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Am J Physiol Cell Physiol 293: C1788-C1796, 2007. First published September 13, 2007; doi:10.1152/ajpcell.00141.2007
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

C/EBPβ phosphorylation rescues macrophage dysfunction and apoptosis induced by anthrax lethal toxin

Martina Buck and Mario Chojkier

Department of Medicine, University of California, and Veterans Affairs Healthcare System, San Diego, California

Submitted 4 April 2007 ; accepted in final form 10 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bacillus anthracis lethal toxin (LT) impairs innate and adaptive immunity. Anthrax lethal factor stimulates cleavage of MAPK kinases, which prevents the activation of antiapoptotic MAPK targets. However, these MAPK targets have not been yet identified. Here, we found that LT induces macrophage apoptosis by enhancing caspase 8 activation and by preventing the activation of ribosomal S6 kinase-2 (RSK), a MAPK target, and the phosphorylation of CCAAT/enhancer binding protein-β (C/EBPβ) on T217, a RSK target. Expression of the dominant positive, phosphorylation mimic C/EBPβ-E217 rescued macrophages from LT-induced apoptosis by blocking the activation of procaspase 8. LT inhibited macrophage phagocytosis and oxidative burst and induced apoptosis in normal mice but not in C/EBPβ-E217 transgenic mice. These findings suggest that C/EBPβ may play a critical role in anthrax pathogenesis, at least in macrophages.

apoptosis; macrophages; mitogen-associated protein kinase; ribosomal S6 kinase-2

ANTHRAX is a consequence of the germination of Bacillus anthracis spores within host macrophages (29). This step is crucial to the pathogenesis of anthrax since it allows the expression of virulence factors such as lethal factor (LF), protective antigen (PA), and edema factor (EF) as well as evasion of the innate immune response (12, 19). Although the spleen is a macrophage-rich organ that develops apoptosis in inhalation anthrax in humans (17), and macrophage apoptosis is also induced in culture by anthrax lethal toxin (LT) (16, 35), the relevance of macrophage cell death in the pathogenesis of anthrax remains uncertain.

Many of the toxic effects of anthrax, including impaired function of dendritic cells regarding adaptive immunity (1), are mediated by LT, which is composed of LF and PA (19). LF is a zinc-dependent protease that cleaves several MAPK kinases (MEKs) in their amino-terminal domains (15, 38, 46, 47), preventing the activation of MAPK pathways (5, 13). Although inhibition of the MAPK signaling cascade is central to the macrophage apoptosis induced by LT, MAPK antiapoptotic targets have not been yet identified (35, 36).

Extracellular stimuli, such as growth factors or extracellular matrix proteins, affect cell survival and the cell cycle through the ERK/MAPK cascade (22, 49). We and others have shown that ribosomal S6 kinase-2 (RSK), which is activated directly by ERK phosphorylation, with the involvement of 3-phosphoinositide-dependent protein kinase (PDK)-1, plays an essential role in the ERK/MAPK signaling pathway regulating cell survival and the cell cycle (6, 7, 10, 11, 18, 23, 41). In turn, phosphorylation of CCAAT/enhancer binding protein-β (C/EBPβ)-T217 by RSK modulates cell proliferation and survival (9, 10). The family of C/EBP proteins regulates transcription but also cell differentiation, proliferation, and survival (25).

An abnormal ERK/MAPK signaling pathway mediates the impaired dendritic cell function after LT treatment (1). However, the disrupted phosphorylation target of the MAPK pathway critical for cell survival (35) and induced by LT has not yet been identified. C/EBPβ is also critical for macrophage differentiation (32), and C/EBPβ–/– macrophages display defective bacterial killing (44).

Our hypothesis was that LT, by disabling the MEK pathway and preventing phosphorylation of C/EBPβ-T217, would block macrophage function and survival (Fig. 1). We postulated that LT would induce macrophage dysfunction and death by preventing phosphorylation of C/EBPβ-T217 in C/EBPβ wild-type (WT) mice but not in C/EBPβ-E217 mice, since this phosphorylation mimic would circumvent the blocking effects of LT on MAPK signaling (Fig. 1). In addition, we expected that nonphosphorylatable C/EBPβ-A217 (by inhibiting RSK activity) and C/EBPβ deficiency (by lacking the target of RSK) would make macrophages prone to macrophage dysfunction and death even in the absence of LT (Fig. 1).


