No abstract

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage‐like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.

SummarySystemic pro-inflammatory cytokine release has been previously implicated as a major death-causing factor in anthrax, however, direct data have been absent. We determined the levels of IL-1 β β β β , IL-6 and TNF-α α α α in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis . More than 10-fold increase in the IL-1 β β β β levels was detected in Ames-challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL-1 β β β β response. Balb/c mice have also responded with higher levels of IL-6. The A/J mice demonstrated IL-1 β β β β and IL-6 systemic response to either Ames or Sterne strain of B. anthracis , whereas no increase in TNF-α α α α was detected in any murine strain. We used RT-PCR for gene expression analyses in the liver which often is a major source of cytokines and one of the main targets in infectious diseases. A/J mice challenged with B. anthracis (Sterne) showed increased gene expression for Fas, FasL, Bax, IL-1 β β β β , TNF-α α α α , TGF-β β β β , MIP-1 α α α α , KC and RANTES. These data favour the hypothesis that apoptotic cell death during anthrax infection causes chemokine-induced transmigration of inflammatory cells to vitally important organs such as liver. Administration of caspase inhibitors z-VADfmk and ac-YVAD-cmk improved survival in Sternechallenged mice indicating a pathogenic role of apoptosis in anthrax.

No abstract

No abstract