1 Division of Cell Biology and Biochemistry, Department of Basic Medical Sciences, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 Division of Bacterial Infection, Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Correspondence
Takashi Nonaka
Shinobu Imajoh-Ohmi
nonakat{at}prit.go.jp
ohmi{at}ims.u-tokyo.ac.jp
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ABSTRACT |
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Present address: Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8, Kamikitazawa, Setagaya-ku, Tokyo, 156-8585, Japan.
Present address: Laboratory of Bacterial Infection, Kitasato Institute for Life Sciences, Kitasato University and the Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan.
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INTRODUCTION |
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With regard to intracellular Shigella and Salmonella, these bacteria have been suggested to be able to induce host cell apoptosis by secreting effector proteins into the host cytoplasm, via the type III secretion system, factors that directly bind and activate caspase-1 (Hilbi et al., 1998; Hersh et al., 1999
). Shigella kills its host macrophage by releasing IpaB, a secreted bacterial protein that is associated with virulence and the ability to invade host cells (Chen et al., 1996
). IpaB directly binds to and activates caspase-1 and is thus thought to be the key molecule in the induction of caspase-1-dependent apoptosis by Shigella infection (Hilbi et al., 1998
; Chen et al., 1996
). Similarly, Salmonella is believed to induce macrophage caspase-1-dependent apoptosis by secreting the SipB protein into the cytoplasm of its host macrophage. The SipB protein is thought to be the Salmonella homologue of IpaB and it also binds directly to and activates pro caspase-1 (Hersh et al., 1999
).
Despite the involvement of caspase-1 in the apparent induction of apoptosis by Shigella and Salmonella, this molecule is usually not involved in most apoptotic processes. Kuida et al. (1995) and Li et al. (1995
, 1997)
demonstrated that caspase-1-deficient mice fail to produce interleukin (IL)-1
and thus could resist endotoxic shock, indicating that caspase-1 plays a major role in cytokine maturation, and the mouse cells did not exhibit significant defects in apoptosis. In contrast, caspase-3 and/or caspase-7 are known to play a central role in driving the classical apoptotic pathways triggered by a variety of stimuli. However, in Shigella-induced macrophage cell death, the activation of caspase-3/-7, or the cleavage of poly(ADP-ribose) polymerase (PARP), one of the specific substrates for these caspases during apoptosis, has not been detected (Hilbi et al., 1998
; Chen et al., 1996
). The lack of caspase-3/-7 involvement in Shigella-induced cell death thus casts doubt on whether the killing truly occurs through apoptosis. Supporting this is the finding by Fernandez-Prada et al. (1997
, 2000)
that human monocyte-derived macrophages infected in vitro with Shigella flexneri undergo a rapid cytolytic event that is similar to oncosis (necrotic cell death) but not apoptosis. They observed that when human monocyte-derived macrophages were infected with virulent Shigella, this resulted in cell death that involved rupture of the plasma membrane, cell swelling, disintegration of the cellular ultrastructure, and generalized karyolysis. Other groups have also reported that the macrophage cell death induced by Salmonella infection does not resemble typical apoptosis (Brennan & Cookson, 2000
; Watson et al., 2000
), as Salmonella-infected macrophages appear to rapidly lose their membrane integrity in a manner similar to Shigella-infected macrophages (Fernandez-Prada et al., 1997
, 2000
; Nonaka et al., 1999
). Thus, the mechanism by which Shigella and Salmonella kill their host macrophages is unclear and controversial (Boise & Collins, 2001
).
In this study, we report that the wild-type S. flexneri strain YSH6000 can induce both types of cell death, necrosis and apoptosis, depending on the type of host cell. We used terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining and measurements of caspase activities to discriminate between apoptotic and necrotic cell death and show that Shigella can induce rapid necrotic cell death in specifically macrophage-like cells. We also found that Shigella can induce apoptosis of particular cells. The mechanism does not require phagocytosis of the bacteria and is not dependent on their virulence. Thus, Shigella can kill host cells through either necrosis or apoptosis depending on the host cell type.
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METHODS |
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Infection of host cells with Shigella.