Figure 1
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  		Fig. 1. Anthrax lethal toxin (LT) inhibits the phosphorylation of CCAAT/enhancer binding protein-β (C/EBPβ)-T217 and macrophage function and survival. The inhibition of MEK signaling by LT prevents the activation of ERK1/2 and ribosomal S6 kinase (RSK), phosphorylation of C/EBPβ-T217, and macrophage function and survival. C/EBPβ-A217 and C/EBPβ–/– macrophage dysfunction and apoptosis are facilitated by blockade of RSK and lack of the target of RSK, respectively. C/EBPβ-E217 rescues the effects of LT by providing a dominant positive mimic of phosphorylated C/EBPβ-T217 that circumvents the absence of phosphorylated C/EBPβ, stimulating macrophage function and survival.

 
Therefore, we investigated the role of C/EBPβ signaling on cell dysfunction and apoptosis induced by LT in macrophages (35, 38) from C/EBPβ WT, C/EBP–/–, transgenic phosphorylation mimic C/EBPβ-E217, and transgenic nonphosphorylatable C/EBPβ-A217 mice.

We found that expression of the phosphorylation mimic C/EBPβ-E217 rescued macrophages from LT-induced inhibition of phagocytosis and bactericidal activity and apoptosis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Construction of C/EBPβ-A217 and C/EBPβ-E217 mice. The animal protocol was approved by the Veterans Affairs Medical Center's Veterinarian Medical Unit. Transgenic mice expressing C/EBPβ-E217, a dominant positive, phosphorylation mimic mutation of the C/EBPβ-T217 phosphoacceptor, were generated as previously described (9) for C/EBPβ-A217 transgenic mice and back-crossed to parental WT inbred FVB mice for more than four generations. Mouse C/EBPβ cDNA was amplified by PCR to mutate T217. The primers used to mutate T217 to A217 were sense: 5'-GCCAAGGCCAAGAAGGCGGTGGACAAGCTGAGC-3' and antisense: 5'-GCTCAGCTTGTCCACCGCCTTCTTGGCCTTGGC -3'. The primers used to mutate T217 to E217 were sense: 5'-GCCAAGGCCAAGAAGGAGGTGGACAAGCTGAGC-3' and antisense: 5'-GCTCAGCTTGTCCACCTCCTTCTTGGCCTTGGC-3'. C/EBPβ was removed from pEVRF0 with ApaI and NheI and cloned into the pHM6 vector (catalog no. 1814664, Boehringer-Mannheim) to add a Rous sarcoma virus (RSV) promoter upstream to the C/EBPβ start site. C/EBPβ was removed from this new construct with BsaI and BspI. The 982-bp insert was cloned into mammalian vector pOP13CAT (Stratagene) from which the CAT portion had been previously removed. DNA for the pronuclear injection was purified over a CsCl gradient and digested with SspI and EclHKI. The 3.6-kb fragment was separated by gel electrophoresis on a 0.8% gel with no ethidium bromide. The appropriate band was removed and electroeluted from the gel using an Elutrap apparatus (Schleicher & Schuell). The eluted DNA was purified with a Qiagen-20 column, precipitated with isopropanol, and dissolved in 7.5 mM Tris (pH 7.4) and 0.15 mM EDTA. All solutions were prepared with tissue-grade water and were endotoxin tested (GIBCO-BRL). Finally, DNA was dialyzed and injected into fertilized ova at a concentration of 1.8 µg/ml at the Transgenic Mouse Core Facility of the University of California (San Diego, CA). The presence of the rsv gene was used to identify these transgenic mice by PCR, and three positive mice resulted. The primer sequences for the RSV PCR were custom designed (RSV.2271: sense, 5'-TAGGGTGTGTTTAGGCGAAA-3' and RSV.2510: antisense, 5'-TCTGTTGCCTTCCTAATAAG-3'). PCR reagents were all from Quiagen. Transgene-bearing founder mice were mated with FVB mice. All founder mice produced viable offspring. This line was bred by mating transgene-positive mice with FVB WT mice and back-crossed to FVB mice for at least four generations.

Animal procedures. In some experiments, adult mice (23–25 g) received a single intraperitoneal injection of LT (12.5 µg PA and 2.5 µg LF) in saline (200 µl) or saline only (200 µl). Two hours later, mice were killed, and immunofluorescence staining was analyzed by confocal microscopy. Macrophages were identified using antibodies specific for F4/80 or CD68 (Abcam) (26, 40).

