Macrophage Apoptosis by Anthrax Lethal Factor Through p38 MAP Kinase Inhibition

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Science  20 Sep 2002:
Vol. 297, Issue 5589, pp. 2048-2051
DOI: 10.1126/science.1073163


The bacterium Bacillus anthracis causes the death of macrophages, which may allow it to avoid detection by the innate immune system. We found that B. anthracis lethal factor (LF) selectively induces apoptosis of activated macrophages by cleaving the amino-terminal extension of mitogen-activated protein kinase (MAPK) kinases (MKKs) that activate p38 MAPKs. Because macrophages that are deficient in transcription factor nuclear factor κB (NF-κB) are also sensitive to activation-induced death and p38 is required for expression of certain NF-κB target genes, p38 is probably essential for synergistic induction of those NF-κB target genes that prevent apoptosis of activated macrophages. This dismantling of the p38 MAPK module represents a strategy used by B. anthracis to paralyze host innate immunity.

Bacillus anthracis, the causative agent of anthrax, has gained notoriety as a potential biowarfare and bioterrorism agent. During inhalation anthrax, the most lethal form of the disease, B. anthracis spores are engulfed by alveolar macrophages (1). The spores, however, survive phagocytosis and germinate within phagosomes, and the bacteria spread to regional lymph nodes and eventually the bloodstream. In this late stage of bacteremia, the infected individual is subjected to fatal systemic shock (2). For successful infection, B. anthracis must evade the host innate immune system by killing macrophages (3), a strategy used by other highly virulent bacteria (4). It is unclear how B. anthracis interacts with macrophages in such a contradictory manner.

Three proteins secreted by B. anthracis are central to its pathogenicity: protective antigen (PA), edema factor (EF), and lethal factor (LF) (5). By binding a specific cell-surface receptor, PA translocates EF and LF into the cytosol (6). EF is an adenylate cyclase that causes tissue edema (7), whereas LF is a metalloprotease that exhibits unique specificity toward MKKs, cleaving between their NH2-terminal extension and the catalytic domain (8). Because the NH2-terminal extension is required for interactions with both MAPKs and MKK kinases (MKKKs) (9), this cleavage prevents MAPK activation (10). Lethal toxin (LT), a complex of PA and LF, is the major factor responsible for the lethality of anthrax (1). Ex vivo, LT exhibits cytotoxicity toward macrophages, an activity likely to be important for evasion of host defenses (1,4). Together, all three toxin components also inhibit neutrophil migration and phagocytosis (11), further helping immune system evasion. A direct causal relation between dismantling of MAPK signaling and LT-mediated cytotoxicity is currently lacking. Although in culture, LT was mostly described as inducing macrophage necrosis (12), necropsy of inhalation anthrax victims revealed extensive macrophage apoptosis (13). Furthermore, mouse strains whose macrophages are resistant to LT-induced necrosis are more susceptible to B. anthracis infection (14), thus questioning the role of the necrotic response.

We sought physiologically relevant conditions under which LT could induce macrophage apoptosis. Treatment of J774A.1 macrophage-like cells with the protein phosphatase inhibitor calyculin A was found to sensitize them to LT-induced apoptosis (12). Treatment of different cells with such phosphatase inhibitors activates numerous protein kinases, including MAPKs (15) and inhibitor of NF-κB (IκB) kinase (IKK) (16). Hence, calyculin A could activate J774A.1 macrophages. We therefore tested the effect of the general macrophage activator lipopolysaccharide (LPS) on the response of J774A.1 cells and bone marrow–derived macrophages (BMDMs) to LT. Titration experiments revealed that in a manner strictly dependent on PA63 (17), a mature and active form of PA (18), LF caused rapid apoptosis of LPS-activated macrophages at 200 ng/ml, a suboptimal concentration for inducing necrosis in J774A.1 and BMDMs from C57BL/6 mice (Fig. 1, A, B, and F). No apoptosis was detected in resting (nonactivated) macrophages. In addition to LPS derived from Gram-negative bacteria, lipoteichoic acids (LTAs) from the Gram-positive bacteria Staphylococcus aureus and B. subtilis also induced apoptosis of LT-treated cells (Fig. 1C), suggesting that a similar component of B. anthracis, a Gram-positive bacterium, can activate macrophages and trigger apoptosis in the presence of LT. Apoptosis induced by LTAs was not inhibited by polymyxin B (17), indicating that it is not mediated by contaminating LPS. At 200 ng/ml, most of the LF-induced cell death in activated J774A.1 cells was apoptotic in nature, whereas in activated BALB/c BMDMs, only 50% of the observed cell death was due to apoptosis (Fig. 1, D and E). At higher concentrations, LT caused the necrotic death of both resting and activated macrophages, and the apoptotic response was attenuated. As previously reported (19), LT did not cause necrosis of C57BL/6 BMDMs. Nonetheless, LT effectively induced the apoptosis of these cells (Fig. 1F). Therefore, unlike necrosis (19), LT-induced apoptosis of activated macrophages is not confined to a subset of mouse strains.