U937 cells were pre-treated with 100 units interferon- ml-1 (IFN; a gift from Shionogi Pharmaceuticals) or 3 µM all-trans-retinoic acid (RA; Sigma) for 48 h to induce them to differentiate into macrophage-like cells before infection as described previously (Nonaka et al., 1999
; Kikuchi et al., 1996
). The host cells (
106 cells per well in 12- or 24-well plates) were cultured with antibiotic-free medium for 1 h before infection. To obtain highly invasive bacteria, cultures incubated overnight at 30 °C were diluted 1 : 50 with BHI broth and incubated at 37 °C for 2 h before use. The cells were then infected with bacteria at the indicated m.o.i., centrifuged at 700 g for 10 min, and incubated for 1 h at 37 °C in a CO2 incubator. Subsequently, 100 µg gentamicin ml-1 (Gm; Sigma) was added to the medium and the cell/bacteria mixture was further incubated at 37 °C in a CO2 incubator for the indicated time to kill the extracellular bacteria.
Measurement of Shigella-induced cytotoxicity.
Cytotoxicity induced by Shigella infection was analysed with the CytoTox 96 Cytotoxicity Assay Kit (Promega). The percentage cytotoxicity was calculated by quantifying the amount of cytoplasmic lactate dehydrogenase (LDH) released by the dying cells according to the following formula: [(experimental LDH activity - spontaneous LDH activity)/(total LDH activity - spontaneous LDH activity)]x100.
Measurement of caspase activity in Shigella-infected cells.
J774 or undifferentiated and differentiated U937 cells were either infected with Shigella or incubated with 0·5 µM of the apoptosis-inducing agent staurosporine (stsp; Sigma) as a positive control for apoptosis. After the indicated period, 107 cells were harvested and washed with 0·15 M NaCl. Cytosolic fractions were prepared as follows. Cells were resuspended in 200 µl lysis buffer (20 mM HEPES/KOH, pH 7·5, 1 mM EDTA), disrupted by sonication, and insoluble material was removed by centrifugation at 15 000 g for 20 min at 4 °C. The supernatant was tested for the measurement of caspase-3/-7 or caspase-1 activity by using the fluorescent synthetic peptide substrates Ac-DEVD-MCA and Ac-WEHD-MCA, respectively (Peptide Institute) according to the established method (Thornberry et al., 1997
). In brief, the samples (5, 10, 20 µl) were mixed with 0·5 ml of assay buffer (20 mM HEPES/KOH, pH 7·5, 1 mM EDTA, 100 mM NaCl and 10 mM 2-mercaptoethanol) and water in a total volume of 1 ml. After pre-incubation at 37 °C for 15 min, 10 µl of 10 mM peptide substrate was added to the reaction mixtures and incubated at 37 °C for 15 min. One millilitre of 2 % (v/v) acetic acid was added to stop the enzymic reaction, and 7-amino-4-methylcoumarin (AMC) release was measured fluorometrically using an excitation wavelength of 365 nm and an emission wavelength of 460 nm. One unit was defined as the amount of enzyme capable of generating 1 nmol AMC in 1 h.
Detection of the cleavage of PARP by immunoblotting.
Infected or stsp-treated cells (106 cells) were harvested and washed with 0·15 M NaCl. Trichloroacetic acid was added to the cell suspension to a final concentration of 10 % (v/v), and the sample was placed on ice for 20 min. Precipitates were recovered by centrifugation at 15 000 g for 5 min at 4 °C, dissolved in 100 µl SDS-sample buffer containing 2·3 % (w/v) SDS, 10 % (w/v) glycerol, 5 % (v/v) 2-mercaptoethanol and 10 µg bromophenol blue ml-1 in 125 mM Tris/HCl, pH 6·8, and heated at 100 °C for 5 min. Samples were run out on a 7·5 % (w/v) polyacrylamide gel containing SDS and electroblotted onto a PVDF membrane (Immobilon, Millipore). The membrane was then soaked for 2 h at room temperature in Tris-buffered saline (TBS: 0·15 M NaCl in 20 mM Tris/HCl, pH 7·5) containing 20 mg bovine serum albumin (BSA) ml-1. The membrane was subsequently incubated overnight at 4 °C with a polyclonal antibody specific for the amino-terminal region of the 85 kDa fragment of human PARP that is generated by caspase-3/-7 (Nonaka et al., 1999
; Kato et al., 2000
). The antibody was diluted 1 : 500 with TBS containing 20 mg BSA ml-1. After washing three times in TBS containing 0·05 % (v/v) Tween 20 (Wako Pure Chemical Industries) for 15 min, the membrane was incubated at room temperature for 1 h with 1 : 7000 diluted anti-rabbit IgG conjugated with alkaline phosphatase (Promega) and then again washed three times in TBS containing 0·05 % Tween 20 for 15 min. Immune complexes were detected by the enzymic reaction of alkaline phosphatase with 5-bromo-4-chloro-3-indolyl phosphate (Nacalai tesque) and nitro blue tetrazolium (Nacalai tesque).