Phagocytosis. Carboxylate-modified blue fluorescent (350/440 nm) polystyrene microspheres (0.1 µm; F8797), opsonized with FCS as described by the manufacturer (Molecular Probes), were used to evaluate phagocytosis (24). C/EBPβ WT, C/EBPβ-E217, and C/EBPβ-A217 mice were given 25 µg PA plus 5 µg LF in a total volume of 200 ml saline. Twenty-four hours later, mice received 500 µl of blue fluorescent microspheres intraperitoneally. Twenty-four hours later, mice were killed. Spleens were removed, embedded in OCT, and frozen in liquid nitrogen. Immunostaining was performed using anti-CD68 (0.2 µg/100 µl) (26) and Alexa 488 secondary antibodies (1:100, Molecular Probes) as previously described (10). Nuclei were stained with propidium iodine.

Analysis of phagocytosis by spleen macrophages was carried out by quantitative scanning confocal microscopy. At least 500 spleen macrophages were analyzed for their ability to phagocyte at least 3 fluorescent microspheres. The degree of phagocytosis was expressed as the percentage of macrophages that contained microspheres.

Macrophage oxidative burst and cytokine production. Macrophage oxidative burst was measured directly in vivo. Animals received a single intraperitoneal injection of LT (12.5 µg PA + 2.5 µg LF) in saline (200 µl) or saline only (200 µl) and, 24 h later, received a cell-permeant indicator for reactive oxygen species. After 20 h, animals received LPS from Escherichia coli 0111:B4 (0.5 mg/kg, Sigma) and were killed 4 h later. We used 5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate mixed isomers (carboxy-H2DCFDA; 1 mg/kg ip). This compound is nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell (51).

We also used the TranSignal Mouse Cytokine Antibody Array (Panomics, Redwood City, CA) to determine mouse cytokine content in serum (21). The cytokine antibody array is based on the sandwich ELISA method for detecting protein. The assay uses strepavidin-horseradish peroxidase to visualize the antibody-protein complexes on the array to determine which active cytokines are present in the sample. The chemiluminescent signal from the enzyme was detected using a Kodak imaging system.

Peritoneal macrophages. Adult FBV male C/EBPβ WT (littermates that do not express any transgene), C/EBPβ–/–, dominant negative, nonphosphorylatable C/EBPβ-A217 transgenic, and dominant positive, phosphorylation mimic C/EBPβ-E217 transgenic mice were used for the isolation of primary peritoneal macrophages. Peritoneal macrophages were obtained from thioglycollate-injected mice and cultured in RPMI media containing 10% FCS as described previously (40). Cells were incubated for 6 h in the presence or absence of recombinant PA (2.5 µg/ml), recombinant LF (0.2 µg/ml, List Biological), and/or LPS from E. coli 0111:B4 (0.1 µg/ml, Sigma). In some experiments, cells were treated with MEK-1 inhibitor U0126 (100 nM, Calbiochem).

Microscopy. Fluorescent labels were observed using a triple-channel fluorescence microscope or a confocal microscope. The fluorochromes utilized were 4',6-diamidino-2-phenylindole (DAPI), TOTO-3, FITC, and Texas red (Molecular Probes). The percentage of annexin V-FITC (R&D Systems)-positive cells was determined by in vivo microscopy (9, 39). At least 100 cells were analyzed per experimental point (10). Nuclear morphology was analyzed by staining cells with DAPI (1:1,500), TOTO-3 (1:1,500), terminal deoxynucleotidyl transferase-FITC (R&D Systems), or propidium B. Confocal microscopy was performed in macrophages using antibodies specific for F4/80 (0.2 µg/100 µl, Abcam), C/EBPβ (0.2 µg/100 µl), procaspase 8 (0.4 µg/100 µl), and active caspase 3 (0.2 µg/100 µl, Santa Cruz Biotechnology).

Immunoprecipitation and immunoblot analysis. C/EBPβ, procaspase 8, RSK, MEK1, ERK1/2, and/or β-actin were detected by immunoblot analysis of cultured macrophage and spleen lysate immunoprecipitates (2 µg antibody/500 µg total protein of cell lysate for immunopurification) (10) following the chemoluminescence protocol (DuPont) using purified antibodies (2 µg antibody/10 ml) specific for C/EBPβ, RSK-2, phospho-RSK-1/2, MEK1 (full length), ERK1/2, phospho-ERK1/2, β-actin (Santa Cruz Biotechnology), and procaspase 8 (PharMingen).

