Salmonella induces macrophage death by caspase- 1-dependent necrosis

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1 Molecular Microbiology (2000) 38(1), 31±40 Salmonella induces macrophage death by caspase- 1-dependent necrosis Molly A. Brennan 1 and Brad T. Cookson 1,2 * Departments of 1 Microbiology and 2 Laboratory Medicine, Box , University of Washington, Seattle, WA 98195, USA. Summary We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis. Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis. In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase- 3-mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADPribose polymerase (PARP). Caspase-3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state. Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase. During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella-induced cytotoxicity. Finally, inhibition of the interleukin (IL)-1-converting enzyme caspase-1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis. Thus, Salmonella-infected macrophages are killed by an unusual caspase-1- dependent mechanism of necrosis. Introduction Salmonella typhimurium infection of mice and S. typhi infection of humans is characterized by inflammation at the site of bacterial entry and in deeper infected tissues (Jones and Falkow, 1996). penetrates the Accepted 13 July, *For correspondence. cookson@u. washington.edu; Tel. (11) ; Fax (11) murine gastrointestinal tract via Peyer's patches (Jones et al., 1994) to colonize underlying mucosal tissue, in which the bacteria preferentially infect phagocytes and consequently disseminate to the spleen and liver (Vazquez-Torres et al., 1999). After infection of phagocytes, Salmonella spp. can survive and eventually destroy these cells (Fields et al., 1986; Lindgren et al., 1996). Cytotoxicity occurs both in vitro and in vivo (Chen et al., 1996a; Lindgren et al., 1996; Richter-Dahlfors et al., 1997) by a mechanism described previously as apoptosis (Monack et al., 1996; Hersh et al., 1999). Apoptosis and necrosis are two forms of eukaryotic cell death that can be distinguished biochemically and morphologically (Majno and Joris, 1995). Apoptosis requires ATP and the activation of specific proteases. Dying cells display membrane blebbing, nuclear condensation and, eventually, cell shrinkage, yet maintain membrane integrity until late in this process. Necrosis results from membrane damage either directly or indirectly because of energy depletion, and yields swollen cells that leak their cytoplasmic contents. As a result, necrosis is strongly proinflammatory in vivo, whereas apoptotic cells are rapidly phagocytosed and thus generate minimal inflammation (Majno and Joris, 1995). Because the mechanism of host cell death can influence the inflammatory response, discriminating apoptosis from necrosis may provide insight into the pathophysiology of infectious diseases caused by intracellular pathogens. The nuclear changes considered characteristic of apoptotic cells include condensation of nuclei and DNA fragmentation (McGahon et al., 1995). However, the latter event was recently also observed to occur during necrosis (Dong et al., 1997; de Torres et al., 1997; Orita et al., 1999), such that detecting DNA cleavage in cells does not necessarily identify apoptosis. Caspases are a family of cysteine proteases important in apoptosis (Cohen, 1997). Caspase-3 activates `executioner' caspases and cleaves other substrate proteins during the apoptotic death programme (Porter and Janicke, 1999). Although caspase-3-independent pathways of apoptosis exist, this enzyme is essential for nuclear condensation and DNA cleavage (Janicke et al., 1998; Woo et al., 1998). Salmonella infection provokes macrophage DNA fragmentation (Chen et al., 1996a; Lindgren et al., 1996; Monack et al., 1996; Richter- Dahlfors et al., 1997), but it is unknown whether caspase-3 Q 2000 Blackwell Science Ltd

2 32 M. A. Brennan and B. T. Cookson A C E B D F Fig. 1. infection stimulates macrophage DNA fragmentation without nuclear condensation. J774A.1 macrophages adherent to glass coverslips were uniformly infected with, mock infected or treated with 5 mm gliotoxin for 4 h to induce apoptosis. Adherent cells were labelled with the TUNEL reaction, detected by fluorescence microscopy (40 objective) and imaged using a digital camera. PBS-treated cells (A), gliotoxin-treated cells (B), SL :1 (C), SL :1 (D), SL1344 prgh 10:1 (E), SL1344 prgh 100:1 (F). A minimum of 200 cells was examined for each experimental condition in seven separate experiments; results from one representative experiment are shown. is required for this event. Caspase-1, an interleukin (IL)-1- beta-converting enzyme required for Salmonella-induced macrophage cytotoxicity, interacts with the Salmonella