Sensitizing Anthrax Lethal Toxin-resistant Macrophages to Lethal Toxin-induced Killing by Tumor Necrosis Factor-alpha *

Sung O. KimDagger §, Qing JingDagger §, Kasper HoebeDagger , Bruce BeutlerDagger , Nicholas S. Duesbery||, and Jiahuai HanDagger **

From the Dagger  Department of Immunology, Scripps Research Institute, La Jolla, California 92037 and the || Van Andel Research Institute, Grand Rapids, Michigan 49503

Received for publication, September 10, 2002, and in revised form, November 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Macrophages from different inbred mouse strains exhibit striking differences in their sensitivity to anthrax lethal toxin (LeTx)-induced cytolysis. Although LeTx-induced cytolysis of macrophages plays an important role in the outcome of anthrax infection, the sensitivity of macrophages in vitro does not correlate with in vivo susceptibility to infection of Bacillus anthracis. This divergence suggests that additional factors other than LeTx are involved in the cytolysis of LeTx-resistant macrophages in vivo. We found that LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis when these cells were activated by bacterial components. Tumor necrosis factor-alpha induced by bacterial components was a key factor that cooperated with LeTx in inducing LeTx-resistant macrophage death. Tumor necrosis factor-alpha /LeTx-induced death of LeTx-resistant macrophages was dependent on mTor (mammalian target of rapamycin), but independent of caspases. Our data indicate that host responses to anthrax infection contribute to cytolysis of LeTx- resistant macrophages.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Anthrax infections are initiated by endospores of Bacillus anthracis (1). These spores germinate after they are phagocytosed by macrophages and begin to express virulence toxins that lead to a systemic immune response, shock, and death (1). It has been shown that the three toxins produced by anthrax bacilli, edema factor (EF),1 lethal factor (LF), and protective antigen (PA), are principally responsible for provoking the host responses (1, 2). PA functions as a molecular transporter by facilitating the entry of LF and EF into cells through endocytosis and translocation of EF and LF into the cytosol (3, 4). LF, a zinc metalloprotease (4), together with PA is termed lethal toxin (LeTx). LeTx is the major contributor of virulence in infected animals (1, 5). EF, an adenylate cyclase (3), functions as an enhancer that enhances the cytotoxicity of LeTx in macrophages (6).

Much is known about the mechanisms of action and cellular entry of LF and EF. PA binds to a cell-surface receptor (7), where it is proteolytically processed on the cell surface (8, 9). The processed PA heptamerizes and binds 3 molecules of either EF or LF, resulting in either a PA·EF or a PA·LF complex, which is internalized through receptor-mediated endocytosis (10). PA then forms a hole in the acidified endocytic vesicle through which EF or LF is delivered to the cytosol (11). Although it is known that EF exerts its effects through a calmodulin-dependent adenylate cyclase activity, the mechanisms of LF intoxication are less clear. LF can be delivered to the cytosol of cells, but cytolysis as a consequence of the intracellular LF is observed only in macrophages from certain mouse strains (5). Conflicting results have been reported regarding whether LeTx induces cytokine production in macrophages (12-14). The toxic activity of LF depends on its protease activity (4), suggesting that proteolysis of one or more cellular protein(s) unleashes a cascade of events resulting in the death of the intoxicated macrophages. Indeed, cleavage of several mitogen-activated protein kinase (MAPK) kinases (MKKs) has been observed (15-17). However, the physiological importance of MKK proteolysis relative to macrophage cytolysis cannot be established because similar cleavage is observed in macrophages that are resistant to LeTx (16, 17).

Inbred mouse strains exhibit striking differences in the sensitivity of their cultured macrophages to the effects of LeTx (5). For example, C3H mouse macrophages lysed by LeTx at 1 ng/ml are 100,000 times more sensitive than A/J mouse macrophages (5). Macrophages from CBA/J and BALB/c mice are sensitive, whereas those from C57BL/6J and DBA/2J mice are resistant. Direct binding studies revealed that the affinity and number of PA receptors/cell are the same in sensitive and resistant cells (5). Proteolytic activation of PA is also the same in both sensitive and resistant macrophages (5). Resistant macrophages are not cross-resistant to other toxins and viruses that, like lethal toxin, require vesicular acidification for activity. This implies that resistance is not due to defects in vesicular acidification (5). As mentioned earlier, the proteolysis of MKKs is comparable in sensitive and resistant cells (16, 17). Thus, resistance is due to a defect at a stage occurring after proteolysis. Watters et al. (17) identified a mutation on a gene named kif1C that is responsible for the different sensitivities of macrophages to LeTx. kif1C encodes a kinesin-like motor protein of the UNC104 subfamily (18). It is clear that proper function of kif1C is required for LeTx resistance, but how this protein is involved in LeTx-elicited changes in macrophages is unknown (17). The kif1C protein has been excluded as a target of LF-mediated proteolysis (17).

