INTRODUCTION

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The Legionnaires' disease bacterium, Legionella pneumophila, is one of the most common etiologic agents of bacterial pneumonia (27). In the aquatic environment, L. pneumophila is a parasite of at least 13 species of amoebae and ciliated protozoa (6). Upon transmission to humans through environmentally generated aerosols, the bacteria invade and replicate within alveolar macrophages and monocytes that have been recruited into the alveolar spaces (see references 1 and 6 for recent reviews). In addition, it has been recently shown that intracellular replication within type I and II alveolar epithelial cells may contribute to the pathogenesis of Legionnaires' disease (18).

Initial bacterial attachment to mammalian macrophages, alveolar epithelial cells, and protozoa is mediated, at least in part, by type IV pili designated CAP (40). Following entry into mammalian and protozoan cells, L. pneumophila replicates within a ribosome-studded phagosome (1, 6). Within this unique niche, the bacterium undergoes dramatic alterations in gene expression, which may be required for interaction with the host cell, for adaptation to the intracellular microenvironment, and possibly for exiting the host cell upon termination of intracellular replication (2-5, 7, 10, 16, 21).

The hallmark of Legionnaires' disease is intracellular replication of L. pneumophila within phagosomes that are blocked from fusion to the lysosomes (23). This alteration in endocytic trafficking has been recently shown to be mediated by L. pneumophila proteins encoded by the pmi, mil, dot, and icm loci (19, 20, 39, 41). Mutations in many of these loci render the bacteria defective in both cytopathogenicity and intracellular replication within macrophages (19, 20, 39, 41). Interestingly, some of the pmi and mil loci that are indispensable for infectivity of macrophages are not required for infectivity of alveolar epithelial cells (18). Although cytopathogenicity of L. pneumophila to macrophages and alveolar epithelial cells has been well documented, the mechanisms of cell death as a result of bacterial infection are not well understood (1, 6, 24, 31, 35).

Apoptosis and necrosis are the two commonly observed types of cell death. While necrosis is characterized as accidental cell death due to physical damage, apoptosis is strictly regulated suicide program within the dying cell manifesting morphological and biochemical features distinct from those of necrosis (8, 14). A cascade of activation of a family of cysteine proteases (caspases) that specifically cleave proteins after aspartate (Asp) residues is required for induction of apoptosis (38). A number of intracellular pathogens are capable of manipulating host cell apoptotic pathways. The obligate intracellular bacteria Chlamydia trachomatis and Rickettsia rickettsii inhibit apoptosis in the host cells, allowing their intracellular growth and persistence (13, 15). The facultative intracellular pathogenic bacterium Shigella flexneri induces apoptosis in macrophages, which is accompanied by activation of caspase-1/ICE (43). In contrast, the bacterium Mycoplasma penetrans fragments DNA of the host cell by secretion of a bacterial nuclease that specifically cleaves host cell DNA into internucleosomal fragments of 180 to 200 bp (9). Whether other intracellular bacterial pathogens induce apoptosis by bacterial nucleases or through the activation of caspases is not known.

Induction of apoptosis in macrophages by L. pneumophila has been observed in HL-60 human-derived macrophages after 24 to 48 h of infection when the cells were infected at a multiplicity of infection (MOI) of 10 to 100 (34). On the other hand, when a high MOI was used for infection, necrosis was evident at 20 to 60 min after infection (24, 26). Apoptosis was not detected during this period when infected cells were examined by electron microscopy (26). Therefore, whether L. pneumophila induces apoptosis in the host cell within 24 h after infection at a lower MOI is not known. Since apoptosis may not be recognized by a single strategy or at a time too early to manifest it, multiple criteria and a broad time frame need to be considered in order to detect it.

In this report, we provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells occurs within a few hours of infection prior to intracellular bacterial replication and correlates with cytopathogenicity. Furthermore, extracellular bacteria are capable of inducing apoptosis and are also cytopathogenic to the host cell. We demonstrate that L. pneumophila-induced apoptosis is mediated by activation of the caspase cascade.

	MATERIALS AND METHODS

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Bacterial strains and growth media. Virulent strain L. pneumophila AA100 and the characterization of the pmi mutants of L. pneumophila have been described previously (19). L. pneumophila strains were grown on buffered charcoal yeast extract (BCYE) agar plates or in buffered yeast extract broth supplemented with 50-µg/ml kanamycin for the mutant strains.

