Anthrax Lethal Toxin Rapidly Activates Caspase-1/ICE and Induces Extracellular Release of Interleukin (IL)-1β and IL-18

Abstract

Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been demonstrated to cleave and to inactivate mitogen-activated protein kinase kinases (MAPKKs) that propagate prosurvival signals in macrophages (1-5). Whether this action of anthrax LT leads to the production of proinflammatory cytokines by macrophages has been more controversial (6, 7). We now report that anthrax LT treatment leads to the specific extracellular release of interleukin (IL)-1␤ and IL-18 by the murine macrophage cell lines, RAW264.7 and J774A.1. Studies of the processing of IL-1␤ reveal that the levels of activated/cleaved IL-1␤ in RAW264.7 and J774.A1 cells are increased following treatment with anthrax LT. Enhanced processing of IL-1␤ directly correlates with increased levels in the activation of its upstream regulator, IL-1␤-converting enzyme/Caspase-1 (ICE). The extracellular release of IL-1␤ and IL-18 in response to anthrax LT is ICE-dependent, as an ICEspecific inhibitor blocks this process. These data indicate that ICE, IL-1␤, and IL-18 are downstream effectors of anthrax LT in macrophages, providing the basis for new bioassays for anthrax LT activity and representing potential therapeutic targets.Patients with anthrax infection recognized at late stages have high mortality even with appropriate antibiotic therapy (8), which is likely due to the effects of bacterial toxins that persist following death of the pathogen. One of these toxins, anthrax LT, 1 comprises anthrax protective antigen (PA) and anthrax lethal factor (LF). Anthrax PA binds target cells and allows entry of the enzymatically active anthrax LF (9). LF, in turn, inactivates mitogen-activated protein kinase kinases (MAPKKs) through cleavage at specific recognition sites (1-5).MAPKKs are critical intermediates in signal transduction cascades that ultimately lead to activation of the NF-B family of transcription factors that promote macrophage survival (4). Although some of the elements underlying the mechanism of action of anthrax LT-induced apoptosis have now been elucidated, the etiology of species-and cell-specific differences in sensitivity to anthrax LT remains unclear. In addition, the role of other downstream effectors, such as cytokines, is disputed. In this regard, seemingly contradictory reports have been published that either support or reject roles for proinflammatory cytokines in responses to anthrax LT in vitro (6 -7). We now report that anthrax LT treatment induces rapid activation of ICE, a caspase family enzyme responsible for the processing of IL-1␤ and IL-18 into active forms. Anthrax LT treatment results in the extracellular release of IL-1␤ and IL-18 by murine macrophage cell lines in a manner dependent upon the activation of ICE. Studies of the action of anthrax lethal toxin on the regulation of IL-1␤ reveal that anthrax lethal toxin likely enhances both the cleavage of cytokine proforms and the release of intracellular stores of preprocessed cytokine. These data provide the basis for developing new anthrax LT bioassays an...

Inflammatory caspases and inflammasomes: master switches of inflammation

“…In mouse macrophages, LeTx can cause rapid apoptosis that requires caspase-1 activation. 98 Macrophages from inbred mice are either susceptible or resistant to apoptosis by LeTx. This trait difference has been mapped to a locus on chromosome 11 named Ltxs1, and was recently associated with NALP1b gene.…”

Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

“…32 However, the theory that Nalp1b and caspase-1 control LT-induced necrosis in murine macrophages is challenged by studies using caspase-1 inhibitors, which failed to block LT killing. 18,36,37 Here we show that macrophages derived from C57BL/6, DBA/2 and AKR strains, previously described as "resistant" to LT-mediated cell killing, are actually susceptible to LT-induced cell death by apoptosis. This finding appears to be applicable in vivo, as LT triggered depletion of splenic macrophages in C57BL/6 mice.…”

“…This is consistent with previous reports of IL-18 processing in LT-treated macrophages. 3,36 Active caspase-1, which has been linked to LT killing, 32,35 is required for IL-18 processing. Notably, IL-18 processing peaked 1-hour post-LT exposure (Fig.…”

Anthrax Lethal Toxin Blocks MAPK Kinase-Dependent IL-2 Production in CD4+ T Cells

“…These protein extracts were electrophoretically separated and then transferred onto 0.2-m nitrocellulose membranes (Bio-Rad). Western blotting was performed using standard techniques as previously described (12). The following primary Abs were used for Western blotting assays: anti-MEK1 (BD Biosciences); anti-MEK2 and anti-␤-actin (Santa Cruz Biotechnology); anti-PLC-␥1, anti-phospho-PLC-␥1, anti-p44/42 MAPK, anti-phospho-p44/42 MAPK, antiphospho-JNK, anti-JNK, anti-phospho-p38 MAPK, and anti-p38 MAPK (Cell Signaling Technology).…”

“…Validated anthrax LT bioassays that are based on human cell parameters would fill a critical void in the development of anthrax therapeutics. Anthrax LT is known to be a potent modulator of cytokine signaling networks, either enhancing (11,12) or inhibiting (7,13,14) production and/or extracellular release of proinflammatory cytokines from APCs depending on the stimulation conditions. Because MAPKK-dependent cytokine production is a feature common to a variety of cell types from anthrax-susceptible species, we investigated whether modulation of cytokine production by anthrax LT in non-APC could form an alternative basis for anthrax LT bioassays.…”