Inhibition of caspase activity and phagocytosis.
Host cells were pre-treated with 100 µM Ac-DEVD-CHO, which inhibits caspase-3/-7, Ac-YVKD-CHO, which inhibits caspase-1, or Z-VAD-fmk, a general caspase inhibitor (Peptide Institute, Osaka, Japan), or with 5 µg ml-1 of the phagocytosis inhibitor cytochalasin D (Sigma) 1 h prior to infection with Shigella. LDH assays were performed 2 and 5 h after infection at an m.o.i. of 50.
TUNEL staining and immunofluorescence microscopy.
J774 cells were grown to 106 cells on a coverslip (18x18 mm). In the case of U937IFN and U937RA,
106 cells were incubated on a poly-L-lysine (Sigma) pre-treated coverslip before infection. Cells on the coverslips were infected with Shigella at an m.o.i. of 50, incubated for 30 min, and then 100 µg Gm ml-1 was added to each sample. After incubation for the indicated times, the infected cells on the coverslips were fixed with 4 % (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. The coverslips were then incubated in 50 mM NH4Cl in PBS for 5 min and cell permeabilization was performed with 0·2 % (v/v) Triton X-100 in PBS for 5 min. After blocking for 30 min in 40 mg BSA ml-1 in TBS, the bacteria on the coverslips were stained by a rabbit anti-S. flexneri 2a lipopolysaccharide (LPS) antibody (Watarai et al., 1997
) followed by the Cy5-labelled goat anti-rabbit IgG (Sigma) as the secondary antibody. After washing, the apoptotic cells were labelled by using the In Situ Cell Death Detection Kit (Roche Diagnostics), according to manufacturer's protocol. The stained cells were observed by confocal laser scanning microscopy (Bio-Rad).
Flow cytometric analysis.
Undifferentiated and differentiated U937 and J774 cells were infected with YSH6000 at an m.o.i. of 50 or treated with 0·5 µM stsp, and then harvested and washed with PBS. Adherent J774 cells were removed from 6-well plates with PBS-EDTA and pooled with the non-adherent cells in the culture medium. Cells were labelled with Annexin-V-FLUOS (Roche Diagnostics) according to the manufacturer's protocol. In brief, 106 cells were washed with incubation buffer (10 mM HEPES/NaOH, pH 7·4, 140 mM NaCl, 5 mM CaCl2), resuspended in 100 µl labelling solution [10 µl Annexin-V-FLUOS and 10 µl 50 µg ml-1 propidium iodide (PI) in 1 ml incubation buffer] and incubated at room temperature in the dark for 15 min. Cells were immediately analysed on a FACScan (Becton Dickinson).
Osmoprotection assay.
U937RA and J774 cells were pre-incubated in RPMI1640 medium supplemented with 20 mM of various osmoprotectants, namely sucrose (Nacalai Tesque), PEG 600 (Tokyo Kasei), PEG 1000 (Nacalai Tesque), and PEG 2000 (Nacalai Tesque) made in PBS. The cells were then infected at an m.o.i. of 50 for 2 h, and LDH release by the infected cells was measured as described above. For the estimation of the functional diameter of inserted pores into host membrane, we used the values for hydrodynamic diameters of the non-electrolytes (Scherrer et al., 1971), where the hydrodynamic diameters were calculated on the viscosity of non-electrolyte solutions.
Measurement of apoptosis-inducing potential of Shigella.
J774 or undifferentiated and differentiated U937 cells were incubated with either the wild-type YSH6000 strain, the avirulent mutant N1411 lacking ipaBCDA, the Gm-treated killed wild-type YSH6000, or E. coli JM109 at the indicated m.o.i.. The Gm-treated YSH6000 were obtained by resuspension in RPMI1640 with 100 µg Gm ml-1 and incubation at 37 °C for 30 min. As a positive control for apoptosis, the cells were incubated with 0·5 µM stsp. The bacterial suspension was added to host cells at the indicated m.o.i., and incubated at 37 °C for 5 h. The cells were harvested and prepared for measurements of caspase activity or immunoblot analyses as described above.
