 12-Episcalaradial inhibits type II sPLA2, the data suggest that cPLA2 is activated by P. aeruginosa.
The cPLA2 inhibitors MAFP and AACOCF3 reduce P. aeruginosa-induced apoptosis. To gain insight into the functional significance of arachidonic acid release, we investigated the role of PLA2 for the induction of apoptosis during P. aeruginosa infection. P. aeruginosa-induced apoptosis of epithelial cells has been previously demonstrated (5, 19, 20) and seems to be critical for the defense of the host against acute infections with these bacteria (19). First signs of apoptosis as determined by breakdown of phosphatidylserine asymmetry in the cell membrane were detected within 15 to 30 min after infection with P. aeruginosa 762. MAFP and AACOCF3 dose dependently inhibited P. aeruginosa-induced apoptosis (Fig. 4A and B), whereas pretreatment with 12-Episcalaradial (sPLA2 inhibitor) (Fig. 4C) was without effect on apoptosis induction. Inhibition of apoptosis by MAFP and AACOCF3, respectively, was not restricted to P. aeruginosa strain 762 and also observed for the P. aeruginosa strains 696, 769, ATCC 27853, and PAO-1 (Fig. 4D), as well as the S. aureus strain ATCC 8325 (not shown).
To test the in vivo role of PLA2 activation for P. aeruginosa-induced apoptosis, we used an in vivo infection model. To this end, we applied the PLA2 inhibitor MAFP into the trachea in vivo, followed by local infection with P. aeruginosa strains 762 or ATCC 27853. The results reveal an induction of apoptosis in respiratory epithelial cells in vivo upon infection with P. aeruginosa, which was prevented by inhibition of cPLA2 (Fig. 5A and B).
DISCUSSION
In the present study we investigated the role of PLA2 in P. aeruginosa infection and its significance in P. aeruginosa-induced epithelial cell death. We show that several P. aeruginosa strains, i.e., the clinical isolates P. aeruginosa 762, 696, and 769, as well as the laboratory strains P. aeruginosa ATCC 27853 and PAO-1, activate cPLA2, release arachidonic acid, and employ this pathway to induce apoptosis of host cells. The use of various subtype-specific inhibitors indicates an activation of cPLA2 by P. aeruginosa-mediating the release of arachidonic acid. Our data further show that cPLA2 activation plays a significant role in P. aeruginosa apoptosis in vitro as well as in vivo, since cPLA2 inhibitors reduce P. aeruginosa-induced cell death. A P. aeruginosa mutant that lacked the type III secretion system failed to stimulate PLA2 and to induce apoptosis. This is consistent with previous findings that demonstrated the failure of a P. aeruginosa type III mutant to induce significant apoptosis of epithelial cells (26).
At present the signals mediating the activation of cPLA2 upon infection with P. aeruginosa are unknown. We have previously shown that P. aeruginosa triggers an activation of the acid sphingomyelinase and the formation of ceramide (19). Ceramide forms membrane platforms that serve to reorganize receptor molecules involved in internalization of the bacteria, as well as the induction of apoptosis (19). However, it is possible that ceramide does not only triggers the reorganization of receptor molecules in ceramide-enriched membrane domains upon infection but also mediates a stimulation of PLA2 (22, 31). In this context it is interesting that ceramide has been shown to bind to the CaLB domain of PLA2 and facilitates membrane docking of and lipid hydrolysis by PLA2 (22, 31). Thus, ceramide might be involved in the spatial reorganization of PLA2 required for the function of this enzyme. In addition, a recent study demonstrated an involvement of ceramide-kinase in the activation of PLA2 by ceramide (36, 37). Since ceramide-1-phosphate was shown to be a direct activator of PLA2 (36, 37), the metabolism of ceramide to ceramide-1-phosphate after infection with P. aeruginosa may also contribute to PLA2 activation. Further, we have previously shown that infection of human and murine epithelial cells and fibroblasts with P. aeruginosa results in the induction of apoptosis by upregulation of the