secreted protein SipB (Hersh et al., 1999). The caspase-1 protease was originally classified as a proapoptotic enzyme based on homology to CED-3, a protein important in Caenorhabditis elegans cell death (Miura et al., 1993). Although caspase-1 is required for inflammation, it has been shown to be dispensable for apoptosis (Colussi and Kumar, 1999): caspase-1-deficient mice fail to produce IL- 1b and resist endotoxic shock, but demonstrate no significant defects in apoptosis (Kuida et al., 1995; Li et al., 1995; 1997). Macrophage apoptosis can be prevented by interactions with activated CD4 1 T cells expressing the CD40L surface receptor and secreting macrophage-activating cytokines interferon (IFN)-g and IL-12 (Suttles et al., 1996; Estaquier and Ameisen, 1997); therefore, CD4 1 T cells responding to Salmonella (Nauciel, 1990; Cookson and Bevan, 1997) may potentially help infected macrophages to avoid apoptosis. To begin investigating this possibility, we studied Salmonella-infected macrophages using methods that discriminate between apoptotic and necrotic cell death. In contrast to chemically induced apoptosis, infected macrophages fragment their DNA but do not activate caspase-3, and rapidly sustain membrane damage that releases their cytoplasmic contents. Exogenous glycine prevents pathological ion fluxes through the plasma membranes of necrotic cells, and correspondingly blocks the death of Salmonella-infected macrophages. In addition, a key enzymatic substrate, normally cleaved during apoptosis to avoid the stimulation of necrotic cell death, remains in its active state in infected cells. However, unlike apoptosis or necrosis, death of infected macrophages requires caspase-1. Thus, Salmonella-infected macrophages are killed by an unusual caspase-1-dependent mechanism of necrosis. Results DNA fragmentation without nuclear condensation in Salmonella-infected macrophages DNA cleavage, previously considered a hallmark of

3 Salmonella induces caspase-1-dependent necrosis 33 Fig. 2. -infected macrophages expose phosphatidylserine as a result of membrane damage. J774A.1 macrophages were infected with strains at an MOI of 10:1, mock infected or treated with 5 mm gliotoxin for 4 h to induce apoptosis. Macrophages were then stained with phosphatidylserine-binding protein FITC-annexin V and vital dye 7-aminoactinomycin D and immediately evaluated by flow cytometry. A minimum of cells was evaluated per sample. A. FITC-annexin V staining of all cells. B. FITC-annexin V staining of cells with intact plasma membranes excluding vital dye (7-AAD-negative cells). One of four representative experiments is shown. apoptotic nuclear collapse (McGahon et al., 1995), can also occur during necrosis (Dong et al., 1997; de Torres et al., 1997; Orita et al., 1999). The nicked DNA ends can be labelled using the TUNEL reaction (terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end-label) and detected in cells by fluorescence microscopy. The condensed nuclear morphology typical of apoptotic cells was demonstrated by macrophages treated with gliotoxin (Fig. 1B), a fungal toxin that induces apoptosis (Waring et al., 1988) and serves as a positive control (Monack et al., 1996) in our experiments. Salmonella infection also stimulated macrophage DNA fragmentation (Fig. 1C and D), in agreement with previous reports (Chen et al., 1996a; Lindgren et al., 1996; Monack et al., 1996; Richter-Dahlfors et al., 1997). However, the diffuse staining of nicked chromatin in infected cells (Fig. 1C and D) contrasts with the condensed pattern in gliotoxin-treated macrophages (Fig. 1B). Significantly, a prgh mutant failed to induce macrophage DNA fragmentation (Fig. 1E and F), in spite of infecting the phagocytes at levels similar to the parent wild-type strain (see Experimental procedures); therefore, bacterial infection per se is not sufficient to induce DNA cleavage. PrgH is an essential component of the Salmonella type III secretion system (TTSS) (Hueck et al., 1995; Pegues et al., 1995) encoded by Salmonella pathogenicity island 1 (SPI1) (Mills et al., 1995). This TTSS is responsible for exporting effector molecules (Hueck, 1998), including the SipB protein (Hueck et al., 1995; Kaniga et al., 1995a,b; Pegues et al., 1995), which is required for cytotoxicity (Chen et al., 1996a; Hersh et al., 1999). The SPI1 TTSS is therefore also required for Salmonella to induce macrophage DNA fragmentation that is morphologically distinguishable from that observed in apoptotic cells. Salmonella infection of macrophages induces rapid membrane damage Fluorescence microscopy is a powerful tool for discerning cellular morphology, but it does not necessarily reveal features of the entire host cell population, because analyses are limited to relatively small numbers of