Protein concentration.
Protein concentrations were determined by the method of Bradford (1976) using BSA as the standard.
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RESULTS |
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Cell death of macrophage-like cells infected with Shigella is not accompanied by caspase activation
Hilbi et al. (1998) reported that Shigella infection induces macrophage apoptosis accompanied by caspase-1 but not caspase-3 activation. This is unusual because caspase-1 is not involved in most apoptotic processes, being more prominent in cytokine maturation. We thus characterized Shigella-induced cell death in macrophage-like cell lines further by directly examining the caspase activities in the infected cells. Host cells (U937UD, U937IFN, U937RA and J774) were infected with wild-type Shigella at an m.o.i. of 50. After subsequent incubation in the presence of Gm for several hours, the cytosolic fractions of the infected cells were prepared and assayed for caspase-1 and caspase-3/-7 activity using the synthetic peptide substrates Ac-WEHD-MCA and Ac-DEVD-MCA, respectively. As shown in Fig. 2
(a), no cleavage of either Ac-DEVD-MCA or Ac-WEHD-MCA by the cytosolic fractions of infected U937RA and J774 cells was detected. In contrast, strong activity against Ac-DEVD-MCA, but not Ac-WEHD-MCA, was seen with the infected U937IFN cells (Fig. 2a
) as well as the stsp-induced apoptotic cells that were used as a positive control for apoptosis (Fig. 2b
).
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Shigella is known to lyse red blood cells (RBCs) when they are brought into close contact with the cells by centrifugation. This is known as contact haemolysis and it is apparently mediated by the Shigella-mediated insertion of a 2·5 nm pore into the red blood cell membrane (Blocker et al., 1999). A similar mechanism is also observed with Yersinia species. Here, the formation of the 1·23·2 nm pore in RBCs requires the yopB gene product (Hakansson et al., 1996
). It is thus possible that the Shigella-mediated rapid cell death of infected host cells may also be due to osmotic lysis caused by the formation of a pore in the host cell membrane. Polyethylene glycols (PEGs) with various molecular masses have been used to measure the size of pores induced by several pathogens (Moran et al., 1992
; Kirby et al., 1998
; Dacheux et al., 2001
). We tested the ability of various osmoprotectants to protect infected U937RA cells from death by pre-incubating host cells with osmoprotectants and then monitoring LDH release 2 h after infection. Although sucrose (0·9 nm diameter) did not prevent the lysis of infected cells, PEGs with a molecular mass of 600 (1·6 nm diameter) and 1000 (2 nm diameter) reproducibly suppressed LDH release, and PEG 2000 (2·8 nm diameter) was the most effective osmoprotectant, as shown in Fig. 7
. From a plot of the relative cell death in the presence of osmoprotectants versus their diameter (not shown), we estimated that the functional diameter of the pore is 2·87 nm (±0·4 nm, standard deviation, n=4). In analyses of Shigella-infected J774 cells, the similar size of pores was also determined (data not shown).
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To determine whether the ability of Shigella to generate apoptosis is dependent on its virulence, we performed infection experiments using the avirulent S. flexneri mutant N1411, which lacks ipaBCDA. As shown in Fig. 1(b), mutant N1411 did not induce rapid necrotic cell death in any cell line used. However, the cleavage of PARP by caspase-3/-7 was seen in infected U937IFN and U937UD cells irrespective of pathogenicity (Fig. 3b
). Furthermore, U937IFN cells were incubated with N1411 at an m.o.i. of 50 for 5 h and caspase-3/-7 and caspase-1 activity in the lysate was measured. Caspase-3/-7 but not caspase-1 activity was detected as summarized in Table 2
. Thus, avirulent strains can also induce apoptosis in U937IFN cells. We then examined whether S. flexneri killed by pre-treatment with antibiotics or the E. coli strain JM109 could also induce apoptosis in U937IFN cells. U937IFN cells were mixed with the bacterial suspensions at the indicated m.o.i., incubated for 5 h, and cytosolic fractions were prepared. Caspase-3/-7 activity was detected in the U937IFN line when it had been exposed to killed wild-type Shigella and even E. coli JM109, as summarized in Table 2
. Caspase-1 activity was not detected at all (data not shown).