It was believed that LeTx-induced cytolysis of macrophages played an important role in the overall outcome of anthrax infection. Injection of LeTx into CBA/J mice, whose macrophages are sensitive to LeTx, results in a more rapid death compared with A/J mice, whose macrophages are resistant to LeTx-induced cytolysis (5). However, the sensitivity of macrophages to LeTx in vitro does not correlate with in vivo susceptibility to infection of either encapsulated or non-encapsulated strains of B. anthracis (5, 16, 17). Because macrophages can be activated by encounters with infected bacteria (19, 20), we addressed whether macrophage activation has an effect on cell viability in LeTx-treated LeTx-resistant macrophages. We found that treatment of macrophages with bacterial components can make LeTx-resistant macrophages become sensitive to LeTx-induced cytolysis. Tumor necrosis factor-alpha (TNF) induced by bacterial components is at least one of the factors that cooperate with LeTx in inducing macrophage death. Our data suggest that the autocrine/paracrine effect of TNF plays a key role in LeTx-resistant macrophage death in vivo.

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EXPERIMENTAL PROCEDURES
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Materials-- Recombinant PA and LF were prepared as described (15). Murine TNF was provided by Dr. Vladimir Kravchenko (Scripps Research Institute). Anti-TNF antibodies were raised in rabbits using recombinant TNF. Lipopolysaccharides, peptidoglycan, and poly-D-glutamic acid were from Sigma. IL-1beta , IL-6, IFN-gamma , and human TNF were from R&D Systems (Minneapolis, MN). Mouse TNF enzyme-linked immunosorbent assay kits were from R&D Systems. All chemicals were from Sigma or as indicated.

Preparations of Peritoneal Macrophages-- Peritoneal macrophages were obtained from mice by saline lavage as previously described (21). Mice received intraperitoneal injections of 3% thioglycolate 4 days before the preparation of peritoneal macrophages. After anesthetization with intramuscular injections of a mixture of ketamine (80 mg/kg of body weight) and xylazine (16 mg/kg of body weight), peritoneal macrophages were harvested from the mice through lavage of the peritoneal cavity with 5 ml of saline. The cells were then washed with RPMI 1640 medium and resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine (supplemented Dulbecco's modified Eagle's medium), and 1 × 105 or 2 × 106 cells were plated on 96-well microtiter or six-well plates, respectively. After incubation at 37 °C for 2 h, the non-adherent cells were removed by washing three times with fresh Dulbecco's modified Eagle's medium and cultured overnight in supplemented Dulbecco's modified Eagle's medium.

Cell Viability Assays-- The extent of cell death was measured using crystal violet uptake and propidium iodide exclusion assays (22). Briefly, the cell culture medium was removed from the plates by immersing them vertically in a beaker containing 2 liters of 0.9% saline. After immersion, all liquid was tapped out onto paper towels. To stain cells, 80 µl of a 5% crystal violet solution containing 25% methanol in saline was added to each well and incubated for 5 min at room temperature. Excess stains were removed by immersing plates twice in saline as described above. After removal of all liquid as described above, 100 µl of 50% acetic acid was added to each well to dissolve all the stained cellular materials. Plates were then placed on a shaker for ~30 min, and the absorbance at 590 nm was measured. Propidium iodide staining was performed using trypsinized cells resuspended in phosphate-buffered saline with 1 µg/ml propidium iodide. The level of propidium iodide incorporation was quantified by flow cytometry on a FACScan flow cytometer.

Hoechst 33258 and Annexin V Staining-- Nuclear condensation and fragmentation were determined by staining the cells with Hoechst 33258 (Sigma) as described previously (22). Phosphatidylserine exposure to the outer leaflet was analyzed by staining cells with fluorescein isothiocyanate-conjugated annexin V (Roche Molecular Biochemicals) as suggested by the manufacturer. Briefly, cells were resuspended in annexin labeling solution containing 10 mM HEPES (pH 7.4), 140 mM NaCl, 5 mM CaCl2, and fluorescein isothiocyanate-conjugated annexin V for 25 min. After washing twice with phosphate-buffered saline, cell pellets were resuspended in propidium iodide (1 µg/ml)-containing phosphate-buffered saline and analyzed by flow cytometry.

DNA Ladder Assay-- Cells were collected, lysed in lysis buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5% SDS, 25 mM EDTA, and 0.1 mg/ml protease K), and incubated at 50 °C overnight. DNA was phenol/chloroform-extracted and precipitated by adding NaCl to 0.3 M and 2 volumes of ethanol. The DNA pellets were resuspended in Tris/EDTA buffer with 0.1 mg/ml RNase I for 1 h at room temperature and then treated with 0.1 mg/ml protease K overnight at 37 °C. DNA was further phenol/chloroform-extracted, precipitated as described above, and resuspended in 20 µl of Tris/EDTA buffer. Equal amounts of DNA were separated on 2% agarose gel and stained with ethidium bromide for visualization.

Total Cell Lysate Preparation and Western Blot Analysis-- Total cell lysate extraction and Western blot analysis for MEK1 were performed as previously described (22). Briefly, cells were lysed in ice-cold cell lysis buffer containing 20 mM MOPS, 15 mM EGTA, 2 mM EDTA, 1 mM Na3VO4, 1 mM dithiothreitol, 75 mM beta -glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1% Triton X-100 and then sonicated on ice. Cell extracts were obtained by centrifuging the homogenate at 13,000 rpm for 10 min. These extracts were electrophoretically resolved in ready-made 10% SDS-polyacrylamide gels (Bio-Rad), followed by transfer onto nitrocellulose membranes. Membranes were subsequently blocked with 5% skim milk for 30 min, immunoblotted with antibodies, and developed using an enhanced chemiluminescence detection system (ECL, Amersham Biosciences). Antibodies raised against the N terminus of MEK1 were purchased from Upstate Biotechnology, Inc.

