Exotoxins produced by anthrax bacilli are believed to be responsible for overt shock symptoms and death in infected animals. Cytolysis of macrophages caused by anthrax lethal toxin (LeTx) is a trigger of shock symptoms and death. LeTx can directly lyse macrophages of some mouse strains. However, the sensitivity of different murine macrophages to LeTx in vitro does not correlate with in vivo susceptibility of corresponding strains to B. anthracis. This suggests that there are factors other than LeTx also contributing to the cytolysis of macrophages. The death of LeTx-resistant macrophages needs to be studied because LeTx alone cannot kill human macrophages in vitro. The long-term goal of our study is to understand the molecular mechanisms that lead to the death of LeTx-resistant macrophages in anthrax infection. We have found that treatment of macrophages with bacterial components can make LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis, suggesting that the death of LeTx-resistant macrophages requires two stimuli. We further determined that tumor necrosis factor-alpha (TNF) induced by bacterial components is at least one of the factors that can cooperate with LeTx in inducing macrophage death. In addition, mTor (mammalian target of rapamycin) signaling was found to be required for the death of LeTx-resistant macrophages. Although anthrax bacilli can escape phagocytosis by macrophages, they should activate macrophages to certain levels. We believed the autocrine effect of TNF plays a key role in LeTx-resistant macrophage death in vivo. In supporting this notion, it has been reported that administration of anti- TNF antibody improved survival of anthrax-infected C57BL/6 mice. This proposal will focus on the mechanisms of the cell death in LeTx-resistant macrophages. The death of LeTx-resistant macrophages will be addressed from the side of macrophage activation. The signaling pathways that cooperatively operate in causing LeTx-resistant macrophage death will be elucidated by biochemical and molecular biology approaches. The information obtained in this study will be very valuable in developing new strategies for the treatment of anthrax infection. [unreadable] [unreadable]