Caspase activity. Caspase 8 activity was determined in cultured macrophages and spleen lysates (500 µg total protein) by the release of the p-nitroaniline colorimetric substrate (Alexis Biochemicals) for caspase 8 within the linear part of the kinetic assay (9, 43).

Statistical analysis. Results are expressed as means ± SE of at least three independent experiments. Student's t-test or Fisher's exact test was used to evaluate the differences in means between groups, with a P value of <0.05 as significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Given that C/EBPβ plays an important role in the MAPK pathway (6, 7, 10) and that LT disrupts this signaling cascade (5, 13), we investigated whether C/EBPβ phosphorylation by RSK is required for LT toxicity in a cellular system relevant to inhalation anthrax in humans (17). To minimize the potential confounding variables of macrophage dedifferentiation and/or contamination by other cells, freshly isolated primary peritoneal macrophages, identified by the expression of F4/80 (40), were used as a model system.

We found that LT induced apoptosis in primary macrophages from normal, C/EBPβ WT mice after 6 h, as determined by the binding of annexin V to exposed phosphatidyl serine within plasma membranes, an early indicator of apoptosis (39) (P < 0.001; Fig. 2, A and B). Because LPS is an important modulator of macrophage activation (31), we assessed its effects in our cell culture system. Treatment of primary macrophages with LPS did not affect the apoptotic response of macrophages to LT (Fig. 2A).


Figure 2
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  		Fig. 2. Macrophages from C/EBPβ-E217 mice are resistant to anthrax LT-induced apoptosis. Cells were incubated for 6 h in the presence or absence of recombinant protective antigen (PA), recombinant lethal factor (LF), and/or LPS from Escherichia coli 0111:B4. A: apoptosis was determined by annexin V-FITC binding in primary macrophages. Data are means ± SE of 8 independent experiments. Cells were treated with LPS (0.1 µg/ml) and/or LT (2.5 µg/ml PA + 0.2 µg/ml LF). P < 0.001 for C/EBPβ-E217 cells treated with LPS + LT or LT alone. B: immunofluorescent microscopy of cells described in A for nuclei [4',6-diamidino-2-phenylindole (DAPI); blue] and annexin V-FITC binding (green). C: active caspase 3 was determined by immunofluorescent microscopy. P < 0.001 for C/EBPβ-E217 cells treated with LPS + LT. D: immunofluorescent microscopy of cells described in C for nuclei (DAPI; blue), macrophages (F4/80; green), and active caspase 3 (red). E: relative caspase 8 activity was induced by LPS + LT in macrophages from C/EBPβ wild-type (WT) mice but not C/EBPβ-E217 transgenic mice (P < 0.01).

 
The presence of active caspase 3 by confocal microscopy confirmed apoptosis of LT-treated macrophages (45) (Fig. 2, C and D). In addition, caspase 8 activity, reflecting induction of the extrinsic apoptotic pathway (4, 45), was stimulated by LT compared with baseline in C/EBPβ WT macrophage lysates (Fig. 2E). Treatment of primary macrophages with LPS did not affect the apoptotic response of macrophages to LT (Fig. 2E).

In addition, macrophages isolated from transgenic mice expressing C/EBPβ-A217, a dominant negative mutant of the RSK phosphorylation site (9), or mice with a targeted deletion of the C/EBPβ gene (10, 42) became rapidly apoptotic, through activation in culture, in the absence or presence of LT ( P < 0.01; Fig. 2, A and B). These findings also support the hypothesis that C/EBPβ phosphorylation by RSK is required for macrophage survival, as we reported for hepatic stellate cells (9).

Next, we assessed the role of C/EBPβ-phospho-T217, the RSK phosphoacceptor (10), on LT-induced cell death. To avoid the confounding variable of DNA transfection onto cell lines, we isolated macrophages from newly developed transgenic mice expressing the C/EBPβ-E217 mutant, a dominant-positive phosphorylation mimic (10). These animals appeared to be phenotypically normal and were fertile. In contrast to C/EBPβ WT macrophages, macrophages isolated from C/EBPβ-E217 transgenic mice were consistently refractory to the LT stimulation of caspase 8 (P < 0.01) and caspase 3 (P < 0.001) activities (Fig. 2, C–E) and apoptosis (P < 0.001; Fig. 2, A and B).