    RESULTS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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LeTx-resistant Macrophages Are Selectively Resistant to LeTx-induced Cell Death-- We used macrophages from C3H/HeN, 129svj, and C57BL/6J mice to repeat published results indicating that macrophages from different mouse strains have different sensitivities to LeTx-induced cell killing in vitro. As shown in Fig. 1A, macrophages from C3H/HeN and 129svj mice were sensitive to LeTx-induced killing, whereas macrophages from C57BL/6J mice were resistant. The maximum cell death of sensitive cells was reached 3-4 h after the cells were treated with LeTx (500 ng/ml PA + 500 ng/ml LF), whereas most of the resistant macrophages were still viable after 16 h of the same treatment (Fig. 1B). The cytolytic dose of LF for the macrophages from C3H/HeN (Fig. 1C) and 129svj (data not shown) mice was ~10 ng/ml when a saturated PA (500 ng/ml) was present. The resistance of C57BL/6J macrophages to LeTx-induced cytolysis was not dependent on the dose of LF used in the experiments because up to a 50-fold increase in LF concentration still could not produce more cytolysis (Fig. 1C). The cell viability was measured using crystal violet uptake of live cells and propidium iodide exclusion (data not shown) with comparable results.


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Fig. 1.   LeTx (PA + LF)-sensitive and -resistant macrophages. A, peritoneal macrophages were isolated from C3H/HeN, 129svj, and C57BL/6J mice and treated with PA (500 ng/ml) plus LF (500 ng/ml). Cell viability was measured 3 h after treatment using crystal violet uptake assay. B, macrophages from C57BL/6J and C3H/HeN mice were treated with PA (500 ng/ml) plus LF (500 ng/ml) for different periods of time, and cell viability was measured. C, macrophages from C57BL/6J and C3H/HeN mice were treated with PA (500 ng/ml) plus different concentrations of LF as indicated for 16 h. Cell viability was measured. D, macrophages from C57BL/6J and C3H/HeN mice were treated with PA (500 ng/ml) plus LF (500 ng/ml) for different periods of time, and MEK1 and ERK1/2 proteins were analyzed by immunoblotting using antibodies raised against the MEK1 N terminus and ERK. E, macrophages from C3H/HeN, 128svj, and C57BL/6J mice were treated with LPS (10 ng/ml) plus Z-VAD-fmk (zVAD; 10 µM), LPS plus IFN-gamma (100 units/ml), sodium nitroprusside (SNP; 500 µM), or etoposide (200 µM). Cell viability was determined 24 h after the treatments. The results represent means ± S.E. (n = 3-6).

It is known that LF cleaves certain intracellular proteins and that proteolysis may be essential for cell death. Recent reports have shown that the cleavage of MEK2 and MKK3 also occurs in LeTx-resistant macrophages (16, 17). This suggests that the resistance to LeTx-induced cytolysis in resistant macrophages is not due to a defect in proteolysis mediated by LF. We tested the cleavage of MEK1 in C57BL/6J and C3H/HeN macrophages. MEK1 was cleaved in the macrophages from both strains when treated with LeTx (Fig. 1D).

It is known that LeTx-resistant macrophages are not resistant to other toxins (5). We further tested whether LeTx-resistant macrophages are generally resistant to death stimuli. Macrophages from C3H/HeN, 129svj, and C57BL/6J mice were treated with lipopolysaccharides (LPS) plus IFN-gamma , LPS plus benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk), sodium nitroprusside, or etoposide. All of these reagents have been shown to cause macrophage death (22, 23). As shown in Fig. 1E, the macrophages from the three strains were killed equally by these death stimuli. Thus, there is no general resistance to death in C57BL/6J macrophages.

Cellular Activation Can Sensitize Resistant Macrophages to LeTx-induced Cell Death-- Available data strongly support the role of macrophage cytolysis in eliciting shock and death in infected animals (1). Because the mice whose macrophages were resistant to LeTx-induced cytolysis in vitro still died after infection, we decided to test whether macrophage activation by bacterial components, which should happen during bacterial infection, has any effect on the viability of LeTx-treated resistant macrophages. Macrophages isolated from C57BL/6J mice (LeTx-resistant macrophages) were used in the experiments. The cells were treated with LeTx (PA + LF) in the presence or absence of poly-D-glutamic acid (PGA; 100 µg/ml), the major component of the B. anthracis capsule (24); peptidoglycan (PG; 10 µg/ml), the cell wall component of Gram-positive bacteria; or LPS (10 ng/ml), cell wall components of Gram-negative bacteria. We also treated the cells with retrovirus (Moloney murine leukemia virus, ~106 plaque-forming units/ml) or zymosan A (100 µg/ml) for comparison. Cell viability was measured 16 h later and is shown in Fig. 2A. The presence of PGA promoted LeTx-induced C57BL/6J macrophage death. LPS and PG also significantly enhanced LeTx-induced cell death. Retrovirus and zymosan A slightly enhanced cell death. The results of an experiment with more controls is shown in Fig. 2B. LPS, PG, or PGA alone did not cause cell death. LPS, PG, or PGA combined with PA or LF did not cause cell death. The cell death of resistant macrophages started at ~5 h of treatment and reached a maximum at ~16 h (Fig. 2C). B. anthracis is a Gram-positive bacteria whose capsule should interact with macrophages, and its cell wall component (PG) can activate macrophages if the capsule is removed. Thus, the death of LeTx-treated resistant macrophages can be promoted by cellular activation with bacterial capsule or cell wall components in vivo.


