The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1–4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.

Patients with late-stage systemic anthrax infection during the bioterrorism attacks of 2001 had high mortality rates (45%), even when treated with appropriate antibiotic therapy (1, 2). These high mortality rates are thought to have been due to the persistence of anthrax toxin components, including those that compose anthrax lethal toxin (LT)3 (3, 4, 5). Animal models reveal that administration of anthrax LT in vivo recapitulates the abrupt shock-like features of late-stage anthrax infection, highlighting the importance of this virulence factor in the pathogenesis of anthrax infection (6, 7). Determining the critical host factors that mediate anthrax LT toxicity is required to develop relevant bioassays for toxin activity, informative biomarkers during toxemia and/or infection, and new therapies for anthrax infection.

A variety of immune cell lineages have been shown to be targets for anthrax LT, including macrophages, dendritic cells, endothelial cells, and T and B lymphocytes (8, 9, 10, 11, 12, 13, 14, 15, 16). With the exception of strain-specific increases in IL-1β and/or IL-18 in mice or murine cells (9, 17, 18), predominantly immunosuppressive effects of anthrax LT have been reported in recent publications (8, 10, 11, 12, 13, 14, 15, 16). Although anthrax LT-mediated immunosuppression likely plays a critical role in the evasion of host immune responses by Bacillus anthracis, the mechanism underlying the severe shock-like illness observed in animal models of toxemia in the absence of infection is not fully understood (19).

One striking feature of murine anthrax LT models is the induction of high levels of circulating neutrophils during toxemia (17). Although neutrophils provide host protection during bacterial infections through the killing and clearance of bacterial pathogens, they have also been implicated as mediators of host damage during systemic inflammatory responses (20, 21). The role of anthrax LT in modulating human neutrophil function has been somewhat controversial; both proinflammatory and immunosuppressive effects have been reported (22, 23, 24, 25, 26, 27, 28). These apparent contradictions could be partially explained by differences in the mode and/or timing of stimulation. In addition, the recent finding that human neutrophil-derived α defensins reversibly neutralize anthrax LT introduces another factor that could confound studies involving human neutrophils (29).

Herein, we describe experiments involving the comprehensive examination of the effects of anthrax LT on bone marrow-derived and circulating murine neutrophils, which lack α defensins that could potentially confound experimental interpretations (30). These experiments demonstrate that anthrax LT only partially disrupts mitogen-activated protein kinase kinase (MKK)-dependent signaling in murine neutrophils, minimally affecting signaling to JNK family members. This anthrax LT-resistant JNK signaling capacity, in the setting of the sustained ERK signaling blockade, supports increased superoxide production in response to stimulation via PMA, TLR-2, or TLR-4. Thus, in contrast to the well-established immunosuppressive effects of LT on murine macrophages, dendritic cells, and T and B lymphocytes, anthrax LT supports a critical proinflammatory function of neutrophils through stimulation pathways expected to be activated during infection.

Neutrophils were isolated from the bone marrow of 6- to 12-wk old BALB/c or C57BL/6 mice, in accordance with an animal protocol approved by the Food and Drug Administration Institutional Animal Care and Use Committee. The neutrophil population was purified using density gradient centrifugation with Histopaque 1119 and 1077 according to the manufacturer’s suggested protocol (Sigma-Aldrich). Purity was assessed by flow cytometry using Gr-1 Ab staining 24 h following isolation. Neutrophils prepared in this manner were found to be routinely 90% Gr-1+. Neutrophils were cultured in RPMI 1640 medium (HyClone) containing 10% FBS (HyClone), 2 mM l-glutamine (Invitrogen), 10 mM HEPES buffer, 1 mM sodium pyruvate (Quality Biological), and 1% antibiotic/antimycotic (Sigma-Adlrich). Cell viability was assessed by trypan blue exclusion.

Lyophilized recombinant anthrax protective Ag (PA) and lethal factor (LF) were purchased from a commercial source (List Biological Laboratories, Inc.) and reconstituted in 1:1 glycerol/water (final: 1 mg/ml in 5 mM HEPES, 50 mM NaCl, pH 7.5) for in vitro studies. Anthrax LT was administered at concentrations of 1.0 μg/ml LF and 2.5 μg/ml PA, unless otherwise indicated. Selected experiments involved coadministration of LPS (200 ng/ml; Sigma-Aldrich) or (Pam)3-Cys-Ser-(Lys)4 (Pam3; 1.0 μg/ml; Calbiochem), a synthetic TLR2 agonist. A FITC-labeled mAb recognizing the murine Ly-6B neutrophil marker (Gr-1, clone NIM P-R14; Hycult Biotechnology) was used for flow cytometry analysis of isolated neutrophils to assess purity.