adherent cells. To evaluate large numbers of Salmonella-infected macrophages, we used a flow cytometric assay that discriminates between apoptosis and necrosis: a fluorescently labelled phospholipid-binding protein, annexin V, was used specifically to detect phosphatidylserine (PS), a membrane lipid that is normally localized to the inner leaflet of the cellular plasma membrane (van Engeland et al., 1998). Early apoptotic cells display PS in their outer leaflet while maintaining membrane integrity. In contrast, necrotic cells expose PS as a result of membrane damage, and therefore exhibit simultaneous uptake of membrane-impermeant dyes such as 7-aminoactinomycin D (7-AAD) (van Engeland et al., 1998). Both Salmonella infection and gliotoxin treatment stimulated macrophage PS exposure to similar levels, as detected by staining with fluorescein isothiocyanate (FITC)-annexin V (Fig. 2A). Unlike apoptotic macrophages, however, Salmonella-infected cells positive for annexin V binding also stained with 7-AAD (Fig. 2B), which indicates membrane disruption of the infected host cells. Examination of Salmonella-infected cells at earlier

4 34 M. A. Brennan and B. T. Cookson % Cytotoxicity PBS gliotoxin prgh Time (h) Fig. 3. Salmonella-infected macrophages rapidly lose membrane integrity. J774A.1 cells were infected with strains at an MOI of 10:1, mock infected or treated with 5 mm gliotoxin to induce apoptosis. Release of the cytoplasmic enzyme lactate dehydrogenase (LDH) was measured in supernatants from these macrophage cultures, and cytotoxicity was calculated as described in Experimental procedures. Each data point represents quadruplicate measurements ^ standard deviation. One of four representative experiments is shown. Indicative of membrane damage, infected and gliotoxintreated macrophages released similar levels of the cytoplasmic enzyme lactate dehydrogenase (LDH) during a 6 h time course (Fig. 3). Rapid LDH release by infected macrophages shows that significant cytoplasmic leakage occurred within 2 h after bacterial invasion (Fig. 3), and similar kinetics were observed using infected thioglycollate-elicited macrophages (data not shown). This was not simply a result of bacterial infection, as the prgh mutant failed to stimulate LDH release (Fig. 3), which confirms the requirement of the SPI1 TTSS for Salmonella to cause macrophage death (Chen et al., 1996a; Hersh et al., 1999). In contrast to infected macrophages, LDH release by apoptotic macrophages was markedly slower and did not reach similar levels until 6 h after gliotoxin treatment (Fig. 3). These observations further define cytotoxicity as a process characterized by diffusely distributed DNA fragmentation and rapid membrane damage, which results in exposure of PS, uptake of vital dye and release of cytoplasmic contents. time points (1 h, 2 h and 3 h) after invasion did not reveal apoptotic populations (FITC-annexin V positive, 7-AAD negative; data not shown), and macrophages infected with a prgh mutant did not show annexin V or 7-AAD staining (Fig. 2). Although membrane damage eventually typifies both apoptosis and necrosis in vitro, the temporal association of the stimulus initiating cell death and detectable membrane damage is distinct. Necrotic cells lose membrane integrity early after injury, whereas apoptotic cells delay this until later stages in their programme of death. Host cell death is independent of apoptotic caspases Apoptosis and necrosis use different enzymatic pathways to execute death, and the caspase-3 protease activated during many apoptotic programmes (Porter and Janicke, 1999) is required for DNA fragmentation (Janicke et al., 1998; Woo et al., 1998). Because infection stimulated macrophage DNA cleavage (Fig. 1), we examined the role of caspase-3 during Salmonella-induced cell death. In contrast to gliotoxin-treated apoptotic cells, infected macrophages did not activate caspase-3 at any time FU Fold Induction A PBS gliotoxin FU Fold Induction B Time (h) 0 PBS gliotoxin prgh + gliotoxin Fig. 4. The apoptotic protease caspase-3 is not activated during Salmonella-induced cytotoxicity. J774A.1 macrophages were infected with S. typhimurium strains at an MOI of 10:1, mock infected or treated with 5 mm gliotoxin to induce apoptosis. Active caspase-3 was detected using a fluorescent substrate as described in Experimental procedures. A. Time course of caspase-3 activation. One of three representative experiments is shown. B. Infection does not inhibit macrophage caspase-3 activation by gliotoxin. J774A.1 cells infected with at an MOI of 10:1 for 2 h were washed and incubated with gentamicin-containing media, with or without gliotoxin, for an additional 2 h before measuring caspase-3 activity. Data represent means 1 SD from three separate experiments.