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Fig. 2.   Bacterial components promote the death of LeTx-treated LeTx-resistant macrophages. A, C57BL/6J (LeTx-resistant) macrophages were treated with PA plus LF (500 ng/ml) alone (Control) or in the presence of Moloney murine leukemia virus (~106 plaque-forming units/ml), zymosan A (100 µg/ml), PG (10 µg/ml), LPS (10 ng/ml), or PGA (100 µg/ml). Cell viability was assayed 16 h after treatment. B, C57BL/6J macrophages were left untreated (Control) or were treated with PA (500 ng/ml), LF (500 ng/ml), or PA plus LF in the presence of LPS, PG, or PGA. Cell viability was measured 16 h after treatment. C, C57BL/6J macrophages were treated with PA (500 ng/ml), LF (500 ng/ml), and LPS (10 ng/ml) for different periods of times, and cell viability was measured.

Phenotype of LeTx-induced Death of Activated Macrophages-- LeTx-induced cytolysis of macrophages is more like necrosis than apoptosis (13, 25, 31), although treatment of macrophages with sublytic amounts of LeTx can trigger some intracellular events of apoptosis (26, 27). To determine whether C3H/HeN and C57BL/6J macrophages treated with LPS plus a cytolytic dose of LeTx have the phenotype of apoptosis, three apoptotic features were examined. Nuclear labeling with Hoechst 33258 is a commonly used method to detect chromatin condensation, a feature of apoptosis. Macrophages from C3H/HeN and C57BL/6J mice were left untreated (control) or were treated with LPS plus LeTx and stained with Hoechst 33258. Chromatin condensation was not seen throughout the course of cell death of both C3H/HeN and C57BL/6J macrophages (Fig. 3A and data not shown). As a positive control, chromatin condensation was observed when C57BL/6J or C3H/HeN macrophages were treated with LPS plus INF-gamma (Fig. 3A and data not shown). DNA fragmentation is a typical feature of apoptosis and was examined in the macrophages treated with LPS plus LeTx. No DNA ladder was detected in C57BL/6J and C3H/HeN macrophages treated with LPS plus LeTx (Fig. 3B). Treatment of C57BL/6J macrophages with LPS plus IFN-gamma was used as positive control for DNA fragmentation (Fig. 3B, last lane). Translocation of phosphatidylserine from the inner part of the plasma membrane to the outer layer is a common early event in apoptosis. Annexin V staining of phosphatidylserine was observed in macrophages treated with LPS plus IFN-gamma , but not in macrophages treated with LPS plus LeTx (Fig. 3C). Thus, LPS/LeTx-induced death of LeTx-resistant macrophages does not have the apoptotic phenotype, and LPS stimulation does not change the mode of cell death induced by cytolytic doses of LeTx.


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Fig. 3.   Phenotype of LeTx-induced death of activated macrophages. A, C3H/HeN and C57BL/6J macrophages were left untreated (Control) or were treated with PA (500 ng/ml), LF (500 ng/ml), and LPS (10 ng/ml). C3H/HeN or C57BL/6J cells were fixed and stained with Hoechst 33258 at 2 or 12 h after treatment, respectively. As a positive control, C57BL/6J macrophages were treated with LPS (100 ng/ml) plus INF-gamma (10 units/ml) for 24 h and stained with Hoechst 33258. B, macrophages from C3H/HeN or C57BL/6J mice were treated with PA and LF or with PA, LF, and LPS as described for A, but for 5 and 24 h, respectively. The positive control was C57BL/6J macrophages treated with LPS plus IFN-gamma for 36 h. Genomic DNA was isolated from C3H/HeN or C57BL/6J macrophages and analyzed by 2% agarose gel electrophoresis followed by ethidium bromide straining. C, macrophages from C3H/HeN and C57BL/6J mice were treated as described for A. The cells were stained with propidium iodide and annexin V. The percentage of propidium iodide-negative and annexin V-positive cells was determined by fluorescence-activated cell sorter analysis (n = 2-3).

TNF Plays a Role in Sensitizing LeTx-resistant Macrophages-- Activated macrophages secrete a number of cytokines, including TNF, IL-1beta , IL-6, and INF-gamma . We treated C57BL/6J macrophages with these cytokines of murine origin in the presence or absence of LeTx and measured cell viability. As shown in Fig. 4A, these cytokines alone did not affect the viability of macrophages. Interestingly, dramatic cell death was induced in LeTx-treated cells in the presence of TNF, but not the other cytokines tested. Thus, TNF can sensitize LeTx-resistant macrophages to LeTx-induced cell death.