U937 macrophages and WI-26 alveolar epithelial cells. The human macrophage-like cell line U937 was maintained and differentiated by using phorbol 12-myristate 13-acetate (Sigma Chemical Co., St. Louis, Mo.) as we described previously (19). Human type I alveolar epithelial cells (WI-26 VA4; American Type Culture Collection) were maintained in supplemented minimal essential medium.

Cytopathogenicity of L. pneumophila to U937 macrophages and WI-26 alveolar epithelial cells. L. pneumophila strains were grown on BCYE plates for 3 days prior to infection of U937 macrophages or WI-26 alveolar epithelial cells. Infection was performed, in triplicate, in 96-well plates containing 10⁵ cells/well at an MOI of 0.5, 5, or 50 for 1 h, followed by three washes of extracellular unattached bacteria and further incubation at 37°C in 5% CO₂. At several time intervals, the monolayers were treated for 4 h with 10% Alamar Blue dye (Alamar Bioscience Inc., Sacramento, Calif.) as we described previously (7). Viability of the monolayers was determined by measurement of the optical density (OD) of Alamar Blue-treated monolayers by using a VMAX Kinetic Microplate Reader (Molecular Devices, Menlo Park, Calif.) and expressed as percent cytopathogenicity compared to uninfected cells by using the formula [1  (mean OD of treated cells/mean OD of nontreated cells)] × 100%. To examine the effect of inhibition of bacterial protein synthesis and intracellular replication by chloramphenicol or erythromycin on the cytopathogenicity of L. pneumophila to U937 macrophages, the bacteria and cells were separately pretreated with chloramphenicol (20 or 100 µg/ml) or erythromycin (8 or 80 µg/ml) (Sigma) for 1 h and the treated bacteria were used to infect the treated cells at an MOI of 50 in the presence of the respective antibiotic. To confirm that protein synthesis of L. pneumophila within the host cells was inhibited by the antibiotics, infected monolayers were pretreated with cycloheximide (200 µg/ml) to inhibit host cell protein synthesis (3) and then incubated with [³⁵S]methionine (150 µCi/ml) (ICN Pharmaceuticals, Irvine, Calif.) to label bacterial proteins in the presence or absence of the antibiotics as we described previously (2). To examine the effect of inhibition of bacterial uptake by cytochalasin D (CytD) on the cytopathogenicity of L. pneumophila to U937 macrophages, the cells were treated with 1-µg/ml CytD and infected at an MOI of 50 as described previously (20). Inhibition of bacterial uptake was confirmed by complete sterilization of the infected monolayers with 50-µg/ml gentamicin after the 1-h infection period as described previously (19, 20).

Growth kinetics of L. pneumophila strains in U937 macrophages and WI-26 alveolar epithelial cells. Infections of U937 macrophages by L. pneumophila strains were performed, in triplicate, in 96-well plates containing 10⁵ cells/well at an MOI of 0.5, 5, or 50 for 1 h. At the end of the infection period, the monolayers were either treated (the initial number represents intracellular bacteria) or not treated (the initial number represents cell-associated bacteria) with gentamicin. The number of bacteria in the monolayers at several time intervals after infection was determined as we described previously (19, 20).

Transmission electron microscopy. U937 macrophages and WI-26 alveolar epithelial cells were infected with L. pneumophila at an MOI of 50 for 1 h, followed by washing of extracellular bacteria. Preparation of ultrathin sections was performed as described previously (20). Briefly, infected macrophages were fixed with 3.5% glutaraldehyde, followed by 1% OsO₄, dehydrated with ethanol, and embedded in Eponate 12 resin (Ted Pella, Redding, Calif.). Ultrathin sections were stained with uranyl acetate, followed by lead citrate, and examined by a Hitachi H-7000/STEM electron microscope (Hitachi Inc., Tokyo, Japan) at 75 kV.