The following primary Abs were used for Western blotting: anti-MKK1 (1:1000; BD Biosciences); anti-MKK2 (N-20), anti-MKK3 (N-20), anti-MKK4 (K-18), anti-MKK5 (H-94), anti-MKK6 (N-19), anti-MKK7 (H-160) (1:250; Santa Cruz Biotechnology); anti-β-actin (I-19, 1:1000; Santa Cruz Biotechnology); and anti-phospho-p44/42 MAPK, anti-total p44/42 MAPK, anti-phospho-JNK, anti-total JNK, anti-phospho-p38 MAPK, anti-total p38 MAPK (1:1000; Cell Signaling Technology). The following HRP-conjugated secondary Abs were used for Western blotting: anti-mouse IgG (1:2000; Amersham), anti-rabbit IgG (1:2000; Amersham), and anti-goat IgG (1:5000; Abcam). Specific inhibitors of JNK (20 μM, SP600125; Sigma-Aldrich), MEK1/2 (2.5 μM, U0126; Sigma-Aldrich), and p38 (5 μM, SB203580; Sigma-Aldrich) were used in selected experiments. These MAPKs inhibitors are poorly soluble in water, and were maintained in 10–20 mM stock solutions using DMSO. Control cultures received identical concentrations of the DMSO-containing carrier.

C57BL/6 and BALB/c mice were treated with varying doses of anthrax LT, using a fixed ratio of LF/PA of 1:2.5. BALB/c mice, which are more susceptible to anthrax LT (17), received 40 μg LF/100 μg PA, whereas the more resistant C57BL/6 mice received 100 μg LF/250 μg PA. Lyophilized LF and PA were resuspended in water (final: 1 mg/ml in 5 mM HEPES, 50 mM NaCl, pH 7.5), then diluted in PBS, and subsequently injected i.p. into mice in a total volume of 0.5 ml. As a negative control, selected mice received PBS alone. Mice were sacrificed 24 h following treatment and bone marrow neutrophils were immediately isolated as described.

Purified neutrophils were cultured with FITC-labeled anthrax PA (25 μg/ml) for 30 min at 4°C, in the presence or absence of unlabeled anthrax PA (150 μg/ml) or BSA (150 μg/ml) to confirm specific binding. Stained cells were washed with PBS and analyzed by flow cytometry. Unstained cells were analyzed in parallel to establish background levels of autofluorescence. Flow cytometry was performed on a FACS Calibur flow cytometer (BD Biosciences) and analyzed using CellQuest Pro software (BD Immunocytometry Systems).

Western blotting was performed using standard techniques as previously described (9). Cells were maintained on ice for 30 min in a buffer containing 20 mM Tris-HCL, 150 mM NaCl, 5 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1% Triton X-100, and a freshly prepared protease inhibitor mixture (Sigma-Aldrich). Following centrifugation, protein lysates were electrophoretically separated on 4–12% NuPage gradient gels (Invitrogen) and subsequently transferred to 0.2-μm nitrocellulose membranes (Bio-Rad). The membranes were incubated with the indicated primary Abs, followed by incubation with the appropriate species-specific HRP-conjugated secondary Abs. Membranes were then treated with Western blotting Luminol Reagent (Santa Cruz Biotechnology), and exposed to Chemiluminescence BioMax film (Kodak).

Bone marrow neutrophils were isolated from untreated animals (in vitro experiments) or mice treated with anthrax LT or PBS (in vivo experiments). Neutrophils were cultured for up to 72 h in the presence or absence of anthrax LT, with or without concomitant treatment with LPS, Pam3, and/or a specific JNK, MEK1/2, or p38 inhibitors (30 min before superoxide determination). Selected cultures received PMA immediately before superoxide determinations (50–250 ng/ml). Superoxide production was then assessed using the LumiMax Superoxide Anion Detection kit (Stratagene), using the manufacturer’s suggested protocol. In brief, neutrophils from cultures were pelleted through centrifugation, washed, and resuspended in 100 μl SOA assay medium mixed with 100 μl SOA-reagent mixture containing 100 μM luminol, 125 μM proprietary chemiluminescence signal enhancer ±100 ng/ml PMA as indicated. Luminosity was recorded using a Microplate Luminometer (EG & G Berthold) at 2–20 min intervals for up to 3 h (until returning to baseline levels). The addition of superoxide dismutase (Stratagene) to the assay mixture eliminated the PMA-induced chemiluminescence signal from either LT-treated or control neutrophils. Moreover, the inclusion of Nω-nitro-l-arginine-methyl ester hydrochloride (l-NAME, an inhibitor of NO production) to the assay mixture had a negligible effect on the PMA-induced chemiluminescence signal. Taken together, these control experiments demonstrated the specificity of this assay for superoxide under our experimental conditions (data not shown).