Treatment of macrophages with LPS activates extracellular signal–regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPKs (20), as well as IKK and NF-κB (21). Titration experiments revealed that LF (together with PA63) inhibited ERK activation by LPS in BMDMs at a concentration as low as 40 ng/ml (Fig. 2A). Inhibition of JNK1 and p38 activation required a higher LF concentration (200 ng/ml), similar to that needed for induction of apoptosis in activated macrophages. However, LF did not inhibit JNK2 nor LPS-induced IκBα degradation, suggesting that it does not inhibit IKK activation. Using inhibitors that are selective for each MAPK cascade [PD98059, a MEK1/MEK2 inhibitor for ERK (22); SP600125 for JNK (23); SB202190 for p38 (24)], we examined the contribution of MAPK inhibition to LF-induced apoptosis of activated macrophages. Only treatment with the specific p38 inhibitor SB202190 induced apoptosis of LPS-treated BMDMs (Fig. 2B). SB202190 was not cytotoxic toward resting macrophages (17).

To determine whether cleavage of the MKKs responsible for p38 activation, MKK3 and MKK6 (25), is required for LF-induced apoptosis, we generated mutant forms of MKK3 and MKK6 that lack specific residues required for recognition by LF (26, 27). Both MKK3bR26Q/I27G(MKK3CR) and MKK6K14Q/I15G (MKK6CR) were resistant to LF cleavage (Fig. 2C). Stable expression of either mutant in RAW264.7 macrophages (which are more amenable to transfection than J774A.1) revealed that only MKK6CR partially protected p38 from inhibition by LF (Fig. 2D). Most importantly, the pooled population of MKK6CR-expressing cells exhibited considerable resistance to LF-induced apoptosis after activation (Fig. 2, E and F). Neither MKK6CR nor MKK3CR protected activated RAW264.7 cells from apoptosis induced by SB202190 (Fig. 2E). Thus, the ability of LT to induce apoptosis of activated macrophages depends on inhibition of p38 activation.

Even though LF does not inhibit the IKK to NF-κB pathway, survival of LPS-activated macrophages depends on IKK and NF-κB activation. Using a conditional Ikkβ allele in which exon 3, which encodes part of the kinase domain, was flanked by binding sites (loxp) for the Cre recombinase, we generated IKKβ-deficient myeloid cells by crossingIkkβloxp/loxp mice with mice that express a lysozyme M promoter–driven Cre recombinase (28). The frequency of Ikkβ deletion in BMDMs ofIkkβloxp/loxp LysM-Cremice was ∼50% (Fig. 3A), resulting in only a partial decrease in IKK (Fig. 3B) and NF-κB (Fig. 3C) activation in the mixed cell population. To circumvent difficulties associated with this heterogeneity, we examined LPS-induced apoptosis in individual macrophages and correlated it with the presence or absence of the p65 NF-κB subunit in the nucleus. These experiments revealed that LPS only induced apoptosis of those cells lacking nuclear p65 (Fig. 3D). No apoptosis was detected upon incubation ofIkkβloxp/loxp BMDMs lacking Cre with LPS. In addition, expression of a degradation-resistant form of IκB in RAW264.7 cells sensitized them to LPS-induced apoptosis (17).

The marked sensitivity of BMDMs that lack either p38 or NF-κB activity to activation-induced death suggests that p38 may be required to activate a transcription factor or recruit a coactivator that synergizes with NF-κB to induce transcription of a gene(s) whose product inhibits apoptosis. Previous analysis of NF-κB–mediated gene expression in dendritic cells revealed that p38 is required to induce some NF-κB target genes (29). To investigate this possibility, we used real-time polymerase chain reaction (PCR) (17) to examine the requirement of p38 for the expression of known NF-κB target genes in J774A.1 cells and BMDMs. Expression of many NF-κB–regulated genes, such as IκBα, iNOS, A20, and GADD45β, was effectively induced by LPS in untreated cells as well as in cells treated with either SB202190 or LT (Fig. 4A). However, expression of other NF-κB target genes, including those encoding interleukin-1α (IL-1α), IL-1β, andCOX-2, was induced by LPS in untreated macrophages but was inhibited by either SB202190 or LT. Expression of only one gene, that encoding tumor necrosis factor–α (TNF-α), was partially inhibited by LT but not by SB202190. Inhibition of LPS-induced TNF-α, IL-1α, and IL-1β expression by LT was observed previously (30). Because p38 is also involved in mRNA stabilization (25), we used nuclear run-off experiments to confirm that the effect of its inhibitor is transcriptional (Fig. 4B). On the basis of these results, we suggest that through phosphorylation of an as-yet unidentified target, p38 synergizes with NF-κB to induce the expression of a subset of target genes, which in macrophages includes inhibitors(s) of activation-induced death. Inhibition of either p38 or NF-κB is sufficient to sensitize macrophages to activation-induced death by preventing induction of this antiapoptotic factor.

Our results uncover a strategy by which B. anthracisparalyzes the innate immune system to promote its undisturbed spread toward systemic infection. By inhibiting activation of p38 MAPK, this deadly pathogen switches the signal for macrophage activation to a trigger of rapid cell death. Selective killing of activated macrophages prevents the secretion of chemokines and cytokines that alert the remainder of the immune system to the presence of the pathogen. This may explain why anthrax infections proceed undetected until the terminal stage, when vast bacteremia occurs. Future research should focus on the balance between macrophage activation and apoptosis, as it seems to play a key role in the pathogenesis of anthrax and other deadly infections.

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