DNA fragmentation analysis. Differentiated U937 macrophages were plated in six-well plates (1.5 × 10⁶ cells/well) and infected with L. pneumophila strains at an MOI of 0.5, 5, or 50 for 1 h. At the end of the infection period, the monolayers were washed three times to remove unattached extracellular bacteria and maintained at 37°C in culture medium. At 3 h postinfection, the cells in each well were lysed in 500 µl of lysis buffer (10 mM Tris [pH 7.5], 20 mM EDTA [pH 8.0], 0.5% Triton X-100) for 30 min on ice. The lysates were treated with 0.5% sodium dodecyl sulfate and 300-µg/ml proteinase K for 2 h and extracted with phenol and chloroform before precipitation with ethanol. The precipitates were solubilized in 10 mM Tris (pH 8.0)-1 mM EDTA containing 0.5-µg/ml RNase, electrophoresed in 1.8% agarose gel, and stained with ethidium bromide. As a positive control for induction of apoptosis, the cells were incubated in culture medium containing 10-µg/ml actinomycin D (Act D; Sigma) for 4 h and DNA fragmentation was examined as described above (28, 34). As a negative control, the cells were incubated in culture medium without bacterial infection. To examine inhibition of DNA fragmentation by the broad-specificity caspase inhibitor Z-VAD-FMK (30) (Oncogene Research Products, Cambridge, Mass.), U937 macrophages were pretreated with the inhibitor (100 µM) for 90 min. The monolayers were infected with strain AA100 at an MOI of 50 for 1 h in the presence of the inhibitor, followed by washing of unattached extracellular bacteria and 3 h of incubation in the presence of the inhibitor.

Development of antiserum. L. pneumophila AA100 grown on a BCYE plate for 3 days was washed three times with saline, and 0.2 ml containing 10⁷ bacteria in complete Freund's adjuvant was injected subcutaneously into a 12-week-old female New Zealand rabbit. Two booster injections were administered at 2-week intervals. The final titer of the antiserum was approximately 1:10⁵, as examined by enzyme-linked immunosorbent assay.

Analysis of apoptosis by fluorescence microscopy. Cells attached to glass coverslips in 24-well plates were infected by L. pneumophila or treated with ActD as described above for the DNA fragmentation studies. For labeling of the bacteria, cells were fixed with 4% paraformaldehyde (Sigma) for 20 min, permeabilized with 0.2% Triton X-100 (Sigma) on ice for 5 min, blocked with 1% bovine serum albumin (Sigma) for 1 h, incubated with rabbit polyclonal antiserum (raised against L. pneumophila AA100) for 1 h, and then incubated for 1 h with a goat anti-rabbit immunoglobulin G secondary antibody conjugated to Alexa red (Molecular Probes, Inc., Eugene, Oreg.). For labeling of apoptotic nuclei, the cells were then subjected to fluorescein isothiocyanate (FITC)-conjugated terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) by using an apoptosis detection kit in accordance with the manufacturer's instructions (Boehringer Mannheim Corporation, Indianapolis, Ind.). Cells were examined with a Zeiss Axiophot Photomicroscope (Carl Zeiss Inc., Oberkochen, Germany) or with a Leica TCS NT confocal laser scanning microscope. A minimum of 100 cells per sample were counted, and apoptosis was quantitated as the percentage of apoptotic cells (TUNEL-positive nuclei) among all of the cells counted by phase-contrast microscopy. Multiple independent samples were examined.

	RESULTS

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Cytopathogenicity of L. pneumophila to macrophages and alveolar epithelial cells during early stages of infection is dose dependent. Infection of U937 macrophages by wild-type L. pneumophila AA100 at an MOI of 50 resulted in cytopathogenicities of 31 and 56% to the monolayers immediately after the 1-h infection period and at 3 h postinfection, respectively, which increased to 100% by 12 h postinfection (Fig. 1A). However, a longer period of time was required for L. pneumophila to induce similar levels of cytopathogenicity to the monolayers when the infection was performed at a lower MOI of 5 or 0.5 (Fig. 1A), suggesting that an increase in intracellular bacterial numbers or accumulation of bacterial products may be required to induce cytopathogenicity. No detectable intracellular replication was observed during the 3-h period after infection at the different MOIs examined (the lag phase is approximately 4 h) (Fig. 1B).