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DISCUSSION |
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Zychlinsky and co-workers previously showed that Shigella induces IpaB-mediated apoptosis in macrophages (Zychlinsky et al., 1992; Chen et al., 1996
; Hilbi et al., 1998
), so we tried to reproduce their results using similar methods. However, we detected only virulence-dependent cell death of infected macrophage-like cells (Fig. 1
), but not the effective suppression of cell death by caspase-1 inhibitor (Fig. 4
), the binding of caspase-1 and IpaB, or the activation of caspase-1 (i.e. processing of pro-caspase-1) in infected cells (data not shown). Recently, it has been reported that the release of mature IL-1
appears to be linked to the processing of precursor forms by caspase-1, although functional caspase-1 has not been detected in monocytes and macrophages (Ayala et al., 1994
; Wewers et al., 1997
; Mehta et al., 2001
). The researchers also suggested that caspase-1 may have a role in release of mature IL-1
that is separate from its function as a protease. Furthermore, it has been also reported that another protease(s) could be involved in the processing of the pro-form of IL-1
(Schonbeck et al., 1998
). These findings raised the possibility that caspase-1 may not be associated with induction of cell death by infection of Shigella. Therefore, we tried to re-analyse cell death of infected macrophages by different methods, including direct assay of caspase activity using synthetic peptide substrates, flow cytometric analysis using annexin V/PI staining, and confocal laser microscopic analysis with TUNEL staining. To examine if the activity of caspase-1 could be required for induction of cell death by Shigella infection, we directly assayed the activity of caspase-1 with a specific synthetic peptide substrate, Ac-WEHD-MCA. However, no activity was detected in lysates from U937RA and J774 cells infected with wild-type Shigella. Similarly, the activity of caspase-3 was not detected at all. We also observed that caspase-1 inhibitor, Ac-YVKD-CHO, had only partial effects on cell death by Shigella early during infection, correlating with the results of the caspase-1 activity assay. We conclude that caspase-1 may have only partial effects on Shigella-induced cell death early during infection under our experimental conditions, but caspase-3/-7 do not contribute to this cell death. Also, we can not rule out the possibility that caspase(s) other than caspase-1, -3 and -7 are associated with induction of Shigella-induced cell death, because Z-VAD-fmk significantly suppressed cell death by Shigella at 2 h post-infection. Furthermore, to distinguish clearly necrosis from apoptosis in infected cells, we examined infected cells with annexin V/PI staining or the TUNEL method. It has been reported that the cleavage of chromatin DNA is caused not only in apoptotic cells but also in necrotic cells (Dong et al., 1997
; de Torres et al., 1997
). In apoptotic cells, the cleavage of chromatin DNA is accompanied by nuclear condensation. On the other hand, necrotic cells showed DNA fragmentation without nuclear condensation. Our results clearly revealed that macrophage-like cell death by infection of Shigella is necrosis rather than classical apoptosis.
A possible mechanism by which Shigella kills macrophage-like cells is that it inserts a pore into the plasma membrane that causes osmotic lysis. This is suggested by the work of Blocker et al. (1999), who showed that Shigella can lyse RBCs by inserting a 2·5 nm pore into the cell membrane. However, it was initially not clear if the macrophage-like cell death induced by wild-type Shigella occurs through a similar mechanism, because Blocker et al. (1999)
examined the causes of Shigella-induced cytotoxicity only with RBCs, not with living macrophage cells. Furthermore, the Shigella-induced haemolysis requires centrifugation (Blocker et al., 1999
), indicating that Shigella can lyse the plasma membrane by contact from the outside of the RBCs. In contrast, we have shown here that the Shigella-induced macrophage-like cell death can be completely inhibited by the pre-treatment of the host cells with cytochalasin D. Thus, phagocytosis is a prerequisite for Shigella-induced macrophage-like cell death, which indicates that only internalized bacteria can lyse the plasma membrane of macrophage-like cells. Nevertheless, we have shown that Shigella kills macrophage-like cells in a manner that resembles haemolysis, at least superficially, in that it forms a pore that could result in osmotic lysis. Although the pore size we measured (2·87±0·4 nm) on macrophage-like cells was very similar to that (2·5 nm) on RBCs (Blocker et al., 1999
), it is not yet clear if the same pore is involved in both phenomena. Supporting the possibility that the same pore is involved is that Blocker et al. (1999)
have found that pore formation in haemolysis is dependent on both IpaB and IpaC. Similarly, macrophage-induced cell death is dependent on the ipaBCDA genes, because Shigella-induced killing does not occur upon infection with the avirulent mutant of the YSH6000 strain, N1411, which lacks ipaBCDA.