Because RSK, which is activated by ERK phosphorylation, plays an essential role in regulating cell survival and the cell cycle (6, 7, 10, 41), we analyzed whether LT inhibits RSK activation in primary macrophages. As expected, treatment of these cells with LT induced cleavage and inactivation of MEK-1, resulting in the absence of active phospho-ERK and phospho-RSK (Fig. 3A). Furthermore, a MEK1-specific inhibitor induced an apoptotic response similar to that of LT in C/EBPβ WT macrophages (P < 0.01; Fig. 3B), indicating that the MEK-1 signaling pathway is required to rescue primary macrophages from apoptosis, as also reported for dendritic cell dysfunction (1). In contrast to normal macrophages, those expressing the phosphorylation mimic C/EBPβ-E217 were refractory to treatment with the MEK-1 inhibitor (Fig. 3B).


Figure 3
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  		Fig. 3. Anthrax LT prevents the phosphorylation of ERK1/2 and RSK. Cells were incubated for 6 h in the presence or absence of recombinant PA, recombinant LF, and/or LPS from E. coli 0111:B4. A: MEK1, ERK1/2, phosphorylated (p)ERK1/2, RSK, and phosphorylated (p)RSK were determined by immunoblot. Lane 1, C/EBPβ; lane 2, C/EBPβ + LPS; lane 3, C/EBPβ + LPS + LT; lane 4, C/EBPβ-E217; lane 5, C/EBPβ-E217 + LPS; lane 6, C/EBPβ-E217 + LPS + LT. Equal loading was confirmed by measuring β-actin. Lanes 3 and 6 showed that LT induced cleavage of MEK1 and decreased pERK1/2 and pRSK. B: apoptosis was determined as described in Fig 2A. Cells were treated with MEK1 inhibitor U0126 (100 nM, Calbiochem). P < 0.01 for C/EBPβ WT cells treated with the MEK1 inhibitor.

 
These results strongly suggest that phosphorylation of C/EBPβ on T217 promotes macrophage survival and that inhibition of C/EBPβ phosphorylation by RSK is critical for the toxic effects of anthrax LT. Because in activated hepatic stellate cells C/EBPβ-phospho-T217 associates with caspase 8, inducing its self-cleavage and activation (9), we analyzed whether C/EBPβ was associated with procaspase 8 in macrophages. We found that C/EBPβ was associated with procaspase 8 in immunoprecipitates from macrophages isolated from C/EBPβ WT and C/EBPβ-E217 transgenic mice (Fig. 4). Inputs from the immunoprecipitates were equal for all samples within a given experiment. LT treatment of C/EBPβ WT macrophages decreased the association of C/EBPβ with procaspase 8 (Fig. 4, lane 3). In contrast, after LT treatment, the C/EBPβ-procaspase 8 association was unchanged in C/EBPβ-E217 macrophages (Fig. 4, lane 6). More important, LT treatment induced the activation of caspase 8 in C/EBPβ WT macrophages but not in C/EBPβ-E217 macrophages (Fig. 2E). Most likely, these findings reflect the fact that C/EBPβ-E217 macrophages are able to circumvent the inhibitory effects of LT on MEK and the subsequent inactivation of the ERK-RSK cascade (Fig. 3A).


Figure 4
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  		Fig. 4. C/EBPβ-E217 associates with procaspase 8 and prevents its activation by anthrax LT. Cells were incubated for 6 h in the presence or absence of recombinant PA, recombinant LF, and/or LPS from E. coli 0111:B4. Macrophage lysates were immunoprecipitated with C/EBPβ or procaspase 8 antibodies. Immunoblots showed decreased association between C/EBPβ and procaspase 8 in C/EBPβ WT macrophages treated with LPS + LT (as described in Fig. 2) but not LPS alone. Immunoblots are representative of 4 independent experiments. Lane 1, C/EBPβ; lane 2, C/EBPβ + LPS; lane 3, C/EBPβ + LPS + LT; lane 4, C/EBPβ-E217; lane 5, C/EBPβ-E217 + LPS; lane 6, C/EBPβ-E217 + LPS + LT. Equal loading was confirmed by measuring the inputs of C/EBPβ, procaspase 8, or β-actin.