View larger version (38K): [in this window] [in a new window]   FIG. 1.   L. pneumophila is cytopathogenic to U937 macrophages (A) and WI-26 alveolar epithelial cells (C) in a dose-dependent manner. The cells were infected by the bacteria at an MOI of 0.5, 5, or 50 for 1 h, and the extracellular bacteria were washed away. The time points indicate the times at which Alamar Blue was added. The values are means of triplicate samples, and the error bars represent standard deviations. Intracellular growth kinetics of strain AA100 within U937 macrophages and WI-26 alveolar epithelial cells are shown in panels B and D, respectively. Infection of the monolayers was performed exactly as for cytopathogenicity assays, except that at the end of the 1-h infection period the monolayers were treated with gentamicin for 1 h to kill extracellular bacteria. At several time intervals, infected cells were hypotonically lysed and the number of intracellular bacteria was determined following growth on agar plates.

Induction of apoptosis by L. pneumophila in macrophages and alveolar epithelial cells is dose dependent. Apoptosis has been observed in L. pneumophila-infected HL-60 macrophages at 24 to 48 h postinfection at an MOI of 10 to 100 (34), but not in L. pneumophila-infected mouse bone marrow-derived macrophages at a high MOI of 500 during the period of 20 to 60 min postinfection (26). Whether L. pneumophila induces apoptosis in host cells within 24 h postinfection at lower MOIs has not been examined. Therefore, we used multiple criteria to examine apoptosis in macrophages and alveolar epithelial cells during early stages of infection by L. pneumophila at different MOIs.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Kinetics of DNA fragmentation in L. pneumophila-infected (lanes 3 to 5) or ActD-treated (lane 2) U937 macrophages examined by agarose gel electrophoresis. The monolayers were infected by strain AA100 at an MOI of 0.5, 5, or 50 exactly as described in the legend to Fig. 1A. DNA isolated from uninfected or infected cells at 3 h post-1-h infection or 4 h after ActD treatment was subjected to electrophoresis and stained with ethidium bromide. Lane M contained a 100-bp molecular size marker (Gibco BRL, Gaithersburg, Md.). Lane 2 contained DNA from cells incubated with 1-µg/ml ActD. NI, noninfected.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Apoptosis in L. pneumophila-infected U937 macrophages (A) and WI-26 alveolar epithelial cells (B) quantitated by TUNEL (see Fig. 4). The monolayers were infected by L. pneumophila as described in the legend to Fig. 2. One hundred cells were counted randomly, and the percentages of TUNEL-positive cells were calculated and expressed as mean percentages of apoptotic cells. NI, noninfected.

View larger version (62K): [in this window] [in a new window]   FIG. 4.   Representative confocal laser scanning images of TUNEL of L. pneumophila-infected U937 macrophages undergoing apoptosis. L. pneumophila infection and ActD treatment of the monolayers were performed exactly as described in the legend to Fig. 2. Monolayers were labeled simultaneously (bottom panels) with FITC-conjugated dUTP (green) and a polyclonal antiserum specific for L. pneumophila detected by a secondary antibody conjugated to Alexa red (red). Panels: B, L. pneumophila infected; D, L. pneumophila infected in the presence of CytD; F, ActD treated; H, uninfected. Phase-contrast images A, C, E, and G correspond to B, D, F, and H, respectively.

View larger version (109K): [in this window] [in a new window]   FIG. 5.   Detection of L. pneumophila-induced apoptosis in U937 macrophages by transmission electron microscopy. Infected cells at 4 h postinfection (B) showed apoptotic nuclear morphology distinct from that of uninfected cells (A). The arrowheads indicate ribosome-surrounded phagosomes containing the bacteria. The arrows indicate intact mitochondria within the vicinity of the phagosomes.

View larger version (46K): [in this window] [in a new window]   FIG. 6.   Representative confocal laser scanning images of annexin-V labeling of L. pneumophila-infected U937 macrophages undergoing apoptosis and comparison to ActD-treated cells. The Monolayers were labeled simultaneously with PI and FITC-conjugated annexin-V. Panels: A, 2 h postinfection; B, 3 h postinfection; C, 4 h post-ActD treatment; D, uninfected cells. The arrowheads indicate double-labeled cells, the arrows in panels B and C indicate cells labeled with PI only, and the rest of the fluorescent cells were with annexin-V only. Quantitation of annexin-V-positive and PI-positive U937 macrophages is shown in panel E. One hundred cells were counted randomly, and multiple samples were examined.