In selected experiments, the effect of anthrax LT on neutrophil superoxide production was also confirmed using a ferricocytochrome C reduction assay (28), modified for our purposes as described below. Purified bone-marrow derived neutrophils from C57BL/6 mice were cultured for 24 h or 48 h in the presence or absence of anthrax LT. Neutrophils (2 × 106) were then resuspended in 100 μl PB buffer (1 mM CaCl2 and 20 mM glucose in PBS). Partially acetylated cytochrome C (50 μM final; Sigma-Aldrich) was added, and the reaction volume brought to a final volume of 200 μl with PB buffer. The assay mixture was incubated at 37°C for 10 min, followed by PMA stimulation (2 μg/ml final; Sigma-Aldrich). Upon stimulation, reduction of cytochrome C was monitored by measuring absorbance at 550 nM using a Versamax microplate reader (Molecular Devices). Superoxide production rates at the time interval from 1000 to 2000 s were calculated using these data and the known extinction coefficients of the substrate and reduced product.

Studies reporting the activity of anthrax LT on human neutrophils have produced seemingly contradictory results, possibly due to differences in timing, stimulation, and assay conditions (22, 23, 24, 25, 26, 27, 28), but perhaps also due to the production of α defensins that reversibly neutralize anthrax LT (29). To overcome this potentially confounding factor, we used murine neutrophils, which do not produce α defensins (30), and investigated the effects of anthrax LT in time-course experiments following stimulation through pathways predicted to be activated during Bacillus anthracis infection.

We initially confirmed that murine neutrophils are targets for anthrax LT. As shown in Fig. 1,A, FITC-labeled anthrax PA bound bone marrow neutrophils derived from either C57BL/6 (left panel) or BALB/c mice (right panel). The specificity of FITC-labeled anthrax PA binding was demonstrated through competitive inhibition with unlabeled PA, compared with a lack of competition by the identical concentration of BSA. Next, we investigated the effect of anthrax LT on its MKK targets in neutrophils. As previously reported in other human and murine primary cells and cell lines, anthrax LT treatment of C57BL/6 neutrophils in vitro led to the cleavage of multiple MKKs (8, 10, 11, 16, 31, 32). These included MKK 1, 2, and 4, as well as the longer MKK3b and MKK6b isoforms (which contain the N-terminal LT cleavage site) (33). MKK5, as reported in other lineages (10) or in over-expression experiments (33, 34), was resistant to cleavage by anthrax LT. However, in contrast to previous reports of the susceptibility of human MKK7 to LT degradation detected in in vitro over-expression systems (33, 34), we observed little, if any, degradation of the expressed short (α) or long (β or γ) isoforms of MKK7 in murine neutrophils following anthrax LT administration, with or without concomitant TLR stimulation (Fig. 1,B). The degradation of targeted MKKs had minimal effect on neutrophil survival during the first 24 h of culture, even in TLR stimulatory conditions evoking those of infection. However, longer term cultures (2–3 days), showed decreased survival rates for LT-treated neutrophils. Moreover, the survival advantage provided by stimulation through TLR2 (with Pam3) or TLR4 (with LPS), which was apparent in 3-day cultures, was completely abrogated by concomitant anthrax LT administration (Fig. 1,C). These data argue against a role for anthrax LT in promoting neutrophil function due to a prosurvival effect. Neutrophils obtained from mice treated with or without LT in vivo showed the identical pattern of differential MKK sensitivity that was observed in vitro; MKK 1–4 and 6, but not MKK 5 or 7, were cleaved in response to LT treatment (Fig. 2 B).

FIGURE 1.

MKK cleavage in anthrax LT-treated neutrophils derived from murine bone marrow. A, Purified bone marrow-derived neutrophils from C57BL/6 (left panel) or BALB/c mice (right panel) were stained with FITC-labeled anthrax PA (25 μg/ml) in the presence or absence of unlabeled anthrax PA (150 μg/ml) or BSA (150 μg/ml) and analyzed by flow cytometry as indicated. Unstained cells were analyzed to establish background autofluorescence as shown. A representative experiment from one of two independent experiments is shown. In addition, two independent experiments with higher FITC-labeled anthrax PA concentrations (50 μg/ml) demonstrated binding as well. B, Purified bone marrow-derived neutrophils from C57BL/6 mice were treated with or without anthrax LT for 3 h, with or without short-term stimulation with Pam3 (10 min) or LPS (15 min), as indicated. Protein levels of MKK1–7 and β-actin were assessed by Western blotting. One of two independent experiments showing the identical qualitative trend is shown. C, Purified bone marrow-derived neutrophils from C57BL/6 mice were cultured for varying time periods (0–3 days) in the presence or absence of anthrax LT, LPS, and/or Pam3 as indicated. Cell viability was assessed by trypan blue exclusion, with the assumption that total cell numbers remained constant. C, Error bars, SD generated from three independent experiments.

FIGURE 1.

