Molecular mechanisms of anthrax toxin-mediated edema

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The objectives of this project, which is supported by a MARGE Career Development Award, are: 1) to dentify the cell types through which ET initiates and sustains the release of mediators, such as histamine, prostanoids and neurokinins, that are involved in ET-mediated edema and; 2) determine if these same mediators are involved in/responsible for ET-induced death in mice. Although we have demonstrated that ET s able to elicit COX-2 expression, using HEK cells, we have not observed an increase in COX-2 mRNA over basal, in human monocyte-derived monocytes. On the other hand, ET elicits a small increase in PGE2 synthesis from RAW264.7 macrophages and the effect is synergistic with LPS-induced prostaglandin production in these cells. To address the hypothesis that the mediator cascade initiated by ET might begin with or include direct stimulation of Substance P (SP) release from neuronal cells, dorsal root ganglion (DRG) cells were isolated from rats and rabbits and exposed to ET in vitro. Thus far, we have not detected a statistically significant release of SP from either of these cells types. Based on our earlier work showing a histamine (H1) antagonist (pyrilamine) and an inhibitor of COX-2 (celecoxib) cause a reduction in edema produced in rabbit skin by intradermal ET, we have tested these to drugs for their effect on ET-induced mortality in of BALB/cJ mice. While neither drug had a significant effect alone, the combination resulted in a reduced mortality and prolonged time to death. In these studies, we noted however, that only ET composed of E. coli-derived EF had this effect and the EF produced in B.anthracis did not cause any mortality. In order to address that discrepancy, we obtained funding through an NIAID/BEI contract, for which we are doing a direct comparison of the ET from these two sources for enzymatic and in vitro toxin activities, as well as in vivo effects in mice and rabbits. This work is ongoing, but there appears to be a significant and reproducible difference in the toxin effects (both in vitro and in vivo) between these two preparations of EF. In the second year of this career development award, we will continue to study the effect of ET on COX-2 induction in different cell types, measure histamine release from mast cells (murine and human) that are exposed to the conditioned medium of ET-treated DRG cells, characterize the effects of pharmacological antagonists on ET-associated clinical signs, laboratory abnormalities, pathological changes, and death in mice and utilize COX-2 and preprotachykinin A-knockout mice, and mast cell-deficient mice to investigate the roles of prostanoids, substance P and mast cells in ET-induced murine pathology/mortality.