In intestinal mucosa, phagocytic cells such as macrophages have a variety of phenotypes. It is known that CD11b is one of the representative surface markers of macrophages, and the expression level is not constant between cell types (i.e. source organs or differentiation stages) (Rogler et al., 1998). Therefore, it has been suggested that phagocytic cells with different levels of CD11b could be a relevant target for Shigella. The U937 cell line can be induced to differentiate into macrophage-like cell lines by treatment with RA or IFN, generating the cell lines we have denoted as U937RA and U937IFN. These two lines differ in that U937RA appears to be more macrophage-like since the cells generate more superoxide and express higher levels of CD11b (Kikuchi et al., 1994
, 1996
). In this study, we used U937IFN and U937RA as reproducibly available cell lines representing the different cell types in the intestinal mucosa, not the partially differentiated myeloid cells. We have shown that Shigella induces necrosis in macrophage-like cells with higher expression of CD11b and apoptosis in cells with lower expression levels. Surprisingly, the inhibition of phagocytosis by cytochalasin D did not suppress Shigella-induced U937IFN apoptosis; rather, it appeared to enhance it. In addition, not only the wild-type YSH6000 strain but also the avirulent N1411 mutant, Gm-killed YSH6000, and the E. coli strain JM109 could induce apoptosis in U937IFN. These observations suggest that extracellular bacteria can transmit apoptotic signals in a manner that is independent of their pathogenicity. Susceptibility to such apoptotic signals was not observed for U937RA and J774 cells. One possible molecule involved in this process could be the Toll-like receptor (TLR)-2 (Kirschning et al., 1998
). Aliprantis et al. (1999)
have reported that bacterial lipoproteins, which are expressed by all bacteria, are potent activators of TLR-2 and that TLR-2 transmits a pro-apoptotic signal. Recently, Y. Niikura and co-workers (personal communication) showed that Fas is up-regulated in U937IFN cells, while the receptor is down-regulated in U937RA cells. U937IFN is more sensitive to apoptotic stimuli through Fas than U937RA. Similar regulation may be seen in the case of other receptor(s) e.g. TLR-2 and porimin (Ma et al., 2001
), which is thought to be a receptor associated with induction of oncosis. These findings suggest that the expression level of such receptors involved in induction of cell death could be associated with Shigella's ability to induce necrosis or apoptosis.
In summary, we have found that infection of macrophage-like cell lines with S. flexneri strain YSH6000 results in death that is due mainly to the rapid induction of necrosis. The mechanism may involve the formation of a pore in the plasma membrane of infected cells, which is dependent on the ipaBCDA virulence genes. Shigella can also induce death in other cells, without even being taken up, through a mechanism that does not involve the ipaBCDA genes and results in typical apoptosis. These observations point to the ability of Shigella to control the way it kills host cells, as it clearly commands mechanisms that lead to either apoptosis or necrosis. This ability most likely plays a crucial role in its initiation of infection, survival, and escape from host immune responses.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan and from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ayala, J. M., Yamin, T. T., Egger, L. A., Chin, J., Kostura, M. J. & Miller, D. K. (1994). IL-1 beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor. J Immunol 153, 25922597.
Blocker, A., Gounon, P., Larquet, E., Niebuhr, K., Cabiaux, V., Parsot, C. & Sansonetti, P. (1999). The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. J Cell Biol 147, 683693.
Boise, L. H. & Collins, C. M. (2001). Salmonella-induced cell death: apoptosis, necrosis or programmed cell death? Trends Microbiol 9, 6467.[CrossRef][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Brennan, M. A. & Cookson, B. T. (2000). Salmonella induces macrophage death by caspase-1-dependent necrosis. Mol Microbiol 38, 3140.[CrossRef][Medline]
Chen, Y., Smith, M. R., Thirumalai, K. & Zychlinsky, A. (1996). A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J 15, 38533860.[Abstract]
Clifton, D. R., Goss, R. A., Sahni, S. K., Antwerp, D. V., Baggs, R. B., Marder, V. J., Silverman, D. J. & Sporn, L. A. (1998). NF-kappa B-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection. Proc Natl Acad Sci U S A 95, 46464651.