 
To ascertain the functional relevance of our findings in primary mouse macrophages, we studied the effects of administration of LT on macrophage apoptosis in mice. Because the spleen is a macrophage-rich organ that develops apoptosis in inhalation anthrax in humans (17), we studied spleens from the various groups of mice. Spleen macrophages were identified with specific antibodies against F4/80 (40). Animals were given a nonlethal dose of LT or PA control intraperitoneally (28) and were killed 2 h later. Because LPS did not affect the response of primary macrophages to LT in any of our experiments in vitro, and to simplify the animal experiments, LPS was not included in the animal experiments except to induce macrophage oxidative burst and cytokine secretion.

LT treatment, but not PA alone, induced macrophage apoptosis in spleens of C/EBPβ WT mice as determined by the presence of active caspase 3 (P < 0.01) and TUNEL assay (Fig. 5, A and B). As expected, LT also induced macrophage apoptosis in spleens of C/EBPβ–/– and C/EBPβ-A217 mice (Fig. 5B). Similar to our findings in primary macrophage cultures, LT blocked the expression of active phospho-ERK and phospho-RSK in C/EBPβ WT mice (Fig. 6A). In contrast, and despite the inhibitory effect of LT on ERK and RSK phosphorylation and activation (Fig. 6A), C/EBPβ-E217 transgenic mice were resistant to in vivo spleen macrophage apoptosis induced by LT (P < 0.001; Fig. 5, A and B), confirming the results observed with primary macrophage cultures isolated from these mice. After LT treatment of C/EBPβ-E217 transgenic mice, C/EBPβ-E217 spleen macrophages remained associated (Fig. 6B, lane 4) and colocalized (Fig. 6C) with procaspase 8.


Figure 5
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  		Fig. 5. C/EBPβ-E217 mice are resistant to macrophage apoptosis induced by anthrax LT. Macrophages were identified using antibodies specific for F4/80 (40). A: 2 h after the intraperitoneal injection of LT (12.5 µg PA + 2.5 µg LF) but not PA alone (12.5 µg), C/EBPβ WT mice but not C/EBPβ-E217 mice displayed an increase in the percentage of spleen macrophages expressing active caspase 3 by confocal microscopy (P < 0.001). Each group was composed of at least 6 animals. Data are representative of 5 independent experiments. B: confocal microscopy for the apoptotic markers [terminal deoxynucleotidyl transferase (TdT; green) and active caspase 3 (red)] was positive in the spleens of C/EBPβ WT, C/EBPβ-A217, and C/EBPβ–/– mice, but not in C/EBPβ-E217 mice, injected with LT.

 

Figure 6
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  		Fig. 6. C/EBPβ-E217 mice are resistant to the impaired MAPK signal transduction induced by anthrax LT in macrophages. Macrophages were identified using antibodies specific for F4/80 (Abcam) (40). A: confocal microscopy for active pERK1/2 and pRSK in spleen macrophages of mice in the experiments described in Fig 5A. pERK1/2 and pRSK (red) were decreased in C/EBPβ WT and C/EBPβ-E217 mice after the administration of anthrax LT. Nuclei (TOTO-3; blue) and macrophages (F4/80; green) were also identified. B: spleen lysates from mice described in Fig 5A were immunoprecipitated with C/EBPβ or procaspase 8 antibodies. Lane 1, C/EBPβ; lane 2, C/EBPβ + LT; lane 3, C/EBPβ-E217; and lane 4, C/EBPβ-E217 + LT. Equal loading was confirmed by measuring β-actin. C/EBPβ associated with procaspase 8 in C/EBPβ-E217 mice, and this association was increased by LT treatment. C: confocal microscopy of spleens from C/EBPβ-E217 mice after LT treatment. Nuclei (TOTO-3; blue), C/EBPβ (green), and procaspase 8 (red) were identified.

 
Next, we assessed to what extent LT-induced macrophage apoptosis in vitro relates to the pathological effects of LT in animals, using phagocytosis, oxidative burst, and the production of cytokines as indicators of activated macrophage function (34, 48). Because C/EBPβ is critical for macrophage differentiation (32) and C/EBPβ–/– macrophages display defective bacterial killing (44), we investigated whether LT would affect phagocytosis, oxidative burst, and cytokine production by macrophages in our mice.

Animals were injected intraperitoneally with opsonized, fluorescent polystyrene microspheres (0.1 µm) to evaluate phagocytosis (24) or with carboxy-H2DCFDA, a cell-permeant indicator for reactive oxygen species. This compound is nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell (51).