endogenous CD95 receptor/CD95 ligand system (5, 19). The critical role of the CD95/CD95 ligand system for induction of apoptosis was demonstrated by the use of CD95- and CD95-ligand-deficient cells. These cells were resistant to P. aeruginosa-induced cell death (19, 25). Recent studies from Cannon et al. confirmed the CD95-dependent induction of apoptosis in mammalian cells by P. aeruginosa (5). CD95 might also mediate the activation of PLA2 upon infection with P. aeruginosa, although the activation of PLA2 by CD95 and the role of this enzyme for CD95-triggered death seem to be controversial. For instance, studies of Ulisse et al. (51) and Cifone et al. (7) demonstrated an activation of PLA2 by CD95 in Sertoli and HuT78 cells that was mediated by a signaling pathway involving extracellular regulated kinases. Activation of PLA2 activity was required, at least in part, for the induction of CD95-induced cell death in these cells. On the other hand, it was shown by Luschen et al. (32), Enari et al. (17), de Valck et al. (14), and Atsumi et al. (2) that an activation of PLA2 is not required for CD95-triggered apoptosis and cPLA2 seems to be even cleaved by proteolysis after CD95 stimulation.
Next, it is also possible that P. aeruginosa triggers an activation of PLA2 independent of the acid sphingomyelinase, ceramide, and the CD95 receptor/CD95 ligand system. In this respect, it is interesting that activation of PLA2 by P. aeruginosa required a functional type III secretion system. Although the exact nature of proapoptotic P. aeruginosa factors that are delivered into infected cells and mediate apoptosis is unknown, it might be possible that bacterial factors directly or indirectly activate PLA2.
Finally, P. aeruginosa expresses enzymes that display PLA2 activity and the observed PLA2 activity could be caused by an activation of a bacterial PLA2 (43, 44). In particular, it was shown that P. aeruginosa exotoxin U exhibits a PLA2 activity that is activated by still unknown mammalian cell factors (38, 49). Moreover, the PLA2 activity of P. aeruginosa exotoxin U was inhibited by the cPLA2 inhibitor MAFP and, accordingly, MAFP also prevented exotoxin U-induced cell death (38). However, we used doses of MAFP of 10 μM, while inhibition of P. aeruginosa exotoxin U by MAFP required concentrations of the inhibitor up to 1.35 mM and prolonged incubation of the enzyme with the inhibitor up to 16 h (38). Therefore, it seems to be unlikely that the observed effects of MAFP are due to an inhibition of the bacterial protein, and our data strongly suggest that a cellular PLA2 is activated by P. aeruginosa to mediate the release of arachidonic acid.
P. aeruginosa-induced PLA2 might be involved in apoptosis by the activation of several proapoptotic pathways. It was demonstrated that arachidonic acid, but not products of the lipoxygenase and the cyclooxygenase pathway, induces a depolarization of mitochondria, a typical event of mitochondrial alterations during apoptosis (29, 30, 34, 35, 46, 50, 52). Furthermore, it was suggested that PLA2 induces the formation of proapoptotic reactive oxygen intermediates and an activation of sphingomyelinases with a concomitant release of ceramide and induction of apoptosis (10, 56). Finally, a recent study demonstrated a change of the nuclear architecture by PLA2 in the course of apoptosis (47). However, the molecular details of arachidonic acid-induced apoptosis are still unknown.
The present data suggest PLA2 and arachidonic acid as novel components of the death machinery in the host cell and the pulmonary host response upon P. aeruginosa infection. Our studies show that P. aeruginosa infection activates cPLA2, resulting in arachidonic acid release. Pharmacological inhibition of PLA2 prevents P. aeruginosa-induced apoptosis, suggesting an important role of PLA2 in the biological response of the lung to infections with P. aeruginosa.
ACKNOWLEDGMENTS
The study was supported by DFG grant Gu 335-10/3 and MO 435/17-3, a BMBF program, and the MANAD program to E.G.
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