Induction of apoptosis by L. pneumophila does not require intracellular bacterial replication or new protein synthesis and correlates with cytopathogenicity. Our data suggested that intracellular replication was not required for L. pneumophila to induce apoptosis (Fig. 1). Two strategies were used to confirm that induction of apoptosis did not require intracellular bacterial replication or new protein synthesis and that apoptosis correlated with cytopathogenicity. First, we examined genetically defined mutants of L. pneumophila that are defective in intracellular replication for the ability to induce apoptosis and cytopathogenicity. We have recently isolated and characterized a collection L. pneumophila mutants that are defective, to various degrees, in intracellular replication within both human macrophages and protozoan cells, and the defective genetic loci have been designated protozoan and macrophage infectivity (pmi) loci (19). As shown in Table 1 and Fig. 7A and B, we examined all of the mutants belonging to pmi mutant groups 1, 2, and 3, all of which are severely defective in intracellular replication within U937 macrophages (19), for the ability to induce apoptosis. Agarose gel electrophoresis of chromosomal DNA of U937 macrophages isolated at 3 h postinfection by the mutants at an MOI of 50 showed that 36 of the 46 mutants induced various degrees of DNA fragmentation (Table 1). Seventeen of these 36 mutants (GB111 to GQ61, Table 1) induced apoptosis at levels similar to that induced by the parental strain and were also highly cytopathogenic to U937 macrophages at 12 h postinfection, despite their intracellular replication defect (Fig. 7). Nineteen of the mutants (Table 1, GN266 to GH37) induced various degrees of apoptosis in U937 macrophages at lower levels than parental strain AA100 and were also less cytopathogenic to these cells (Table 1), and an example of these (GO128) is shown in Fig. 7. In contrast to the 36 mutants that induced apoptosis, the other 10 mutants were completely defective in the induction of DNA fragmentation in U937 macrophages (Table 1 and Fig. 7D). Correlated with their defect in apoptosis induction, these 10 mutants were also severely defective in cytopathogenicity to these cells (Fig. 7F). All three dotA and icmWXYZ mutants (GG105, GL10, and GS95) (19) were completely defective in both apoptosis induction (Fig. 7D, lanes 3, 5, and 7) and cytopathogenicity (Fig. 7F). Importantly, the level of apoptosis consistently correlated with the level of cytopathogenicity in multiple experiments.

View larger version (57K): [in this window] [in a new window]   FIG. 7.   Ability of pmi mutants of L. pneumophila to induce apoptosis in U937 macrophages correlates with cytopathogenicity to the cells. Intracellular growth kinetics of the mutants (A and B) was examined as described in the legend to Fig. 1B but without gentamicin treatment. The ability of these mutants to induce DNA fragmentation in U937 macrophages was examined at 3 h postinfection (C and D). Infection of the monolayers was performed at an MOI of 50 exactly as described in the legend to Fig. 1A. Percent cytopathogenicity of the wild-type and mutant strains of L. pneumophila to U937 macrophages infected at an MOI of 50 at 12 h postinfection is shown in panels E and F and was determined as described in the legend to Fig. 1A. Lane M contained a 100-bp molecular size marker. NI, noninfected.

View larger version (30K): [in this window] [in a new window]   FIG. 8.   Inhibition of intracellular bacterial replication and inhibition of bacterial uptake do not block L. pneumophila-induced apoptosis and cytopathogenicity to U937 macrophages. (A) Some monolayers were treated with chloramphenicol at 20 (lane 7) or 100 (lane 8) µg/ml for 1 h prior to infection at an MOI of 50 and during the 1-h infection period and the subsequent 3-h incubation time. Some monolayers were pretreated with 1-µg/ml CytD for 30 min and infected at an MOI of 50 for 30 min in the presence of CytD before gentamicin treatment. CytD was either removed (lane 5) or maintained (lane 6) after the 1-h gentamicin treatment. Uninfected monolayers were incubated with chloramphenicol at 0 (lane 1), 20 (lane 9), or 100 (lane 10) µg/ml for 5 h or with CytD at 1 µg/ml for 1 h (lane 2) or 5 h (lane 3). Formalin-killed bacteria were also used to infect U937 macrophages to examine their ability to induce DNA fragmentation (lane 11). Lane M contained a 100-bp molecular size marker. (B) Cytopathogenicity of L. pneumophila to U937 macrophages under different treatment conditions. Infection or treatments were performed as described in the legend to Fig. 7 for detection of DNA fragmentation. Percent cytopathogenicity was determined as described in the legend to Fig. 1A.