MKK cleavage in anthrax LT-treated neutrophils derived from murine bone marrow. A, Purified bone marrow-derived neutrophils from C57BL/6 (left panel) or BALB/c mice (right panel) were stained with FITC-labeled anthrax PA (25 μg/ml) in the presence or absence of unlabeled anthrax PA (150 μg/ml) or BSA (150 μg/ml) and analyzed by flow cytometry as indicated. Unstained cells were analyzed to establish background autofluorescence as shown. A representative experiment from one of two independent experiments is shown. In addition, two independent experiments with higher FITC-labeled anthrax PA concentrations (50 μg/ml) demonstrated binding as well. B, Purified bone marrow-derived neutrophils from C57BL/6 mice were treated with or without anthrax LT for 3 h, with or without short-term stimulation with Pam3 (10 min) or LPS (15 min), as indicated. Protein levels of MKK1–7 and β-actin were assessed by Western blotting. One of two independent experiments showing the identical qualitative trend is shown. C, Purified bone marrow-derived neutrophils from C57BL/6 mice were cultured for varying time periods (0–3 days) in the presence or absence of anthrax LT, LPS, and/or Pam3 as indicated. Cell viability was assessed by trypan blue exclusion, with the assumption that total cell numbers remained constant. C, Error bars, SD generated from three independent experiments.

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FIGURE 2.

Anthrax LT disrupts MKK-dependent signaling in murine neutrophils both in vitro (A) and ex vivo (B). A, Purified bone marrow-derived neutrophils from C57BL/6 mice were treated with anthrax LT or vehicle control for 5 h, followed by Pam3 stimulation for varying time periods as indicated. Protein levels of phosphorylated ERK, total ERK, phosphorylated JNK, total JNK, phosphorylated p38, total p38, MKK1–7, and β-actin in cell lysates obtained from these cultures were assessed by Western blotting. B, Purified neutrophils were isolated from the bone marrow of C57BL/6 mice, which had been treated for 24 h in vivo with either intraperitoneal anthrax LT (100 μg LF and 250 μg PA) or PBS control. Neutrophil cultures were subsequently stimulated with Pam3 for varying time periods as indicated. Protein levels of phosphorylated ERK, total ERK, phosphorylated JNK, total JNK, phosphorylated p38, total p38, MKK1–7, and β-actin were assessed by Western blotting. Representative experiments from one of two independent experiments showing the same qualitative trend are shown for both A and B.

FIGURE 2.

Anthrax LT disrupts MKK-dependent signaling in murine neutrophils both in vitro (A) and ex vivo (B). A, Purified bone marrow-derived neutrophils from C57BL/6 mice were treated with anthrax LT or vehicle control for 5 h, followed by Pam3 stimulation for varying time periods as indicated. Protein levels of phosphorylated ERK, total ERK, phosphorylated JNK, total JNK, phosphorylated p38, total p38, MKK1–7, and β-actin in cell lysates obtained from these cultures were assessed by Western blotting. B, Purified neutrophils were isolated from the bone marrow of C57BL/6 mice, which had been treated for 24 h in vivo with either intraperitoneal anthrax LT (100 μg LF and 250 μg PA) or PBS control. Neutrophil cultures were subsequently stimulated with Pam3 for varying time periods as indicated. Protein levels of phosphorylated ERK, total ERK, phosphorylated JNK, total JNK, phosphorylated p38, total p38, MKK1–7, and β-actin were assessed by Western blotting. Representative experiments from one of two independent experiments showing the same qualitative trend are shown for both A and B.

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We next examined the effect of anthrax LT on MKK-dependent signal transduction. MKKs phosphorylate the MAPKs, which comprise the ERK family, the JNK family, and the p38 family (35). MAPK signaling is required for a variety of host cell functions, including proliferative and cytokine responses during immune activation (8, 10, 35). As has been demonstrated with other types of human and murine primary cells and cell lines, pretreatment of murine neutrophils with anthrax LT in vitro had a marked effect on signal transduction (8, 10, 12, 16, 31, 32). For example, though rapid phosphorylation of p38 and ERK p42/p44 was observed in control cultures following treatment with the TLR2 agonist, Pam3, phosphorylation of these signal transduction intermediates was blocked in neutrophils that had been pretreated with LT for 5 h in vitro. In contrast, LT pretreatment had only a modest effect on TLR2-dependent phosphorylation of JNK p46/54. As was observed in control neutrophil cultures, phosphorylation of JNK p46/54 in anthrax LT pretreated neutrophils peaked 15 min following Pam3 stimulation, though peak phosphorylation levels were reduced (Fig. 2,A). Neutrophils isolated from mice 24 h following treatment with anthrax LT in vivo showed a similar pattern of disruption in MKK-dependent signal transduction. Paralleling in vitro results, TLR2-dependent phosphorylation of ERK p42/p44 was blocked by prior in vivo treatment with anthrax LT. Although markedly reduced, trace p38 phosphorylation was detected following TLR2 stimulation. As observed in vitro, TLR-dependent phosphorylation of JNK p46/p54 was readily detectable ex vivo despite prior in vivo treatment with anthrax LT, peaking simultaneously with control cultures that had not been exposed to anthrax LT (15 min; Fig. 2 B).