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Fig. 4.   Sensitizing LeTx-resistant macrophages to LeTx-induced death by TNF. A, C57BL/6J macrophages were left untreated (Control) or were treated with murine TNF (200 pM), IL-1beta (10 units/ml), IL-6 (250 units/ml), or IFN-gamma (100 units/ml) in the presence or absence of PA (500 ng/ml) plus LF (500 ng/ml). Cell viability was determined 16 h after treatment. B, C57BL/6J macrophages were left untreated or were treated with PG, PGA, or LPS in the presence or absence of PA plus LF for 16 h. TNF concentrations in culture medium were measured by enzyme-linked immunosorbent assay. C, C57BL/6J macrophages were treated with PA, LF, and TNF; PA, LF, and LPS; PA, LF, and PG; or PA, LF, and PGA in the presence or absence (control (Ctrl)) of anti-TNF antiserum (1:500 dilution) for 16 h. The extent of cell death was analyzed. The percentage of inhibition of cell death was calculated by ((cell death in the absence of antibody) - (cell death in the presence of antibody)/cell death in the absence of antibody). D, macrophages from C57BL/6J mice were treated with human TNF (hTNF; 500 pM) or murine TNF (mTNF) in the presence of PA plus LF. Cell viability was determined 16 h after treatment. E, macrophages from C57/ScSn or C57/10ScCr mice were treated with PA and LF; PA, LF, and LPS; or PA, LF, and murine TNF. Cell viability was determined 16 h after treatment.

We measured TNF production in C57BL/6J macrophages after stimulation with LPS, PG, and PGA. LPS stimulation led to the highest production of TNF (Fig. 4B). The PG-stimulated TNF production was about half of the LPS-induced TNF production, and PGA was less potent in inducing TNF production. The treatment of cells with LeTx reduced (but did not abolish) TNF production. The level of TNF production (Fig. 4B) correlated with the death of LeTx-treated resistant macrophages promoted by these bacterial components (Fig. 2A), suggesting that TNF is a mediator of bacterial component-promoted death of LeTx-treated cells.

To determine whether the sensitization of LeTx-resistant macrophages to LeTx-induced death by bacterial components is mediated by TNF, we used a neutralizing antibody to block TNF. As shown in Fig. 4C, ~50% inhibition of the TNF/LeTx-induced cell death was observed when anti-TNF antiserum was included in the cell culture medium. Pre-bled serum had no effect, indicating that the antibody can block the function of TNF. This antibody also inhibited LPS/LeTx, PG/LeTx, or PGA/LeTx-induced cell death, supporting the idea that TNF is at least one of the factors responsible for sensitizing the resistant macrophages to LeTx-induced cytolysis. Thus, the autocrine/paracrine effect of TNF plays a role in LeTx-resistant macrophage death.

The bioactivity of TNF is mediated by two TNF receptors, TNF-RI and TNF-RII. Although human TNF and murine TNF are very homologous, human TNF binds only to murine TNF-RI (28). We treated C57BL/6J macrophages with human TNF plus LeTx and found that, in contrast to murine TNF, human TNF cannot sensitize LeTx-resistant macrophages to LeTx-induced cell death (Fig. 4D), suggesting that TNF-RII-mediated signaling is required for sensitizing LeTx- resistant macrophages.

C57/10ScCr is a mouse strain that has a deletion of the toll-like receptor-4 (TLR4) gene and will not respond to LPS (29). A comparable strain with the wild-type TLR4 gene is C57/ScSn. The macrophages isolated from these two strains of mice were resistant to LeTx (Fig. 4E). Treatment of LPS in the presence of LeTx led to cell death in C57/ScSn macrophages, but not in C57/10ScCr macrophages, confirming that TLR4 signaling is required for LPS-promoted death of LeTx-treated cells. In contrast, TNF plus LeTx caused cell death in both macrophages, which is consistent with the idea that bacterial components promote the death of LeTx-treated cells through the induction of TNF.

TNF can induce the death of a number of different cells. Although TNF alone had no effect on the viability of macrophages, it is possible that intracellular signaling activated by TNF can augment the death of C3H/HeN macrophages induced by LeTx. To examine this possibility, we tested whether TNF can promote the death of macrophages treated with sublytic doses of LF. Macrophages from C3H/HeN and C57BL/6J mice were treated with 500 ng/ml PA and different doses of LF in the presence or absence of TNF. The levels of cytolysis of cells were analyzed. As shown in Fig. 5A, TNF stimulation did not enhance the cell death of C3H/HeN macrophages treated with sublytic or cytolytic doses of LF. In contrast, TNF increased the cell death of C57BL/6J macrophages treated with cytolytic doses (Fig. 5B). The sensitization of LeTx-induced cell death by TNF appears to be a unique feature of LeTx- resistant macrophages.


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Fig. 5.   Dose curve of LF in mediating the cell death of TNF-activated C3H/HeN and C57BL/6J macrophages. C3H/HeN (A) or C57BL/6J (B) macrophages were treated with PA (500 ng/ml) and different doses of LF in the presence or absence of TNF (500 pM) for 16 h. Cell viability was measured.