Extracellular L. pneumophila induces apoptosis in U937 macrophages. The actin microfilament depolymerizer CytD completely inhibits uptake of L. pneumophila by U937 macrophages (25). We observed by transmission electron microscopy and confocal laser scanning microscopy that numerous cells in the infected monolayers that did not contain bacteria were apoptotic (data not shown). Therefore, we examined whether extracellular L. pneumophila induced apoptosis in U937 macrophages during inhibition of bacterial uptake by CytD. We confirmed complete blockage of bacterial uptake by CytD-treated cells by sterilization of the infected monolayers following gentamicin treatment (19, 20). Our data showed that CytD by itself did not have detectable effects on DNA fragmentation and association of the bacteria with the U937 macrophages (Fig. 8A) (19, 20). Inhibition of bacterial uptake by CytD reduced but did not block the ability of L. pneumophila to induce apoptosis in U937 macrophages, as examined by agarose gel DNA fragmentation (Fig. 8A, lanes 5 and 6). This observation was further confirmed by TUNEL assays (Fig. 4D). Viability of L. pneumophila was essential for induction of apoptosis in U937 macrophages since formalin-killed bacteria completely lost the ability to induce apoptosis (Fig. 8A, lane 11). A viable bacterium-cell contact may be required for induction of apoptosis, since bacterial culture supernatants or supernatants of infected monolayers were not able to induce apoptosis (data not shown). In addition, correlated with their ability to induce apoptosis in U937 macrophages, extracellular bacteria were also cytopathogenic to these cells (Fig. 8B), further supporting our observations of a correlation between induction of apoptosis and cytopathogenicity.

L. pneumophila-induced apoptosis is mediated by activation of the caspase cascade. Activation of the cascade of caspases is required for DNA fragmentation in various types of cells induced by a variety of apoptotic stimuli (38). While S. flexneri has been shown to induce DNA fragmentation in the host cell by activation of caspase-1, Mycoplasma penetrans induces DNA fragmentation in the host cell by secretion of a bacterial nuclease that specifically cleaves host cell DNA (9). To examine whether DNA fragmentation in L. pneumophila-infected U937 macrophages was mediated by a bacterial nuclease or by activation of the host cell caspases, a broad-specificity, cell-permeable caspase inhibitor, Z-VAD-FMK, was used to examine whether it could block L. pneumophila-induced apoptosis. Monolayers were pretreated with Z-VAD-FMK for 90 min, infected with strain AA100 at an MOI of 50 for 1 h in the presence of the inhibitor, washed to remove unattached extracellular bacteria, and incubated for an additional 3 h in the presence of the inhibitor. DNA was isolated at the end of the 3-h incubation period and examined by agarose gel electrophoresis for detection of DNA fragmentation. As shown in Fig. 9, DNA fragmentation in L. pneumophila-infected U937 macrophages was completely blocked by Z-VAD-FMK. Similar results were obtained for macrophages infected by L. pneumophila at a lower MOI of 5 (data not shown). Incubation of L. pneumophila with Z-VAD-FMK did not affect bacterial viability or the ability to induce apoptosis in U937 macrophages (data not shown), indicating that inhibition of DNA fragmentation was specifically due to inhibition of the activity of caspases.

View larger version (41K): [in this window] [in a new window]   FIG. 9.   Inhibition of DNA fragmentation in L. pneumophila-infected U937 macrophages by the caspase inhibitor Z-VAD-FMK. Monolayers were pretreated with a 100 µM concentration of the inhibitor for 90 min and infected with strain AA100 at an MOI of 50 in the presence of the inhibitor, extracellular bacteria were washed off, and monolayers were incubated for an additional 3 h in the presence of the inhibitor before DNA isolation. Lane M contained a 100-bp molecular size marker. NI, noninfected.

