Anthrax lethal toxin induces ketotifen-sensitive intradermal vascular leakage in certain inbred mice

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.