As MAPK signal transduction pathways are known to be important regulators of the production of reactive oxygen species (36, 37, 38, 39), we subsequently studied the effect of anthrax LT on this critical neutrophil function. We first compared bone marrow-derived neutrophils from C57BL/6 mice cultured with or without anthrax LT. Time-course experiments examining PMA-induced superoxide revealed that the kinetics of superoxide induction was altered by exposure to anthrax LT (Fig. 3). Although vehicle-treated neutrophils produced slightly greater amounts of superoxide compared with LT-treated neutrophils at very early time points, induction of superoxide in LT-treated cells was delayed compared with untreated controls. Moreover, superoxide production in LT-treated cells was markedly increased and more sustained compared with vehicle-treated control cultures, resulting in much greater overall superoxide production in anthrax LT-treated neutrophils compared with that in control cultures not receiving the toxin (observe area under the curves; Fig. 3). The prolongation of superoxide production was evident 24 h following LT treatment (Fig. 3upper panel), but was still readily detectable in 48-h (middle panel) and 72-h cultures (lower panel). Moreover, similar results were observed at PMA concentrations ranging from 50 to 250 ng/ml, indicating that this effect was not due to a shift in PMA responsiveness (data not shown). Increased superoxide production in LT-treated neutrophils was confirmed using the classical cytochrome C reduction assay as well. Neutrophils pretreated with LT produced superoxide at much higher initial rates than vehicle-treated control cells (>65% greater following either 24 or 48 h pretreatment with LT; data not shown). However, in contrast to the classical assay, which was deleterious to cells in longer-term cultures, the luminol-based assay allowed long term measurement of superoxide, as well as the detection of intracellular superoxide production. For these reasons, the luminol-based assay was determined to be more appropriate for subsequent experiments.

FIGURE 3.

Anthrax LT supports increased neutrophil superoxide production in vitro. Purified bone marrow-derived neutrophils from C57BL/6 mice were cultured for 24–72 h with anthrax LT or vehicle alone before PMA stimulation as indicated. Measurements of superoxide anion production were made at frequent intervals for 120–180 min using a luminol-based assay. Representative time-course results from one of five independent experiments are shown.

FIGURE 3.

Anthrax LT supports increased neutrophil superoxide production in vitro. Purified bone marrow-derived neutrophils from C57BL/6 mice were cultured for 24–72 h with anthrax LT or vehicle alone before PMA stimulation as indicated. Measurements of superoxide anion production were made at frequent intervals for 120–180 min using a luminol-based assay. Representative time-course results from one of five independent experiments are shown.

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Parallel results were observed in bone marrow-derived neutrophils obtained from mice treated with anthrax LT in vivo. Bone marrow-derived neutrophils from either LT-treated C57BL/6 (Fig. 4,A) or BALB/c mice (Fig. 4,B) showed increased PMA-induced superoxide production compared with PBS-treated controls. Moreover, prior in vivo LT treatment led to more sustained production of superoxide, resulting in a substantial increase in overall superoxide production. This prolongation in superoxide production was observed in neutrophils 24–72 h following treatment with anthrax LT, as previously shown in vitro (Fig. 3). Similar to bone marrow-derived neutrophils, peripheral blood neutrophils obtained from LT-treated mice in vivo showed markedly greater superoxide production than neutrophils obtained from PBS-treated control animals. PMA-stimulated superoxide in neutrophils from LT-treated animals was sustained above baseline for >3 h, resulting in much greater overall superoxide production than that in control cultures, which returned to baseline levels within 1 h (Fig. 4,C). To address the limitation that PMA is a nonphysiological stimulus, we also examined the effect of LT treatment on superoxide induced through the TLR2 and TLR4 pathways predicted to be activated during infection, using Pam3 and LPS, respectively. As observed in experiments with PMA, bone marrow-derived neutrophils obtained from LT-treated mice showed enhanced superoxide production in response to TLR stimulation, which persisted for at least 3 days in ex vivo cultures (Fig. 4 D).

FIGURE 4.