	DISCUSSION

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It has been well documented that L. pneumophila replicates within and is cytopathogenic to macrophages, monocytes, and epithelial cells (1, 6, 24, 31, 35). The mechanisms of cell death due to L. pneumophila infection are not well understood. Both apoptosis and necrosis have been recently observed in L. pneumophila-infected macrophages (24, 26, 34). Müller et al. reported apoptosis in L. pneumophila-infected HL-60 macrophages at 24 and 48 h postinfection when the cells were infected at an MOI of 10 to 100 (34). On the other hand, when a high MOI of 500 was used for infection of mouse bone marrow-derived macrophages, L. pneumophila induced rapid necrosis within 20 to 60 min after infection, without signs of apoptosis during this period, as examined by electron microscopy (26). In this study, we have used multiple criteria to examine apoptosis and shown that, at an MOI of 0.5, 5, or 50, L. pneumophila induces apoptosis in macrophages and alveolar epithelial cells within a few hours of infection in a dose-dependent manner. We also provide genetic and biochemical evidence that induction of apoptosis by L. pneumophila does not require intracellular bacterial replication or new protein synthesis and that apoptosis correlates with cytopathogenicity. Therefore, apoptosis plays an important role in L. pneumophila-induced cell death of macrophages and alveolar epithelial cells during early stages of infection.

Our data show that apoptosis is exhibited in L. pneumophila-infected cells during the first few hours of infection. The ability of L. pneumophila to induce apoptosis is independent of the bacterial growth phase (17). In addition to induction of apoptosis, L. pneumophila becomes cytotoxic and induces rapid necrosis in macrophages and alveolar epithelial cells only upon exiting the exponential phase and entering the postexponential phase of growth (1, 10, 21). Therefore, we propose a model by which L. pneumophila promotes biphasic death of the host cell. In the first phase, host cells are exposed to a low dose of the bacteria, which is what occurs naturally (6). During this phase, the cells undergo apoptosis when the bacterial number reaches a certain threshold, regardless of the growth phase. The second phase is manifested during late stages of the infection, when there is a large number of postexponential-phase bacteria. During this phase, the bacteria become cytotoxic, infected cells become necrotic, intracellular bacteria are released, and the neighboring cells also undergo necrosis upon exposure to a large number of cytotoxic bacteria released from the lysed cells. This second phase of necrotic death is most probably mediated by temporal expression of the pore-forming toxin upon entry of L. pneumophila into the postexponential phase of growth (10, 21, 24, 26). The two strategies (necrosis and apoptosis) utilized by L. pneumophila to kill the host cell may be required to ensure exploitation of host cell resources for intracellular replication and subsequent efficient killing to exit the spent host cell upon termination of intracellular replication.

Different strategies can be used by different species of facultative intracellular bacterial pathogens to induce apoptosis in specific host cells. S. flexneri induces apoptosis in macrophages after escape from the phagosome into the cytoplasm (42) but does not induce apoptosis in epithelial cells (43). Yersinia enterocolitica, which induces apoptosis in macrophages from an extracellular location, also does not induce apoptosis in epithelial cells (37). In contrast, we have demonstrated that extracellular L. pneumophila is capable of inducing apoptosis in both macrophages and alveolar epithelial cells. This induction of apoptosis prior to entry of L. pneumophila may be required for formation of the intracellular niche and alteration of the host cell endocytic pathway.

While S. flexneri has been shown to induce DNA fragmentation in the host cell by activation of caspase-1, M. penetrans induces DNA fragmentation in the host cell by secretion of a bacterial nuclease that specifically cleaves host cell DNA (9). Inhibition of caspase activity by the broad-specificity caspase inhibitor completely blocks nuclear apoptosis in L. pneumophila-infected U937 macrophages. This is further confirmed by our recent observations that activation of caspase-3, but not caspase-1, is essential for nuclear apoptosis induced upon infection by L. pneumophila (17). Therefore, activation of the caspase cascade, but not a bacterial nuclease activity, is responsible for apoptosis induced by L. pneumophila.

Based on our observations that live bacteria are required to induce apoptosis in macrophages from an extracellular location, we speculate that L. pneumophila exerts its apoptotic effect on the host cell through contact-mediated export of a bacterial factor(s) that results in activation of the cascade of caspases. This speculation is supported by our finding that pmi mutants of L. pneumophila with mutations in various dot or icm loci, which encode proteins thought to be involved in the assembly of a type IV-like secretion apparatus (39, 41), are completely defective in the induction of apoptosis in U937 macrophages. It is likely that some of these mutants are defective in components of the secretion apparatus (such as the cytoplasmic membrane protein DotA) (36) or in expression of the apoptosis-inducing factor(s). Further characterization of the mechanisms of induction of apoptosis by L. pneumophila will elucidate these possibilities.