Dacheux, D., Goure, J., Cabert, J., Usson, Y. & Attree, I. (2001). Pore-forming activity of type III system-secreted proteins leads to oncosis of Pseudomonas aeruginosa-infected macrophages. Mol Microbiol 40, 7685.[CrossRef][Medline]
de Torres, C., Munell, F., Ferrer, I., Reventos, J. & Macaya, A. (1997). Identification of necrotic cell death by the TUNEL assay in the hypoxic-ischemic neonatal rat brain. Neurosci Lett 230, 14.[CrossRef][Medline]
Dong, Z., Saikumar, P., Weinberg, J. M. & Venkatachalam, M. A. (1997). Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases. Am J Pathol 151, 12051213.[Abstract]
Fan, T., Lu, H., Hu, H., Shi, L., McClarty, G. A., Nance, D. M., Greenberg, A. H. & Zhong, G. (1998). Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation. J Exp Med 187, 487496.
Fernandez-Prada, C. M., Hoover, D. L., Tall, B. D. & Venkatesan, M. M. (1997). Human monocyte-derived macrophages infected with virulent Shigella flexneri in vitro undergo a rapid cytolytic event similar to oncosis but not apoptosis. Infect Immun 65, 14861496.[Abstract]
Fernandez-Prada, C. M., Hoover, D. L., Tall, B. D., Hartman, A. B., Kopelowitz, J. & Venkatesan, M. M. (2000). Shigella flexneri IpaH7·8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages. Infect Immun 68, 36083619.
Fratazzi, C., Arbeit, R. D., Carini, C. & Remold, H. G. (1997). Programmed cell death of Mycobacterium avium serovar 4-infected human macrophages prevents the mycobacteria from spreading and induces mycobacterial growth inhibition by freshly added, uninfected macrophages. J Immunol 158, 43204327.[Abstract]
Gao, L. Y. & Abu Kwaik, Y. (2000). Hijacking of apoptotic pathways by bacterial pathogens. Microb Infect 2, 17051719.[CrossRef][Medline]
Hakansson, S., Schesser, K., Persson, C., Galyov, E. E., Rosqvist, R., Homble, F. & Wolf-Watz, H. (1996). The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity. EMBO J 15, 58125823.[Abstract]
Hersh, D., Monack, D. M., Smith, M. R., Ghori, N., Falkow, S. & Zychlinsky, A. (1999). The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc Natl Acad Sci U S A 96, 23962401.
Hilbi, H., Moss, J. E., Hersh, D. & 7 other authors (1998). Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J Biol Chem 273, 3289532900.
Kato, M., Nonaka, T., Maki, M., Kikuchi, H. & Imajoh-Ohmi, S. (2000). Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells. J Biochem 127, 297305.[Abstract]
Kikuchi, H., Fujinawa, T., Kuribayashi, F., Nakanishi, A., Imajoh-Ohmi, S. & Kanegasaki, S. (1994). Induction of essential components of the superoxide generating system in human monoblastic leukemia U937 cells. J Biochem 116, 742746.[Abstract]
Kikuchi, H., Iizuka, R., Sugiyama, S., Gon, G., Mori, H., Arai, M., Mizumoto, K. & Imajoh-Ohmi, S. (1996). Monocytic differentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor alpha in human monoblast U937 cells. J Leukoc Biol 60, 778783.[Abstract]
Kirby, J. E., Vogel, J. P., Andrews, H. L. & Isberg, R. R. (1998). Evidence for pore-forming ability by Legionella pneumophila. Mol Microbiol 27, 323336.[CrossRef][Medline]
Kirschning, C. J., Wesche, H., Ayres, T. M. & Rothe, M. (1998). Human Toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide. J Exp Med 188, 20912097.
Kuida, K., Lippke, J. A., Ku, G., Harding, M. W., Livingston, D. J., Su, M. S. & Flavell, R. A. (1995). Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267, 20002003.[Medline]
Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G. & Earnshaw, W. C. (1994). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371, 346347.[CrossRef][Medline]
Li, P., Allen, H., Banerjee, S. & 7 other authors (1995). Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell 80, 401411.[Medline]
Li, P., Allen, H., Banerjee, S. & Seshadri, T. (1997). Characterization of mice deficient in interleukin-1 beta converting enzyme. J Cell Biochem 64, 2732.[CrossRef][Medline]
Ma, F., Zhang, C., Prasad, K. V., Freeman, G. J. & Schlossman, S. F. (2001). Molecular cloning of Porimin, a novel cell surface receptor mediating oncotic cell death. Proc Natl Acad Sci U S A 98, 97789783.
