Contributions of histamine, prostanoids, and neurokinins to edema elicited by edema toxin from Bacillus anthracis

Abstract

Bacillus anthracis edema toxin (ET), composed of protective antigen and an adenylate cyclase edema factor (EF), elicits edema in host tissues, but the target cells and events leading from EF-mediated cyclic-AMP production to edema are unknown. We evaluated the direct effect of ET on several cell types in vitro and tested the possibility that mediators of vascular leakage, such as histamine, contribute to edema in rabbits given intradermal ET. ET increased the transendothelial electrical resistance of endothelial monolayers, a response that is mechanistically inconsistent with the in vivo vascular leakage induced by ET. Screening of several drugs by intradermal treatment prior to toxin injection demonstrated reduced ET-induced vascular leakage with a cyclo-oxygenase inhibitor (indomethacin), agents that interfere with histamine (pyrilamine or cromolyn), or a neurokinin antagonist (spantide). Systemic administration of indomethacin or celecoxib (cyclo-oxygenase inhibitors), pyrilamine, aprepitant (a neurokinin 1 receptor antagonist), or indomethacin with pyrilamine significantly reduced vascular leakage associated with ET. Although the effects of pyrilamine, cromolyn, or aprepitant on ET-induced vascular leakage suggest a possible role for mast cells (MC) and sensory neurons in ET-induced edema, ET did not elicit degranulation of human skin MC or substance P release from NT2N cells in vitro. Our results indicate that ET, acting indirectly or directly on a target yet to be identified, stimulates the production/release of multiple inflammatory mediators, specifically neurokinins, prostanoids, and histamine. These mediators, individually and through complex interactions, increase vascular permeability, and interventions directed at these mediators may benefit hosts infected with B. anthracis.

FIG. 1.

ET-induced dye leakage is dose/time dependent. The mean BLSA (▪) for each ET dose is shown; error bars represent SEM. (A) ET dose response. Three rabbits were used, with six sites per dose. P was <0.05 for comparisons between all ET doses except with the 10-ng and 100-ng responses. (B) ET-induced dye leakage increases over time. The ET dose at each time point was 10 μg. Numbers above data points represent the number of independent observations at each time point. P was <0.05 for comparisons between all time points. (C) Transilluminated skin demonstrating the appearance of ET-induced dye leakage 180 min after toxin injection. Top lesion, ET (1 μg); bottom lesion, ET (10 μg). The white bar represents 1 cm.

FIG. 2.

ET and ACT both increase TER in HUVEC monolayers. Data points, means; error bars, SEM. Five experiments were performed. iET, PA plus EF (K346R); ACT, AC toxin from Bordetella pertussis. P was <0.05 for the following comparisons: ET versus the control at 90 and 120 min; ACT versus the control at 20, 30, 60, 90, and 120 min; and ET versus ACT at 20 and 30 min only. Results for iET versus the control were not significant at any time point.

FIG. 3.

ET-induced dye leakage is associated with, but not dependent on, perivascular neutrophil infiltration. (A) Photomicrograph of dermis (H+E; magnification, ×20) 180 min after i.d. injection of ET (10 μg). Arrows highlight PMN margination and infiltration in the perivascular space. (B) Graph of BLSAs over time in neutropenic (▴) (n = 3) and nonneutropenic (□) (n = 5 at 120 min, n = 31 at 180 min) rabbits. Data points, means; error bars, SEM; *, P was not significant.

FIG. 4.

Effects of local (i.d.) antagonists (A) and systemic (oral or i.v.) antagonists (B) on ET-induced BLSA. IND, indomethacin; PYR, pyrilamine; CROM, cromolyn; RAN, ranitidine. See Results for specific drug doses/routes. Numbers above bars represent the number of independent observations for each condition. P values refer to each individual condition in comparison to ET alone (control). See Results for specific P values. All ET doses were 10 μg/site.

FIG. 5.

ET does not elicit human skin MC degranulation. The y axis represents the percentage of β-hexosaminidase release from primary human skin MCs for each condition. 22E7 mAb, MAb for FcɛRI; 22E7 mAb+ET, MCs exposed to ET (1 μg/ml) for 45 min prior to addition of 22E7 antibody. *, P was 0.0007 for comparison to vehicle control; #, P was not significant for comparison to vehicle control; @, P was not significant for comparison to 22E7 MAb alone. Two experiments were performed, each in triplicate.