TNF/LeTx-induced Cell Death in LeTx-resistant Macrophages Requires the Proteolytic Activity of LF and mTor Signaling-- LF(E687C), an inactive LF mutant, has been shown to be unable to mediate the proteolysis of proteins in vitro and in cells. Also, LF(E687C) is incapable of causing cytolysis of LeTx-sensitive macrophages (15). To determine whether the protease activity of LF is required for TNF/LeTx-induced death of resistant macrophages, we compared the effect of wild-type LF and LF(E687C) on the cell viability of resistant macrophages in the presence of TNF (Fig. 6A). No cell death was observed in LF(E687C)-treated cells, indicating that the protease activity of LF is required for TNF/LeTx-induced death of resistant macrophages.


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Fig. 6.   TNF/LeTx-induced death of resistant macrophages requires the protease activity of LF and is independent of caspases. A, C57BL/6J macrophages were left untreated (Control) or were treated with PA, LF, LF(E687C), PA plus LF, or PA plus LF(E687C) in the presence of TNF (500 pM). Cell viability was measured 16 h after treatment. B, C57BL/6J macrophages were left untreated or were treated with TNF, Z-VAD-fmk (zVAD), PA plus LF, and their combinations. Cell viability was measured 16 h after treatment. C, C57BL/6J macrophages were treated with TNF, PA, and LF in the presence of U0162 (10 µM), SB203580 (20 µM), JNK inhibitor-2 (8 µM), bisindolylmaleimide II (3 µM), genistein (50 µM), herbimycin (100 ng/ml), wortmannin (1 µM), LY294002 (10 µM), or rapamycin (20 nM). Cell viability was measured 16 h after treatment. D, C57BL/6J macrophages were treated with or without TNF in the presence of U0162 (10 µM), SB203580 (20 µM), JNK inhibitor-2 (8 µM), NF-kappa B SN50 cell-permeable inhibitor peptide (18 µM), bisindolylmaleimide II (3 µM), LY294002 (10 µM), and their combinations. Cell viability was measured 16 h after treatment.

Caspases have been implicated to play an important role in TNF-induced cell death of many different cells (30). To test whether caspase is involved in TNF/LeTx-induced death of LeTx-resistant macrophages, we used a general caspase inhibitor, Z-VAD-fmk. As shown in Fig. 6B, Z-VAD-fmk itself did not have an effect on the viability of resistant macrophages, nor did Z-VAD-fmk together with LeTx have any effect on cell viability. Therefore, caspases are not required for TNF/LeTx-induced death of LeTx-resistant macrophages.

TNF activates a number of signaling pathways that are involved in cell survival or the death of a number of different cells (32-35). To understand how TNF sensitizes LeTx-resistant macrophages, we initiated our investigation on whether the known signaling pathways that are activated by TNF are involved in sensitizing LeTx-resistant macrophages. We used available inhibitors to inhibit the signaling pathways activated by TNF. Inhibitors (U0126, c-Jun N-terminal kinase (JNK) inhibitor-2, and SB203580) of three MAPK pathways (extracellular signal-regulated kinase (ERK), JNK, and p38) did not have any inhibitory effect on TNF/LeTx-induced cell death, but rather enhanced cell death (Fig. 6C). The other inhibitors that enhanced cell death were the protein kinase C inhibitor bisindolylmaleimide II, the tyrosine kinase inhibitors genistein and herbimycin, and the phosphatidylinositol 3-kinase inhibitor wortmannin. Because an oxidative burst was implicated to be responsible for LeTx-induced cell death (13), we also tested butylated hydroxyanisole, a scavenger of free radicals, and found no effect on the death of resistant macrophages (data not shown). The only inhibitor we tested that inhibited cell death was rapamycin (Fig. 6C). The maximum inhibition of TNF/LeTx-induced cell death by rapamycin was reach at 10 nM. The known target of rapamycin is mTor (mammalian target of rapamycin; also named FRAP and RAFT1), and no nonspecific effect of rapamycin has been reported at 10 nM. Thus, mTor is most likely involved in cell death.