Mehta, V. B., Hart, J. & Wewers, M. D. (2001). ATP-stimulated release of interleukin (IL)-1b and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage. J Biol Chem 276, 38203826.
Monack, D. M., Raupach, B., Hromocky, A. E. & Falkow, S. (1996). Salmonella typhimurium invasion induces apoptosis in infected macrophages. Proc Natl Acad Sci U S A 93, 98339838.
Moran, O., Zegarra-Moran, O., Virginio, C., Gusmani, L. & Rottini, G. D. (1992). Physical characterization of the pore forming cytolysine from Gardnerella vaginalis. FEMS Microbiol Immunol 5, 6369.[Medline]
Nonaka, T., Kuwae, A., Sasakawa, C. & Imajoh-Ohmi, S. (1999). Shigella flexneri YSH6000 induces two types of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937. FEMS Microbiol Lett 174, 8995.[CrossRef][Medline]
Orth, K., Palmer, L. E., Bao, Z. Q., Stewart, S., Rudolph, A. E., Bliska, J. B. & Dixon, J. E. (1999). Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia effector. Science 285, 19201923.
Rogler, G., Hausmann, M., Vogl, D., Aschenbrenner, E., Andus, T., Falk, W., Andreesen, R., Scholmerich, J. & Gross, V. (1998). Isolation and phenotypic characterization of colonic macrophages. Clin Exp Immunol 112, 205215.[CrossRef][Medline]
Ruckdeschel, K., Roggenkamp, A., Lafont, V., Mangeat, P., Hessemann, J. & Rouot, B. (1997). Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis. Infect Immun 65, 48134821.[Abstract]
Sasakawa, C., Kamata, K., Sakai, T., Murayama, S. Y., Makino, S. & Yoshikawa, M. (1986). Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun 51, 470475.[Medline]
Sasakawa, C., Kamata, K., Sakai, T., Makino, S., Yamada, M., Okada, N. & Yoshikawa, M. (1988). Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a. J Bacteriol 170, 24802484.[Medline]
Scherrer, R. & Gerhardt, P. (1971). Molecular sieving by the Bacillus megaterium cell wall and protoplast. J Bacteriol 107, 718735.[Medline]
Schesser, K., Spiik, A. K., Dukuzumuremyi, J. M., Neurath, M. F., Pettersson, S. & Wolf-Watz, H. (1998). The yopJ locus is required for Yersinia-mediated inhibition of NF-kappaB activation and cytokine expression: YopJ contains a eukaryotic SH2-like domain that is essential for its repressive activity. Mol Microbiol 28, 10671079.[CrossRef][Medline]
Schonbeck, U., Mach, F. & Libby, P. (1998). Generation of biologically active IL-1 beta by matrix metalloproteinases: a novel caspase-1-independent pathway of IL-1 beta processing. J Immunol 161, 33403346.
Thornberry, N. A., Rano, T. A., Peterson, E. P. & 9 other authors (1997). A combinatorial approach defines specificities of members of the caspase family and granzyme B. J Biol Chem 272, 1790717911.
Watarai, M., Kamata, Y., Kozaki, S. & Sasakawa, C. (1997). rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells. J Exp Med 185, 281292.
Watson, P. R., Gautier, A. V., Paulin, S. M., Bland, A. P., Jones, P. W. & Wallis, T. S. (2000). Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis. Infect Immun 68, 37443747.
Wewers, M. D., Dare, H. A., Winnard, A. V., Parker, J. M. & Miller, D. K. (1997). IL-1 beta-converting enzyme (ICE) is present and functional in human alveolar macrophages: macrophage IL-1 beta release limitation is ICE independent. J Immunol 159, 59645972.[Abstract]
Zychlinsky, A., Prevost, M. C. & Sansonetti, P. J. (1992). Shigella flexneri induces apoptosis in infected macrophages. Nature 358, 167169.[CrossRef][Medline]
Received 11 March 2003;
revised 27 May 2003;
accepted 4 June 2003.