Anthrax Lethal Toxin Induces Ketotifen-Sensitive Intradermal Vascular Leakage in Certain Inbred Mice

Abstract

Bacillus anthracis lethal toxin (LT) is a bipartite toxin composed of protective antigen (PA) and lethal factor (LF). Injection of LT produces clinical signs characteristic of anthrax infection, including pleural edema and vascular collapse in various animal models. We utilized the classic Miles leakage assay to quantify vascular leakage in mice. LT injected intradermally induced leakage as early as 15 to 25 min in some inbred mouse strains, but not in others, whereas PA or LF individually did not induce leakage. A third component of anthrax toxin, edema factor, did not induce leakage alone or with PA. Leakage was quantified in eight mouse strains, and no correlation was found between sensitivity to intradermal leakage and sensitivity to the lethality of systemically administered LT. The leakage could be inhibited by ketotifen, an inhibitor of mast cell degranulation, but not by azelastine, a histamine receptor 1 antagonist, or by ketanserin, a serotonin 5-HT2A receptor antagonist. LT was cytotoxic to MC/9 mast cells (in vitro) by 7 h after toxin treatment but did not induce histamine release from these cells. Mast cell-deficient mice exhibited the leakage event and had no increased resistance to systemic LT. Human umbilical vein endothelial cells were resistant to LT over 12 h, with only 20% of cells succumbing by 24 h, suggesting that endothelial cell killing is not the cause of the rapid LT-mediated leakage event. We describe here a ketotifen-sensitive vascular leakage event induced by LT which is the most rapid in vivo or in vitro LT-mediated effect reported to date.Anthrax, the disease caused by Bacillus anthracis, is a worldwide bioterrorism concern. Anthrax toxin, a major virulence factor of this organism, consists of three polypeptides: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is required for binding and translocation of EF and LF into target cells (5). The injection of lethal toxin (LT is LF plus PA) into animals is sufficient to induce some symptoms of anthrax infection, including pleural effusions indicative of vascular leakage and lethality (3,4,6,10,23,24,28). Early studies suggested that LT kills animals by inducing nonspecific shocklike manifestations (3,36), and recent studies with mice and rats have confirmed an LT-mediated cytokine-independent vascular collapse (6, 28). Humans and primates infected by aerosol exposure to spores present pleural effusions as the most common symptom of disease (8,20,40). Histopathological analyses of human subjects with inhalational anthrax infections as well as studies of nonhuman primates and other animals show hemorrhaging in various organs resulting from destruction of both large and small vessels (1,7,11,15,16,38). However, while observations of pulmonary edema, inflammation, endothelial necrosis, vessel inflammation, increased vascular permeability, and hemorrhage have been associated with bacterial infection, they are not seen in systemic models of mouse and rat LT toxicity (6, 28). Clearly, LT is a single virulence factor in t...

Anthrax lethal toxin induces cell death-independent permeability in zebrafish vasculature

“…Recent studies using endothelial cells in culture have reported LeTx induced apoptosis or barrier dysfunction, depending on the particular endothelial cell type and treatment (12,16,17). Our model exposes endothelial cells to LeTx within the context of intact, lumenized, and functional blood vessels.…”

“…Early reports indicated that EdTx did not produce significant mortality (9), but recently robust effects have been reported, including lethality in rodents (10,11). However, the ability to induce loss of vascular integrity and leakage has been consistently associated with LeTx (7,(10)(11)(12).…”

“…Both receptors mediate anthrax toxin internalization and intracellular delivery of LF, and are expressed in many cell types including endothelial cells (12,16,17).…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…Vascular leakage in mouse models shows similar results using a Miles assay, which uses intravenously-delivered Evan's blue dye to quantify macromolecular vascular leakage. Subcutaneous injection of purified LT results in a rapid (15-25 minutes) and dose dependent response in mice [255]. This rapid response to LT suggests that vascular leakage is due to a transcription/translation independent event.…”

“…The fact that ketotifen, a histamine inhibitor, can greatly reduce the amount of leakage lends credence to this idea, though it is uncertain via which mechanism or on which cell ketotifen is functioning. The ability and degree of leakage varies between inbred mice strains and has no correlation to strain susceptibility to LT-mediated death [255]. Therefore, LT mediated death may be due both to vascular leakage and the host's response to the leakage.…”