To assess macrophage oxidative burst and cytokine production in vivo, animals received E. coli 0111:B4 LPS, an inducer of macrophage activity, 4 h before death. Microspheres or carboxy-H2DCFDA were injected 24 h after LT administration, and analysis of in vivo phagocytosis and oxidative burst by spleen macrophages were carried out 24 h later by quantitative scanning confocal microscopy. Macrophages were identified by the expression of CD68 (26), and phagocytosis or oxidative burst were measured as the percentage of macrophages that incorporated the microspheres (blue fluorescence) or expressed the oxidative indicator (green fluorescence). In C/EBPβ WT mice, LT inhibited macrophage phagocytosis (Fig. 7 A and Supplemental Fig. S1A)1 1 and the oxidative burst required for bactericidal activity (Fig. 7B and Supplemental Fig. S1B). In contrast, phagocytosis (P < 0.01) (Fig. 7A and Supplemental Fig. S1A) and the induction of oxidative burst (P < 0.05; Fig. 7B and Supplemental Fig. S1B) were increased at baseline and remained within normal limits after LT treatment in spleen macrophages of C/EBPβ-E217 mice. As expected, C/EBPβ-A217 mice had low phagocytosis (Fig. 7A and Supplemental Fig. S1A) and oxidative burst (Fig. 7B and Supplemental Fig. S1B) before and after LT treatment. These findings suggest that phosphorylation of C/EBPβ on T217 is indispensable for these differentiated macrophage functions in vivo.


Figure 7
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  		Fig. 7. C/EBPβ-E217 mice are resistant to the macrophage dysfunction induced by anthrax LT. Animals were injected with LT (25 µg PA + 5 µg LF) in a total volume of 200 µl saline. Microspheres or 5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate mixed isomers were injected 24 h after LT administration, and analysis of in vivo phagocytosis and oxidative burst by spleen macrophages was carried out 24 h later by quantitative scanning confocal microscopy. To assess macrophage oxidative burst in vivo, animals received E. coli 0111:B4 LPS, an inducer of macrophage activity, 4 h before death. Phagocytosis and oxidative burst were analyzed as described in MATERIALS AND METHODS. A: the degree of phagocytosis was quantified and expressed as a percentage of spleen macrophages capable of phagocytosis. It was higher in C/EBPβ-E217 mice than in C/EBPβ WT or C/EBPβ-A217 mice (P < 0.01). B: the degree of oxidative burst was expressed as the percentage of spleen macrophages containing the oxidative burst indicator. It was higher in C/EBPβ-E217 mice than in C/EBPβ WT or C/EBPβ-A217 mice (P < 0.05).

 
Although the production of macrophage cytokines has not been clearly correlated with LT toxicity (14, 38), we assessed the levels of TNF-{alpha}, IFN-{gamma}, IL-1{alpha}, IL-5, IL-6, and IL-12 under the experimental conditions used to evaluate macrophage oxidative burst in vivo. These cytokines were modestly affected by LT, with no significant differences in C/EBPβ WT and C/EBPβ-E217 mice (Supplemental Table 1). IFN-{gamma} and IL-6 serum levels were higher in C/EBPβ-A217 mice at baseline (P < 0.05), and although IFN-{gamma} increased with LT treatment, IL-6 decreased with LT treatment (P < 0.05; Supplemental Table 1).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Collectively, our results support the main hypothesis that LT, by inhibiting the MEK/ERK/RSK signaling cascade and preventing phosphorylation of C/EBPβ on T217, preempts critical antibacterial and survival responses to anthrax in macrophages. It remains to be determined whether these findings are also relevant to the effects of LT on other target cells.

An abnormal ERK/MAPK signaling pathway also mediates the impaired dendritic and macrophage cell function after LT treatment (1, 34). Furthermore, small molecules that activate the MAPK pathway, including ERK1/2 phosphorylation, inhibited LT-induced macrophage apoptosis, suggesting that this signaling cascade is essential for LT toxicity (34). However, the disrupted phosphorylation target of the MAPK pathway critical for cell survival and induced by LT (35) has not yet been identified (1, 34).

The proposed role of unphosphorylated and phosphorylated C/EBPβ on the modulation of macrophage survival following exposure to LT is in agreement with the documented role of C/EBPβ on differentiation and cell arrest/proliferation not only of macrophages (32) but also other cells (10, 27, 33). In contrast, under other conditions, both ERK1/2 and RSK-mediated survival pathways were activated concurrently with apoptosis in RAW 264.7 macrophages (52). The type of macrophage response initiated by toxins will likely depend on whether the balance is toward apoptotic or survival pathways (52).

