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

Authors:
Roesslein M, Schibilsky D, Muller L, Goebel U, Schwer C, Humar M, Schmidt R, Geiger KK, Pahl HL, Pannen BH, Loop T
In:
Source: J Pharmacol Exp Ther
Publication Date: (2008)
Issue: 325(1): 217-25
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Jurkat
Species: human
Tissue Origin: blood
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
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%.
Abstract
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.