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 immune 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 resistant to sepsis-induced T cell apoptosis, and mortality was decreased in sepsis. The purpose of this study was to identify key initiator and executioner "caspases" involved in sepsis-induced lymphocyte apoptosis and to determine 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 activity 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 conjugated 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 activation of caspases -2, -3, -6, and -9 in comparison with thymocytes of sham-operated mice. Caspase-1 was not activated. BCL-2 prevented sepsis-induced thymocyte apoptosis and inhibited activation of all caspases. We conclude that sepsis causes activation of multiple caspases and that BCL-2 acts upstream as an inhibitor of caspase activation. The pattern of caspase activation suggests a mitochondrial mediated pathway.
Tinsley
K W
KW
Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Cheng
S L
SL
Buchman
T G
TG
Chang
K C
KC
Hui
J J
JJ
Swanson
P E
PE
Karl
I E
IE
Hotchkiss
R S
RS
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002432:Cecum; D004789:Enzyme Activation; D005260:Female; D019254:Genes, bcl-2; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D018805:Sepsis / Q000473:pathology / Q000503:physiopathology*; D013601:T-Lymphocytes / Q000473:pathology / Q000502:physiology*
D017304:Annexin A5; D019253:Proto-Oncogene Proteins c-bcl-2; C505409:Casp3 protein, mouse; C505516:Casp6 protein, mouse; C506434:Casp9 protein, mouse; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6; D053453:Caspase 9; D020169:Caspases