Anthrax LT supports increased neutrophil superoxide production in neutrophils isolated from mice treated with LT in vivo. A and B, C57BL/6 (A) or BALB/c mice (B) were treated i.p. with either anthrax LT (C57BL/6, 100 μg LF/250 μg PA; BALB/c, 40 μg LF/100 μg PA) or PBS control. Following 24 h of treatment, animals were sacrificed and bone marrow neutrophils were isolated. Neutrophils isolated from C57BL/6 (A) or BALB/c (B) mice treated in vivo were cultured for 24–72 h as indicated. Following stimulation with PMA, superoxide was measured at frequent intervals until returning to baseline levels as shown. C, Peripheral blood neutrophils isolated from C57BL/6 mice treated with anthrax LT or PBS control for 24 h in vivo were stimulated with PMA. Measurements of superoxide were made at frequent time intervals for 3 h as shown. D, Bone marrow-derived neutrophils isolated from C57BL/6 mice treated in vivo with anthrax LT or PBS control were cultured in the presence or absence of the TLR agonists, LPS (left panel) or Pam3 (right panel). Baseline superoxide production (i.e., without PMA restimulation) was assessed at various time points for up to 72 h as shown. Representative time-course results of these three independent experiments are shown for A and B. C, A representative experiment from one of two experiments is shown. D, Error bars, SD generated from three independent experiments.

FIGURE 4.

Anthrax LT supports increased neutrophil superoxide production in neutrophils isolated from mice treated with LT in vivo. A and B, C57BL/6 (A) or BALB/c mice (B) were treated i.p. with either anthrax LT (C57BL/6, 100 μg LF/250 μg PA; BALB/c, 40 μg LF/100 μg PA) or PBS control. Following 24 h of treatment, animals were sacrificed and bone marrow neutrophils were isolated. Neutrophils isolated from C57BL/6 (A) or BALB/c (B) mice treated in vivo were cultured for 24–72 h as indicated. Following stimulation with PMA, superoxide was measured at frequent intervals until returning to baseline levels as shown. C, Peripheral blood neutrophils isolated from C57BL/6 mice treated with anthrax LT or PBS control for 24 h in vivo were stimulated with PMA. Measurements of superoxide were made at frequent time intervals for 3 h as shown. D, Bone marrow-derived neutrophils isolated from C57BL/6 mice treated in vivo with anthrax LT or PBS control were cultured in the presence or absence of the TLR agonists, LPS (left panel) or Pam3 (right panel). Baseline superoxide production (i.e., without PMA restimulation) was assessed at various time points for up to 72 h as shown. Representative time-course results of these three independent experiments are shown for A and B. C, A representative experiment from one of two experiments is shown. D, Error bars, SD generated from three independent experiments.

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We next investigated the relative roles of the three MAPK signal transduction pathways in mediating the effects of anthrax LT. To this end, bone marrow-derived neutrophils were pretreated for 24 h in vitro with or without anthrax LT. Selected cultures were also treated with specific inhibitors of MAPK family signal transduction pathways. As shown in Fig. 5,A (upper panel), inhibition of p38 had no effect on superoxide following PMA stimulation. The LT-induced delay and prolongation of superoxide production was not affected by p38 inhibition; however, overall superoxide levels were slightly reduced, perhaps due to inhibition of the trace p38 signaling that persists despite LT. In this regard, low level p38 signaling was noted in PMA-stimulated neutrophils pretreated with LT for 24 h (data not shown) and in Pam3-stimulated neutrophils pretreated with LT in vivo (Fig. 2,B). The inhibition of ERK signaling with the MEK1/2 inhibitor, U0126, led to delayed and prolonged superoxide production following PMA stimulation, as was observed with LT treatment. However, MEK1/2 inhibition of LT-treated cultures led to a modest decrease in the amplitude of the superoxide signal, without changing the general shape of the curve (Fig. 5,A, middle panel). We cannot exclude the possibility that the small reduction of superoxide production observed in LT-treated cultures results from the known weak inhibitory effect of this inhibitor on other kinases, including JNK (40). In contrast to p38 and ERK p42/44 signaling, however, substantial JNK signaling occurred despite the presence of the toxin, both in vitro and ex vivo (Fig. 2, A and B). Following stimulation with PMA, both vehicle control- and LT-treated cultures showed a sharp decline in superoxide production in the presence of a specific chemical inhibitor of JNK (Fig. 5,A, lower panel). Overall superoxide production was comparable between PBS-treated and anthrax LT-treated cultures following pretreatment with the JNK inhibitor. More marked results were observed in experiments ex vivo, where inhibition of JNK signaling with a specific inhibitor equalized superoxide production by neutrophils obtained from mice treated with anthrax LT in vivo with that observed in neutrophils obtained from PBS-treated controls (Fig. 5 B). Taken together, these chemical inhibition data indicate that superoxide production in LT-treated neutrophils clearly requires JNK signaling, while the delay and prolongation of superoxide production correlates with effects due to the blockade of MEK1/2/ERK signaling by LT. The overall increase in amplitude of PMA-induced superoxide, however, likely results from the incomplete blockade of MAPK signaling by LT that is not reproduced with chemical MAPK inhibitors.

FIGURE 5.