5 Salmonella induces caspase-1-dependent necrosis 35 Fig. 5. Poly ADP-ribose polymerase (PARP), a mediator of necrotic cell death, remains in its active, uncleaved state in Salmonellainfected macrophages. J774A.1 macrophages were infected with S. typhimurium at an MOI of 10:1, mock infected or treated with 5 mm gliotoxin for 4htoinduce apoptosis. Approximately 10 6 cells were analysed for PARP inactivation by cleavage using Western blotting with an anti-parp monoclonal antibody. Lane 1, PBS; lane 2, gliotoxin; lane 3, ; lane, 4 SL1344 prgh. Results from one of four representative experiments are shown. during a 6 h infection (Fig. 4A). Similar results were obtained with infected peritoneal macrophages (data not shown). However, Salmonella-infected cells remained competent for caspase-3 upregulation by gliotoxin treatment (Fig. 4B), demonstrating that infection neither inhibits activated caspase-3 nor obscures its experimental detection. This suggests that infection-stimulated DNA cleavage and cytotoxicity are caspase-3-independent events. As caspase-3 functions downstream of caspase- 9 and is also required for caspase-2, -6, -8 and -10 activation during apoptosis (Slee et al., 1999), the absence of caspase-3 activation in infected macrophages % Cytotoxicity PBS media YVAD glycine prgh gliotoxin ATP depletion Fig. 6. Inhibition of either non-specific ion fluxes or caspase-1 blocks Salmonella-induced cytotoxicity. J774A.1 cells were pretreated with 5 mm glycine to prevent non-specific ion fluxes through the plasma membrane, or with 100 mm YVAD, a peptide inhibitor of caspase-1. The pretreated macrophages were infected with at an MOI of 10:1 for 2 h, mock infected, treated with 5 mm gliotoxin for 4 h to induce apoptosis or induced to undergo necrosis as a result of ATP depletion, as described in Experimental procedures. LDH released into the supernatant from one of three representative experiments is shown. strongly suggests that Salmonella induces cell death by a mechanism distinct from apoptosis. During apoptosis, cleavage and inactivation of poly ADP-ribose polymerase (PARP) by one or more caspases (Rosen and Casciola-Rosen, 1997) is critical for preserving cellular ATP and therefore the energy required to complete programmed cell death (Ha and Snyder, 1999). DNA damage, including strand breakage, activates PARP to consume substrate NAD1, which leads to ATP depletion and cellular necrosis (Ha and Snyder, 1999). In contrast to apoptotic macrophages, PARP remained in its active, uncleaved state in infected cells (Fig. 5). This suggests that the apoptotic pathways leading to PARP inactivation do not function during Salmonella-induced macrophage death. Together with the kinetics of membrane damage and release of cytoplasmic contents, and the distribution of DNA fragmentation without detectable caspase-3 activation, the mechanism of Salmonellainduced cytotoxicity is most consistent with necrosis. Salmonella-induced macrophage death is prevented by inhibition of either caspase-1 or non-specific ion fluxes through the plasma membrane Necrotic cell death resulting from ATP depletion can be prevented by treatment with exogenous glycine (Dong et al., 1997), which blocks the formation of non-specific plasma membrane leaks for small ions such as sodium (Frank et al., 2000). Glycine treatment also blocks necrosis induced by mitochondrial poisons, cold ischaemia and hypoxia (Weinberg et al., 1987; Dong et al., 1997; Frank et al., 2000). Our observations suggest the possibility that the attendant DNA damage of Salmonella infection activates PARP to deplete ATP and subsequently cause death by necrosis. Consistent with this hypothesis, glycine completely prevented cell death induced by Salmonella infection or ATP