It is known that the proteolysis by LF impairs the two MAPK pathways, the ERK and p38 pathways. It was proposed that disruption of certain intracellular pathways by LF impairs cell survival mechanisms and thus promotes cell death (15-17, 27). To test whether disruption of the ERK and/or p38 pathway by LeTx is involved in TNF/LeTx-induced death of LeTx-resistant macrophages, we used chemical inhibitors to mimic the inhibitory effect of LF on the ERK and p38 pathways. Inhibition of the ERK pathway by U0126 and the p38 pathway by SB203580 either independently or together did not influence the cell viability of TNF-treated LeTx-resistant macrophages (Fig. 6D and data not shown), suggesting that cleavage of MEK1/2 and MKK3/6 by LF either is not involved in TNF/LeTx-induced cell death or is insufficient to reduce the cell viability. We further tested inhibitors of other pathways. We used JNK inhibitor-2 to inhibit the JNK pathway (another MAPK pathway) and did not find any change in the viability of TNF-treated LeTx-resistant macrophages (Fig. 6D). Similarly, inhibition of the NF-kappa B, protein kinase C, or phosphatidylinositol 3-kinase pathways had no effect on the cell viability of TNF-treated macrophages (Fig. 6D). We tested whether inhibition of all three MAPK pathways had an effect on the viability of macrophages treated with TNF. As shown in Fig. 6D, ~20% cell death was observed when ERK, p38, and JNK were inhibited at the same time. These data suggest that simultaneous inhibition of multiple MAPK pathways may have a role in TNF/LeTx-induced macrophage death. However, the possible nonspecific effect of these inhibitors in influencing cell viability cannot be excluded. Inhibition of NF-kappa B or protein kinase C and the three MAPK pathways at the same time did not further enhance the death of TNF-treated C57BL/6J macrophages (Fig. 6D). Inhibition of the phosphatidylinositol 3-kinase pathway together with the three MAPK pathways further enhanced cell killing (Fig. 6D). Collectively, these data suggest that impairing any one of the known pathways is not sufficient for sensitizing LeTx-resistant macrophages. However, it is possible that disruption of multiple signaling pathways by LF can impair cell survival mechanisms, although a conclusion cannot be made due to limited information on the cellular targets of LF.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have examined the effect of cellular activation of macrophages on the cell viability of LeTx-treated LeTx-resistant macrophages. We found that treatment of macrophages with different bacterial components made LeTx-resistant murine macrophages susceptible to LeTx-induced cell death. TNF produced by activated macrophages is a key mediator that sensitizes LeTx-resistant macrophages to LeTx-induced death. We have determined that the protease activity of LF and mTor activity in cells are required for TNF/LeTx-induced resistant macrophage death and that this type of cell death is caspase-independent. Sensitizing LeTx-resistant macrophages to LeTx-induced cytolysis suggests that host responses to anthrax infection participate in the macrophage death in vivo.

LeTx-induced cytolysis of macrophages plays an important role in the outcome of anthrax infection. Lethality in mice of different strains resulting from direct injections of LeTx mimics the in vitro sensitivity of macrophages to LeTx-induced cell death (5). A contribution of host responses to the lethality of anthrax infection can also be deduced from the study of Welkos et al. (5). They showed that C3H/HeN and C3H/HeJ mouse strains are killed by B. anthracis Vollum 1B strain (wild-type) infection with similar LD50 values (five to six spores). However, the LD50 of the non-encapsulated toxin-producing strain Sterne is significantly different. The LD50 of C3H/HeN mice is 8 × 106 spores, whereas C3H/HeJ mice are completely resistant to the Sterne strain at the highest dose used (2 × 107 spores). These two strains have similar genetic backgrounds, except that C3H/HeJ mice have a point mutation in TLR4 that impairs signaling initiated by some bacterial cell wall components such as LPS. It is possible that capsulated B. anthracis can escape TLR4 recognition and therefore that the TLR4 mutation does not alter the host responses to anthrax infection. However, "naked" B. anthracis can be detected by TLR4, and the TLR4 deficiency impairs macrophage responses, thereby increasing levels of LD50. Although wild-type B. anthracis does escape phagocytosis by macrophages and some other host defense reactions, certain levels of host response should occur. The capsule should interact with macrophages, and we have shown here that the major components of the capsule such as PGA can stimulate macrophages to produce TNF. Although the capsule may prevent PG from interacting with the macrophages, this interaction would occur if the capsule was released or damaged. Because macrophages are producers of TNF, the local concentration of TNF around macrophages can be high. We suggest that macrophage-produced TNF may be responsible for cytolysis of LeTx-resistant macrophages in vivo. This contention is consistent with two published in vivo studies using neutralizing antibodies of TNF. When the anti-TNF antibody is administered to BALB/c mice, there is no protective effect from an injection of LeTx (14). In contrast, administration of the anti-TNF antibody delays the death of C57BL/6J mice infected with B. anthracis (36). When the macrophages from BALB/c and C57BL/6J mice were analyzed for their sensitivity to LeTx in vitro, BALB/c macrophages were found to be sensitive to LeTx, but C57BL/6J macrophages were not. It is possible that because LeTx can directly trigger cytolysis of macrophages in BALB/c mice, inhibition of TNF does not help the survival of the mice. Although TNF is involved in the cytolysis of macrophages in anthrax-infected C57BL/6J mice, blocking TNF delays death.

Life or death of a cell is determined by a balance between death and survival pathways. TNF is a pleiotropic cytokine produced mainly by macrophages. TNF is also recognized by macrophages and regulates the pattern of gene expression (35). TNF-induced cellular responses are mediated by either one of the two TNF receptors, TNF-RI (p55) and TNF-RII (p75) (37). Macrophages express both receptors (33). Because TNF-RI knockout mice have the same sensitivity to anthrax infection as wild-type mice (36), the sensitization of LeTx-resistant macrophages to LeTx-induced death by TNF is most likely through TNF-RII. This notion is consistent with the observation that human TNF, which is able to bind only to TNF-RI on murine cells, could not promote the death of LeTx-resistant macrophages (Fig. 4D). TNF can induce caspase-dependent and -independent cell death (38-40). It is clear that TNF/LeTx-induced cell death is independent of caspases because Z-VAD-fmk did not inhibit cell death (Fig. 6B). Because the mechanism of caspase-independent cell death by TNF is largely unknown, it is unclear whether the same method is used in TNF/LeTx-induced macrophage death.