Anthrax LT supports increased neutrophil superoxide production through differential effects on MAPK signaling. A, Bone marrow-derived neutrophils from C57BL/6 mice were treated with anthrax LT or vehicle control for 48 h, with or without specific p38 (upper panel), MEK1/2 (middle panel), or JNK inhibitors (lower panel) as indicated. Following PMA stimulation, superoxide was assayed at various time intervals for 130 min as shown. B, C57BL/6 mice were treated for 24 h with either intraperitoneal anthrax LT (100 μg LF and 250 μg PA) or PBS control. Neutrophils isolated from the bone marrow of sacrificed animals were cultured for 48 h without additional LT, and then treated for 30 min with or without a specific JNK inhibitor and subsequently stimulated with PMA. Superoxide production was assayed at various time intervals for 3 h as shown. A and B, Representative experiments from one of two independent experiments.

FIGURE 5.

Anthrax LT supports increased neutrophil superoxide production through differential effects on MAPK signaling. A, Bone marrow-derived neutrophils from C57BL/6 mice were treated with anthrax LT or vehicle control for 48 h, with or without specific p38 (upper panel), MEK1/2 (middle panel), or JNK inhibitors (lower panel) as indicated. Following PMA stimulation, superoxide was assayed at various time intervals for 130 min as shown. B, C57BL/6 mice were treated for 24 h with either intraperitoneal anthrax LT (100 μg LF and 250 μg PA) or PBS control. Neutrophils isolated from the bone marrow of sacrificed animals were cultured for 48 h without additional LT, and then treated for 30 min with or without a specific JNK inhibitor and subsequently stimulated with PMA. Superoxide production was assayed at various time intervals for 3 h as shown. A and B, Representative experiments from one of two independent experiments.

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Most studies to date investigating the effects of anthrax lethal toxin on neutrophils have involved the use of circulating human neutrophils, which produce α defensins (22, 23, 24, 25, 26, 27, 28, 29). α Defensins have been shown to reversibly inhibit the activity of anthrax LT, representing at least one of several potential confounding factors underlying seemingly contradictory results in published reports (29). In this respect, our studies using both bone marrow-derived and circulating neutrophils from mice have an advantage that murine neutrophils do not produce α defensins (30). In addition, our conclusions differ from those derived from the single other published report involving the direct effects of LT on superoxide production by murine neutrophils. This study was limited in that it only addressed the short-term (2 h) effects of LT, reporting variable and strain-specific effects of short-term LT treatment on superoxide production (41). In contrast, we also examined longer term effects of LT (e.g., 1–3 day of exposure), at which times we observed a marked enhancing effect on superoxide production in both C57BL/6 and BALB/c mice. Through a series of in vitro and ex vivo experiments using multiple stimuli in time-course experiments, we believe that we have provided clear evidence that the overriding effect of anthrax LT is to cause a substantial increase in the capacity of murine neutrophils to produce superoxide.

In addition to their implications regarding animal models for anthrax infection and toxemia, our findings have potential relevance for the understanding of human disease as well. Because early myeloid precursors do not express α defensins (42), they would be predicted to have a window of susceptibility to anthrax LT, whose effects could persist through maturation. Indeed, we find that neutrophil precursors differentiated in human bone marrow are susceptible to LT and are currently studying the effect of the toxin on their cellular functions (data not shown). Moreover, even in conditions where α defensins would be expected to be present, MKK cleavage has been reported in human circulating neutrophils treated with anthrax LT (28). Extended kinetics studies (i.e., involving long-duration LT treatment and long-duration superoxide measurement) in human neutrophils are warranted to determine whether there exists a parallel between our findings in murine neutrophils and effects on human neutrophils.

The effect of anthrax LT on neutrophil superoxide production occurs in cells from both relatively toxin-susceptible (BALB/c) and -resistant (C57BL/6) mouse strains, and it is generally characterized by a delayed, but more sustained increase in superoxide production in LT-treated neutrophils compared with vehicle-treated controls. In addition, it is notable that superoxide production in LT-exposed neutrophils is generally less than that in unexposed control neutrophils at very early time points following stimulation. This suggests that, in addition to differences in the species and stimuli studied, the timing of superoxide measurements could be another explanation for differences in our results compared with previous studies.

The observation that residual JNK signaling is required for superoxide production in LT-treated murine neutrophils provides a major basis for this toxin activity. Albeit reduced by ∼50%, this residual JNK signaling, in the context of ERK and p38 signaling blockade, supports markedly more superoxide production than that observed in untreated control cells. These findings are consistent with recent reports demonstrating a critical role for the JNK family in mediating TNF-α- and TLR2-dependent superoxide induction (38, 39, 43). The differential effects of anthrax LT on the primary MKKs upstream of the JNK family members, MKK4 (cleaved) and MKK7 (not cleaved), could alter the signal transduction activities characteristics of JNK via differential phosphorylation of Tyr185 vs Thr183 by MKK4 and MKK7, respectively (44), leading to increased superoxide production. Another mechanism for altering superoxide production involves the toxin’s effects on ERK MAPK signaling pathways. Chemical blockade of the MEK1/2/ERK pathway leads to delayed, but more sustained production of superoxide, similar to superoxide production kinetics in LT-treated cultures. These findings not only have relevance in the setting of anthrax toxemia, but they introduce a new scientific tool to dissect the relative roles of these MAPK signal transduction pathways in regulating superoxide production in settings outside of anthrax infection or toxemia.