depletion, but negligibly affected apoptosis induced by gliotoxin (Fig. 6). Recent work has demonstrated that Salmonellainduced cytotoxicity requires active caspase-1 (Hersh et al., 1999), which, unlike other homologous caspases, is not required for apoptosis (Kuida et al., 1995; Li et al., 1995; 1997). Pretreatment with a caspase-1 peptide inhibitor, YVAD, prevented infection-induced death of macrophages (Fig. 6), including thioglycollate-elicited peritoneal macrophages (data not shown). This confirmed a caspase-1-dependent mechanism of cytotoxicity in our experimental system, which was observed previously by Hersh et al. (1999) using RAW and peritoneal macrophages. Significantly, caspase-1 inhibition did not prevent experimentally induced apoptosis or necrosis (Fig. 6). We conclude that infected macrophages release their cytoplasmic LDH and probably die as a result of nonspecific leaks in their plasma membrane, which can be

6 36 M. A. Brennan and B. T. Cookson Table 1. Summary of observations. Apoptosis Necrosis Salmonella-induced cytotoxicity DNA fragmentation Yes Yes Yes Nuclear condensation Yes No No Caspase-3 activation Yes No No Membrane damage Yes (late in death) Yes (early in death) Yes (early in death) Phosphatidylserine exposure Yes Yes Yes Active PARP No Yes Yes Blocked by glycine No Yes Yes Requires caspase-1 No No Yes blocked by exogenous glycine. Unlike conventional necrosis, this process requires the IL-1-converting enzyme caspase-1, and thus Salmonella induces cytotoxicity by a unique mechanism. Discussion We demonstrated that induces macrophage death via an unusual caspase-1-dependent mechanism of necrosis. The evidence supporting this conclusion is presented in Table 1. An intact SPI1 TTSS system, responsible for secreting SipB, was required for Salmonella to stimulate macrophage DNA fragmentation. Caspase-3, the protease required for DNA fragmentation during apoptosis, was not activated after Salmonella infection, nor did infected macrophages display the condensed nuclei typical of apoptotic cells. Salmonellainfected macrophages demonstrated rapid loss of membrane integrity, release of cytoplasmic LDH and uptake of vital dye concomitant with PS exposure. Infected cells retained PARP in its active uncleaved state, suggesting the possibility that PARP activation by damaged macrophage DNA caused ATP depletion and death by necrosis (Ha and Snyder, 1999). Experimental ATP depletion causes necrotic membrane damage, which is blocked by exogenous glycine (Dong et al., 1997), and glycine treatment also blocked Salmonella-induced cytotoxicity. Conversely, gliotoxin treatment of macrophages induced bona fide apoptosis, as demonstrated by DNA fragmentation, nuclear condensation, caspase-3 activity, PS exposure without membrane damage and PARP proteolysis. Finally, Salmonella-induced cytotoxicity required active caspase-1, unlike apoptosis induced by gliotoxin or necrosis induced by ATP depletion. The mechanism of Salmonella-induced cytotoxicity was examined in a macrophage cell line and primary macrophages at several time points during infection using multiple criteria to distinguish between apoptosis and necrosis. Controls for both types of cell death were included, as were experiments with a non-cytotoxic bacterial mutant to exclude the possibility that our observations were simply the result of bacterial invasion or infection. Others have also observed features of Salmonella-infected macrophages that were consistent with apoptosis (Chen et al., 1996a; Lindgren et al., 1996; Monack et al., 1996; Richter-Dahlfors et al., 1997; Hersh et al., 1999), such as DNA cleavage and caspase-1- dependent cell death; however, neither of these events are apoptosis specific, as revealed in our investigation (Table 1) and supported by a growing body of evidence in the literature (Kuida et al., 1995; Li et al., 1995; 1997; Dong et al., 1997; de Torres et al., 1997; Orita et al., 1999). Further, it is unlikely that small populations of cells in the early or late stages of apoptosis would go undetected in our experiments using sensitive methods such as flow cytometry (Fig. 2) and Western blotting for PARP cleavage (Fig. 5). Thus, our results extend previous studies by demonstrating that infected macrophages die by a caspase-1-dependent mechanism of necrosis, a conclusion at odds with the current model of Salmonella-induced apoptosis. Our data indicated that necrotic death of infected macrophages can be prevented by glycine (Fig. 6), which blocks non-specific ion fluxes through the plasma membrane (Frank et al., 2000). We conclude that an unusual mechanism accounts for Salmonella-induced cytotoxicity: necrotic cell death experimentally induced by ATP depletion was also prevented by glycine, but did not depend upon the activity of caspase-1 (Fig. 6). Inflammation at sites of infection in vivo (Jones and Falkow, 1996) may therefore result from both the production of IL-1b and IL-18 by caspase-1 and the release of cytoplasmic contents from infected, necrotic cells. Collectively, these data suggest that caspase-1 activation triggers non-specific plasma membrane leaks, possibly by using effector pathways common to other forms of necrosis, such as cold ischaemia, hypoxia, mitochondrial poisons and ATP depletion (Weinberg et al., 1987; Dong et al., 1997; Frank et al., 2000). The requirement for other enzymes during Salmonellainduced macrophage death remains largely unknown. For example, are serine proteases required for infected cell death? DNA fragmentation during necrosis results from serine protease activation, in contrast to caspase

7 Salmonella induces caspase-1-dependent necrosis 37 (cysteine protease)-mediated DNA fragmentation during apoptosis (Dong et al., 1997). Interestingly, exogenous glycine also inhibits degradative intracellular proteolytic activity in necrotic cells during ATP depletion (Dickson et al., 1992). PARP may also function during Salmonellainduced cytotoxicity. This polymerase acts in a polar manner during cell death: inactivation of PARP by cleavage preserves cellular energy levels and allows apoptosis to proceed, whereas damaged DNA activates uncleaved PARP to deplete ATP, leading to membrane damage and necrosis (Ha and Snyder, 1999). The Salmonella-induced DNA damage may therefore activate PARP, resulting in ATP depletion and the formation of plasma membrane leaks that can be blocked by glycine (Fig. 6). Fibroblasts from PARP-deficient mice are protected against ATP depletion and necrotic death, but remain sensitive to apoptotic stimuli (Ha and Snyder, 1999). Thus, PARP±/± macrophages may also be resistant to Salmonella-induced cytotoxicity if death requires polymerase activity. Numerous bacterial pathogens, including Salmonella, Shigella and Yersinia spp. (Zychlinsky et al., 1992; Monack et al., 1996; 1997; Mills et al., 1997; Ruckdeschel et al., 1997), have been described that induce apoptosis in eukaryotic cells (Weinrauch and Zychlinsky, 1999). These pathogens use TTSS to kill macrophages in vitro: the secreted proteins SipB (Salmonella), IpaB (Shigella), YopJ (Y. pseudotuberculosis) and YopP (Y. enterocolitica) are required to induce the death of infected cells (Zychlinsky et al., 1994; Chen et al., 1996a; Mills et al., 1997; Monack et al., 1997). Caspase-1 stimulation may be a common feature of TTSS-mediated cytotoxicity, as both SipB and IpaB bind and activate caspase-1 (Chen et al., 1996b; Hersh et al., 1999). These similarities suggest that Shigella also kills macrophages by a