Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70

Roesslein M, Schibilsky D, Muller L, Goebel U, Schwer C, Humar M, Schmidt R, Geiger KK, Pahl HL, Pannen BH, Loop T
Source: J Pharmacol Exp Ther
Publication Date: (2008)
Issue: 325(1): 217-25
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Species: human
Tissue Origin: blood
Nucleofectorâ„¢ I/II/2b
Jurkat T cells in logarithmic growth phase were washed in phosphate buffered saline. 2.5 x 10^6 - 5 x 10^6 cells were transfected with 6 μg hspA1A siRNA directed against hsp70-mRNA or nonsilencing siRNA. Transfection efficiency was determined by Alexa fluor 488 labelled hspA1A-siRNA and was > 90%.
Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. While all barbiturates inhibit T cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor kappaB (NF-kappaB). Various pharmacologic inhibitors of the NF-kappaB pathway are concomitant non-thermal inducers of the heat shock response (HSR), a cellular defense system, which is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3(+) T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering (si) RNA targeting hsp70 before thiopental incubation. Apoptosis was induced by staurosporine. DNA-binding activity of HSF-1 was analyzed by "Electrophoretic Mobility Shift Assay", mRNA-expression of hsp-27, -32, -70 and -90 by "Northern Blot" and protein-expression of Hsp70 by "Western Blot" and flow cytometry after fluorescein isothiocyanate (FITC)-Hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp-27, -70 and -90 but not hsp-32 mRNA expression as well as Hsp-70 protein expression. Thiopental dose-dependently activated the DNA-binding activity of HSF-1, while other substances investigated had no effect. In addition, pre-treatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental-treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response and mediates cytoprotection via the expression of Hsp70 in human T lymphocytes.