It is also notable that our finding of the resistance of neutrophil MKK7 to anthrax LT-dependent cleavage in vitro and in vivo is in apparent conflict with earlier reports involving overexpression of MKK substrates (33, 34). Nevertheless, our observation is consistent with a previous study involving MAPK signaling in murine T cells. In this particular study, JNK signaling in T cells derived from mice treated with anthrax LT in vivo persisted, whereas the ERK and p38 pathways were blocked (12). As MKK4 and MKK7 are responsible for JNK phosphorylation (45), it would follow that at least one of these MKKs would have residual function despite anthrax LT exposure. The observed susceptibility of MKK4 to anthrax LT-mediated cleavage (Fig. 1,B) leaves MKK7 as the most likely candidate responsible for anthrax LT-resistant JNK signaling. Our data suggest the possibility that subtle differences in MKKs, which were not detectable in initial studies that involved over-expression of MKK targets, are nevertheless relevant in physiological settings with native protein. Another possibility is that differential susceptibility of MKK7 isoforms to cleavage by LT underlies the apparent discrepancy in MKK7 susceptibility (prior overexpression studies used murine MKK7β/human MKK7α, but not MKK7γ) (33, 34). In contrast to MKK7β, MKK7γ lacks one of two predicted LT cleavage sites. Our Western blots experiments (Fig. 2, A and B) likely do not resolve these isoforms, as they differ by only 16 aa. We can nevertheless conclude, however, that one or both of these isoforms is relatively resistant to LT cleavage and available to transduce signals to JNK.

Taken together, our results indicate that anthrax LT enhances the capacity of murine neutrophils to produce superoxide through differential effects on MAPK signaling. This conclusion leads to many questions, including the role of superoxide during anthrax infection and/or toxemia. Despite its well-recognized role in bacterial killing, there is evidence that superoxide might not always be host-protective during anthrax infection. Superoxide dismutase is a major constituent of the exosporium of Bacillus anthracis (46), suggesting the potential of pathogen resistance to this killing mechanism. Moreover, the flux of superoxide concentrations has been shown to promote endospore germination (47). Likewise, one might speculate that superoxide production during toxemia could have effects that are either beneficial or detrimental to the host. Although excessive superoxide production has been implicated in host damage in a variety of inflammatory settings (48), it is plausible that superoxide could be beneficial during toxemia (e.g., through neutralization of the toxin or down-regulation of other destructive neutrophil factors). It is also possible that neutrophil superoxide could have differential effects depending on both the level and the timing of its production during toxemia. In future studies, we plan to address these questions directly through experiments with mice that are deficient in the genes encoding the protein components of NADPH oxidase (e.g., p47 phox) (49). In addition, it is important to note that the finding that anthrax LT enhances superoxide production suggests the likely possibility that the production of other host-toxic neutrophil factors could also be increased by anthrax LT via the persistent JNK signaling capacity. Moreover, the role of LT-resistant JNK signaling in promoting proinflammatory effects on other cell lineages warrants investigation as well.

Anthrax LT treatment leads to a rapid shock-like death in animal models, yet no strain- and species-independent association of toxemia with the induction of the cytokines usually associated with septic shock has been demonstrated (IL-1β, TNF-α, IL-6, etc.) (17, 19, 50). This can be explained mechanistically by the LT-dependent disruption of MKK-dependent pathways regulating the induction of these cytokines. We herein have identified the mechanistic basis through which anthrax LT enhances the superoxide production capacity of neutrophils, via selective effects on MAPK signaling pathways. The next challenge will be to ascertain whether this dysregulation of MAPK signaling promotes other potentially toxic activities of neutrophils as well. Most importantly, it will be critical to determine whether LT-resistant MKK7/JNK signaling, not balanced by signaling through parallel MAPK pathways, represents a pathway mediating the pathogenic effects of this virulence factor.

We thank Julianne Cyr, Chikako Torigoe, Wen Jin Wu, Mate Tolnay, and Barbara Rellahan for helpful advice during the preparation of this manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

The information presented here reflects the views of the authors and does not necessarily represent the policy of the U.S. Food and Drug Administration.

3

Abbreviations used in this paper: LT, lethal toxin; PA, protective Ag; LF, lethal factor; MKK, mitogen-activated protein kinase kinase; Pam3, (Pam)3-Cys-Ser-(Lys)4.

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