mechanism distinct from apoptosis: macrophages lacking the apoptosis effector proteins caspase-3, caspase-11 or p53 and macrophages overexpressing the cell death inhibitor proteins Bcl-2 or Bcl-X L remain susceptible to Shigella-induced cytotoxicity, whereas caspase-1-deficient macrophages are resistant (Hilbi et al., 1998). While this manuscript was in preparation, Salmonella was shown to induce cytotoxicity of infected macrophages despite inhibition of caspase-3 (Watson et al., 2000). Collectively, these data and our observations suggest that these Gram-negative bacteria induce cell death by a unique pro-inflammatory mechanism most closely resembling necrosis. Infections with Salmonella, Shigella and Yersinia result in macrophage death and extensive inflammation (Weinrauch and Zychlinsky, 1999), and the production of IL-1 and IL-18 by caspase-1 is required for host inflammatory responses (Li et al., 1995). This suggests that caspase-1- dependent cell death specifically leads to inflammation, which is central to the pathogenesis of these bacteria in vivo. Supporting this hypothesis, the pathophysiology of disease is altered in caspase-1 knockout mice infected with Shigella or Salmonella: the acute inflammatory response and macrophage death were reduced during Shigella infection (Sansonetti et al., 2000), whereas Salmonella was unable to colonize tissues deeper than Peyer's patches after oral infection (Monack et al., 2000). The strong correlation between bacteria-induced cytotoxicity and inflammation supports our model of cell death, as conventional apoptosis does not stimulate an inflammatory response in vivo (Ren and Savill, 1998). This raises the question as to how to describe bacterial-induced host cell death that appears to be inflammatory in nature. We propose that kills macrophages by an unusual mechanism: this pro-inflammatory process requiring the Salmonella SPI1 TTSS system and host caspase-1 ultimately gives rise to necrotic cell death resulting from pathological ion fluxes in the plasma membrane. Experimental procedures Bacterial strains, macrophages and growth conditions strain SL1344 and its prgh1::tnphoa derivative (Behlau and Miller, 1993) were used for all experiments. Bacteria were grown as described previously (Chen et al., 1996a). Briefly, overnight cultures back-diluted 1:15 into L-broth containing 0.3 M sodium chloride were grown at 378C with shaking for 3 h, washed and resuspended in cold sterile PBS, and kept on ice before macrophage infections. The macrophage-like cell line J774A.1 was obtained from the American Type Culture Collection. Peritoneal exudate macrophages were selected as the plasticadherent population from C3H/HeJ mice 3 days after 1 ml of Brewer's thioglycollate intraperitoneally (Cookson and Bevan, 1997). Macrophages were cultured at 378C in 7% CO 2 in Dulbecco's minimal essential medium (DMEM; Gibco BRL) supplemented with 10% FCS, 5 mm HEPES, 0.2 mg ml 21 L-glutamine and 0.05 mm b-mercaptoethanol. Reagents Gliotoxin (Sigma) was used at 5 mm to induce macrophage apoptosis (Waring et al., 1988; Monack et al., 1996). To deplete cells of ATP and induce necrosis, macrophages in glucose-free Ringer's buffer were treated for 3 h with ionomycin (Calbiochem) and carbonylcyanide-m-chlorophenylhydrazone (Sigma) at concentrations of 5 mm and 15 mm respectively (Dong et al., 1997). Caspase-1 was inhibited with acetyl-tyr-val-ala-asp-choloromethyl ketone (ac-yvadcmk, effective concentration 100±200 mm; Calbiochem) for 1 h before infection or induction of apoptosis or necrosis. Glycine protection was tested in macrophages pretreated with 5 mm glycine (Fisher Chemicals) for 1 h (Weinberg et al., 1987; Dong et al., 1997) before infection or induction of necrosis or apoptosis.