We also found an inhibitory effect of LT on macrophage phagocytosis and the oxidative burst required for bactericidal activity. Moreover, after LT treatment, macrophages of C/EBPβ-E217 mice had a level of phagocytosis and oxidative burst in vivo comparable with that of C/EBPβ WT mice not treated with LT. These findings are relevant to understanding the pathogenesis of anthrax since defective phagocytosis and bactericidal activity would block the innate immunity against B. anthracis. Because LT-induced macrophage cell death is highly dependent on the mouse strain (30), it is important to emphasize that our C/EBPβ transgenic and knockout mice have the same FVB background as control C/EBPβ WT mice. In contrast to our results, cultured nonhuman primate alveolar macrophages, despite MEK1 cleavage, impairment of cytokine secretion, and loss of bactericidal activity following LT treatment, were still capable of phagocytosis (38). The differences in the effects of LT on macrophage phagocytosis could be related to differences between in vivo and in vitro studies and/or the variable susceptibility of nonhuman primate and mouse cells to LT. In any case, the primary function of LT in anthrax pathogenesis may be to facilitate B. anthracis survival (38).

Signaling through double-stranded RNA-activated protein kinase (PKR) is important for apoptosis of cultured macrophages induced by heat-inactivated B. anthracis, but these experiments did not involve LT (20). Although both PKR and C/EBPβ can be activated by LPS signaling (2, 3, 20), and C/EBPβ plays an important role in macrophage differentiation (32), it remains to be determined whether there is a functional interaction between PKR and C/EBPβ in LT-induced macrophage dysfunction and apoptosis.

The novel finding of this study is that the MAPK survival signaling cascade in macrophages treated with LT requires phosphorylation of C/EBPβ on T217 by RSK and, most likely, RSK activation. However, definitive proof of the role of activated RSK will require extensive genetic and molecular manipulations of RSK. MEK-1-specific inhibitors also induced apoptosis of C/EBPβ WT macrophages but not C/EBPβ-E217 macrophages isolated from transgenic mice, further supporting the main hypothesis.

Our results suggest a mechanism by which anthrax LT stimulates macrophage dysfunction and apoptosis. Proteolytic inactivation of MEK with the consequent inactivation of ERK and RSK and prevention of C/EBPβ phosphorylation on T217 contributes substantially to macrophage dysfunction and apoptosis. Thus, newly developed dominant positive C/EBPβ-E217 transgenic mice are resistant to LT-induced macrophage dysfunction and apoptosis. In support of our findings, expression of C/EBPβ-E217 enhances the survival of cultured progenitor neuronal cells (27), whereas C/EBPβ–/– macrophages display defective bacterial killing and tumor cytotoxicity (44).

Although C/EBPβ-E217 mice were resistant to macrophage dysfunction and apoptosis induced by LT, the precise molecular mechanisms by which phospho-C/EBPβ prevents LT-induced dysfunction and apoptosis have not yet been characterized. Phospho-C/EBPβ could induce the inhibition of proapoptotic proteins, such as p53 (50), or the activation of survival proteins, such as MnSOD (37) or FLIP (8). Alternatively, granzyme B rather than caspase 8 could be the major target of C/EBPβ-phospho-T 217, as we suggested previously for hepatic stellate cell apoptosis/survival (9).

Thus, our study indicates a potential role of altered macrophage immune responses in infections, such as anthrax, that disable the MEK-ERK-RSK-C/EBPβ signaling cascade.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health (NIH) Grants DK-38652, R37-DK-46971, and CA-96932 and a Department of Veterans Affairs Merit Review. M. Buck was supported by the Howard Temin Award from the NIH.


   ACKNOWLEDGMENTS
 
We thank Antimone Dewing, Melissa Mitrou, and Marcus Kouma for the macrophage isolations and cultures and Smith Iuli for the preparation of figures.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Buck, Dept. of Medicine, Univ. of California-San Diego, and Veterans Affairs Healthcare System, San Diego, CA 92161 (e-mail: mbuck{at}ucsd.edu)

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.

1 Supplemental material (Supplemental Fig. 1 and Table 1) for this article is available at the American Journal of Physiology-Cell Physiology website. Back


   REFERENCES
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