Monocytes are highly sensitive to Clostridium difficile toxin A-induced apoptotic and nonapoptotic cell death

In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes to Clostridium difficile toxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to > 100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochrome c was released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 mug/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated that C. difficile toxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.