8 38 M. A. Brennan and B. T. Cookson Invasion protocol Macrophages in supplemented DMEM were allowed to adhere and mature in vitro for 24 h before infection. Peritoneal macrophages were cultured overnight in antibiotic-free media before infection. Using a previously described invasion protocol (Chen et al., 1996a), bacteria were added to macrophages at the indicated multiplicity of infection (MOI) in antibiotic-free media and allowed to invade for 2 h. After washing twice with Hanks' buffered salt solution (HBSS), media containing 15 mg ml 21 gentamicin were added to kill extracellular bacteria. The infected cells were allowed to incubate until the time points specified and then evaluated experimentally. Macrophage infection was confirmed by microscopy or lysing infected macrophages and determining the number of gentamicin-protected intracellular bacteria. At an MOI of 10, multiple experiments demonstrated that 98% of macrophages were infected by SL1344 (average of 10 bacteria/macrophage), whereas 78% were infected by the prgh1::tnphoa derivative (average of 3 bacteria/macrophage). TUNEL staining J774A.1 adherent to 12 mm 2 glass coverslips were washed five times with PBS, fixed, permeabilized, and fluoresceinated nucleotide was incorporated enzymatically (In Situ Death kit; Boehringer Mannheim) to label nicked DNA. Cellular fluorescence was evaluated by microscopy (40 objective), and images captured using a digital camera were equally adjusted for brightness and contrast and reduced in size for publication using Adobe Photoshop. Flow cytometry Adherent J774A.1 cells were removed from 25 mm 2 flasks using PBS±EDTA and pooled with any non-adherent cells in the culture media. Cells were analysed for FITC-annexin V and 7-AAD (Sigma) staining as described previously (Li and Tait, 1998). Cells washed twice in annexin V binding buffer (10 mm HEPES-Na, 133 mm NaCl, 5.8 mm KCl, 5 mm glucose, 0.1% BSA, 2.5 mm CaCl 2, ph 7.4) were resuspended to cells ml 21, and 10 6 cells were incubated with 100 nm FITC-annexin V for 15 min on ice. After two washes in binding buffer, 7-AAD was added to 2 mg ml 21 final concentration, and cells were immediately analysed on a Coulter EPICS MCL flow cytometer. Lactate dehydrogenase release Supernatants from macrophages grown in 96-well plates in the presence of 5% FCS were evaluated for the presence of cytoplasmic enzyme LDH using the Cytotox 96 kit (Promega). Percentage cytotoxicity ˆ 100 (experimental LDH±spontaneous LDH)/(maximum LDH release±spontaneous LDH). Caspase-3 assays Adherent macrophages removed from 25 mm 2 flasks using PBS±EDTA and pooled with non-adherent cells from culture media were washed once in PBS20.1% BSA, and resuspended to 10 7 cells ml 21. Lysates from 10 6 cells were assayed for active caspase-3 using the substrate aspartateglutamate-valine-aspartate-7-amino-4-tri-fluoromethyl coumarin (DEVD-AFC; ApoAlert Caspase-3 assay kit; Clontech). AFC release was measured fluorometrically at 505 nm. Fluorescent units (FU) fold induction was calculated as the fluorescence of the experimental sample incubated with substrate divided by the fluorescence of the PBS-treated control sample incubated with substrate. Active caspase-3 could be detected in a population with a minimum of 25% apoptotic cells (data not shown). PARP immunoblotting Adherent J774A.1 cells were removed from 25 mm 2 flasks using PBS±EDTA and pooled with non-adherent cells from culture media. Total protein from 10 6 cells washed once in PBS20.1% BSA was separated by 10% SDS±PAGE, and PARP cleavage was assessed by Western blotting using anti-parp monoclonal IgG1 antibody clone C-2-10 (Clontech). Acknowledgements We thank Dr John Tait for generous use of recombinant fluorescein-annexin V and helpful comments regarding the manuscript, Dr Michael Jacobson and Philip Bergman for critical review of the manuscript, Dr Samuel Miller for kindly providing the prgh mutant, and Kathy Allen for excellent technical support in flow cytometry. B.C. was supported as a Pfizer Scholar during this work and by a Pilot Research Grant from the Howard Hughes Medical Institute. References Behlau, I., and Miller, S.I. (1993) A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells. J Bacteriol 175: 4475±4484. Chen, L.M., Kaniga, K., and Galan, J.E. (1996a) Salmonella spp. are cytotoxic for cultured macrophages. Mol Microbiol 21: 1101±1115. Chen, Y., Smith, M.R., Thirumalai, K., and Zychlinsky, A. (1996b) A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J 15: 3853±3860. Cohen, G.M. (1997) Caspases: the executioners of apoptosis. 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