Caspases-2,-3,-6, and-9, but not caspase-1, are activated in sepsis-induced thymocyte apoptosis

Sepsis induces extensive lymphocyte cell death that may contribute to immun e depression and morbidity/mortality in the disorder, bcl-2 is a member of a new class of oncogenes that prevents cell death from an array of noxious stimuli. Transgenic mice that overexpress BCL-2 in T lymphocytes are resist ant to sepsis-induced T cell apoptosis, and mortality was decreased in seps is, The purpose of this study was to identify key initiator and executioner "caspases" involved in sepsis-induced lymphocyte apoptosis and to determin e if BCL-2 acts prior to caspase activation. Thymi were removed 5-22 h post -cecal ligation and puncture (CLP) or sham surgery. Apoptosis was evaluated in thymocytes by annexin-V FITC labeling and flow cytometry, Caspase-1 act ivity was determined by western blot analysis of the procaspase protein and p20 subunit of the activated caspase; activities of caspases -2, -6, and - 9 were determined by colorimetric assays using specific substrates conjugat ed to a color reporter molecule. Caspase-3 activity was determined both by western blot and by a fluorogenic assay in which a fluorescent compound was generated. Thymocytes from CLP mice had markedly increased apoptosis and a ctivation of caspases -2, -3, -6, and -9 in comparison with thymocytes of s ham-operated mice. Caspase-1 was not activated. BCL-2 prevented sepsis-indu ced thymocyte apoptosis and inhibited activation of all caspases. We conclu de that sepsis causes activation of multiple caspases and that BCL-2 acts u pstream as an inhibitor of caspase activation. The pattern of caspase activ ation suggests a mitochondrial mediated pathway.