Thiopental-induced apoptosis in lymphocytes is independent of CD95 activation.
BACKGROUND
Barbiturate coma is used in patients with traumatic brain injury whenever increases in intracranial pressure remain unresponsive to less aggressive therapeutic regimens. However, barbiturate-mediated neuroprotection correlates with lymphopenia, which increases the risk of infection. The mechanisms by which barbiturates lead to lymphopenia remain to be determined.


METHODS
Freshly isolated human lymphocytes and Jurkat cells were incubated with the barbiturate thiopental for 24 and 48 h. Apoptosis was measured by fluorescein isothiocyanate-Annexin and propidium iodide staining, rhodamine 123 staining, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Caspase-3 activity was detected by Western blot and substrate cleavage assay.


RESULTS
Thiopental dose-dependently (5-500 microg/ml) increased apoptosis in Jurkat cells from basal levels (4.4 +/- 1.9%) to 29.7 +/- 2.8% after 24 h and 39.7 +/- 3.2% after 48 h, whereas in lymphocytes, thiopental-induced necrosis was observed. Parallel to apoptosis, thiopental dose-dependently increased caspase-3-like activity in Jurkat cells. However, the pan-caspase inhibitor z-VAD-fmk (20 microm) only marginally reduced thiopental-induced (250 microg/ml) apoptosis in Jurkat cells (20.2 +/- 2.5 to 17.2 +/- 2.5%) and necrosis in lymphocytes (39.2 +/- 7.5 to 30.7 +/- 14%). In contrast, anti-CD95-induced apoptosis in Jurkat cells (27.0 +/- 2.0%) was completely blocked by z-VAD-fmk (8.1 +/- 1.8%). Neither expression of CD95 on Jurkat cells nor pretreatment with a neutralizing anti-CD95 antibody influenced thiopental-induced apoptosis, indicating that thiopental induces apoptosis independently of the CD95 system. The nuclear factor kappaB inhibitor gliotoxin accelerated both thiopental- and CD95-mediated apoptosis, indicating a mutual control mechanism of thiopental- and CD95-induced apoptosis.


CONCLUSIONS
Thiopental directly induces cell death in lymphocytes and Jurkat cells by a CD95-independent mechanism.
Keel
Marius
M
Department of Trauma Surgery, Division of Trauma Surgery, University Hospital Zurich, Switzerland. marius.keel@usz.ch
Mica
Ladislav
L
Stover
John
J
Stocker
Reto
R
Trentz
Otmar
O
Härter
Luc
L
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes / Q000187:drug effects* / Q000473:pathology; D008928:Mitochondria / Q000187:drug effects / Q000473:pathology; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D013874:Thiopental / Q000494:pharmacology*; D019014:fas Receptor / Q000502:physiology*
D016328:NF-kappa B; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D013874:Thiopental