Even though TNF induces cell death in many cells, some cell types such as macrophages proliferate in response to TNF (33). TNF activates various kinases of the MAPK family and induces various transcription factors such as NF-kappa B in macrophages (32-35). Activation of MAPK has been shown to be involved in both apoptosis and cell survival of macrophages depending on the cell death stimuli (41, 42). Recently, Park et al. (27) showed that LPS stimulation of macrophages treated with sublytic doses of LF and saturated PA63 (cleaved active form of PA) results in apoptosis. The dismantling of the p38 MAPK pathway by sublytic doses of LF was implicated to be partly responsible for the apoptosis of activated macrophages (27). We have shown here that inhibition of ERK, p38, JNK, protein kinase C, tyrosine kinase, and phosphatidylinositol 3-kinase pathways enhanced TNF/LeTx-induced death of LeTx-resistant macrophages (Fig. 6C), which supports the idea that damage to certain intracellular signaling pathways by LF may impair cell survival mechanisms (27). Inhibition of any single pathway did not have an effect on the viability of TNF-treated macrophages (Fig. 6D), suggesting that disruption of a single signaling pathway cannot mimic the LF effect on survival mechanisms. This speculation was supported by the observation that a combination of inhibitors of MAPKs and phosphatidylinositol 3-kinase significantly induced cell death in TNF-treated macrophages (Fig. 6D). In contrast to the reported effect of sublytic doses of LF (27), apoptotic features could not be found in macrophages treated with cytolytic doses of LeTx in the presence of LPS stimulation (Fig. 3). It appears that the phenotype of cell death is determined by the level of LF damage rather than cellular activation because sublytic doses of LeTx alone already triggers apoptotic features (26).

Rapamycin was the only inhibitor we tested that could prevent TNF/LeTx-induced macrophage death (Fig. 6C). The rapamycin target mTor is a controller of regulatory metabolic responses (43). The phosphatidylinositol 3-kinase/Akt pathway can regulate mTor in some systems (43). It is unlikely that the phosphatidylinositol 3-kinase/Akt pathway is responsible for mTor regulation in our system because wortmannin or LY294002 did not have the same effect as rapamycin, but rather had an opposite effect. Because rapamycin did not inhibit LeTx-induced cytolysis of LeTx-sensitive macrophages (data not shown), mTor is most likely involved in TNF signaling in TNF/LeTx-induced death of LeTx-resistant macrophages. How mTor is regulated and how mTor functions in TNF/LeTx-induced LeTx-resistant macrophage death are a subject for further investigation.

LeTx causes the proteolysis of certain cellular proteins by triggering the cytolysis of LeTx-sensitive macrophages. Because of a difference of three amino acids in the kinesin-like motor protein kif1C, this proteolysis cannot cause cell death in LeTx-resistant macrophages. kif1C was suggested to be involved in retrograde vesicle transport, and TNF may influence this transport to sensitize LeTx-resistant macrophages. It is also possible that TNF sensitizes LeTx-resistant macrophages through a mechanism independent of kif1C. LeTx leads to proteolysis of certain proteins that may damage some cellular functions. TNF-induced cellular responses under this condition could be imbalanced and result in cell death. It was reported that human macrophages are sensitive to LeTx-induced cytolysis in vitro (13). However, we have tested human macrophages from two donors and found that both were resistant to LeTx-induced cell death and could be sensitized through TNF treatment, as was observed in LeTx-resistant murine macrophages (data not shown). This result fits with a predication that human macrophages should be resistant to the effect of LeTx because the human KIF1C gene has the same amino acid sequence as C57BL/6J and other resistant strains in the region that determines LeTx sensitivity (17). It appears that different human populations exhibit differences in the sensitivity of their macrophages due to the effect of LeTx. Thus, our study on LeTx-resistant macrophage death is relevant to anthrax infection in humans and provides valuable insight into the anthrax pathogenesis.

    ACKNOWLEDGEMENTS

We thank Dr. R. J. Collier for helpful discussion and Tonya Thomson for excellent secretarial work.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant AI-41637 and a grant from the California Cancer Research Program (to J. H.). This is Publication 15156-IMM from the Department of Immunology, Scripps Research Institute (La Jolla, CA).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

Supported by a Canadian Institutes of Health Research fellowship.

** To whom correspondence should be addressed: Dept. of Immunology, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8704; Fax: 858-784-8665; E-mail: jhan@scripps.edu.

Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M209279200

    ABBREVIATIONS

The abbreviations used are: EF, edema factor; LF, lethal factor; PA, protective antigen; LeTx, lethal toxin; MAPK, mitogen-activated protein kinase; MKK, mitogen-activated protein kinase kinase; TNF, tumor necrosis factor-alpha ; IL, interleukin; IFN-gamma , interferon-gamma ; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MOPS, 4-morpholinepropanesulfonic acid; LPS, lipopolysaccharide(s); Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; PGA, poly-D-glutamic acid; PG, peptidoglycan; TNF-R, tumor necrosis factor receptor; TLR4, toll-like receptor-4; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.

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