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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Thiopental Protects Human T Lymphocytes from Apoptosis in Vitro via the Expression of Heat Shock Protein 70

Department of Anesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany (M.R., D.S., L.M., U.G., C.S., M.H., R.S., K.K.G., H.L.P., T.L.); and Department of Anesthesiology and Critical Care Medicine, University Hospital, Duesseldorf, Germany (B.H.J.P.)

Received for publication October 16, 2007
Accepted January 22, 2008.

	Abstract

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Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-B (NF-B). Various pharmacologic inhibitors of the NF-B pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that 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 RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed 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 hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment 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 it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.

Although the association between barbiturate therapy, decreased intracranial hypertension, and anti-inflammatory effects is clinically well documented, it is unclear whether the protective actions reflect a specific effect of barbiturates. Moreover, the underlying molecular mechanisms remain to be identified.

The so-called heat shock response (HSR) is a cellular defense system that is highly conserved throughout evolution (Welch, 1992; Malhotra and Wong, 2002). It can be found in a wide spectrum of organisms ranging from prokaryotes to human beings. The underlying molecular mechanism is based on the ability of the transcription factor heat shock factor (HSF)-1 to display inducible DNA binding activity to the consensus heat shock element (HSE) and involves multiple steps, including nuclear translocation, oligomerization, and inducible serine phosphorylation of HSF-1, the latter being an important determinant of the transactivating potency of HSF-1 (Westerheide and Morimoto, 2005). The HSR is characterized by the expression of heat shock proteins (hsps), which are synthesized by cells in response to heat, hence the name, as well as to various other stressful stimuli (De Maio, 1999; Kregel, 2002). Expression of hsps, which are classified by their function and size, has been shown to protect cells from a broad range of cellular stressors, such as hypoxia, oxygen radicals, endotoxin, infections, and fever (De Maio, 1999). The cytoprotective capacity of heat shock proteins may be attributed in part to their ability to stabilize intracellular protein structures, which allows cells and organisms facing life-threatening insults resumption of normal cellular and physiological activities (De Maio, 1999).

Several links have been established between the HSR and the NF-B pathway. The existing reciprocity between these two pathways is underlined by the fact that various pharmacologic inhibitors of the NF-B pathway, such as geldanamycin, dexamethasone, acetylsalicylic acid, and bimoclomol are inducers of the HSR (Malhotra and Wong, 2002). In contrast, induction of the HSR by either hyperthermia or nonthermal inducers before a proinflammatory stimulus was shown to inhibit subsequent inflammatory responses such as the mononuclear cell expression of tumor necrosis factor- and interleukin-1β (Schmidt and Abdulla, 1988; Snyder et al., 1992).

We have recently reported that thiopental inhibits the activation of NF-B in human CD3⁺ T cells (Loop et al., 2002, 2003). Therefore, the hypothesis of this study was to determine whether thiopental would also be able to mediate cytoprotection in these cells by inducing the HSR.

	Materials and Methods

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Reagents. The following anesthetics were used: etomidate (Braun, Melsungen, Germany), s-ketamine (Parke Davis, Berlin, Germany), midazolam (Hoffman-La Roche, Grenzach-Wyhlen, Germany), propofol (Astra-Zeneca, Plankstadt, Germany), and thiopental (Altana, Konstanz, Germany). The remaining reagents were purchased from Sigma Chemie (Deisenhofen, Germany) unless specified otherwise.

Isolation of CD3⁺ T Lymphocytes and Cell Culture. Peripheral blood mononuclear cells were isolated from blood bank product buffy-coats obtained from healthy donors requiring no further Institutional Review Board consent. The cells were acquired by using density centrifugation on Ficoll-Hypaque (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's recommendations. The cells were microscopically analyzed, and then they were counted in a Neubauer chamber. For the isolation of CD3⁺ T lymphocytes, the peripheral blood mononuclear cells (3–4 x 10⁸) were incubated for 15 min on ice with anti-CD3 antibodies conjugated to magnetic beats (Miltenyi Biotec, Bergisch-Gladbach, Germany). Separation of CD3⁺ cells was performed using an L/S column (Miltenyi Biotec), and it was confirmed by flow cytometry (>85% purity).

Cell Culture and Transfection of Jurkat T Cells. Jurkat T cells (ACC 282; DSMZ, Braunschweig, Germany) were cultured in RPMI 1649 medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum, 2 mM glutamine, and 100 U/ml penicillin G and streptomycin (all obtained from Invitrogen, Carlsbad, CA) each. Jurkat T cells in logarithmic growth phase were washed in phosphate-buffered saline. Cells (2.5 x 10⁶–5 x 10⁶) were transfected with 6 µg of hspA1A small interfering RNA (siRNA) directed against hsp70-mRNA or nonsilencing siRNA (both obtained from QIAGEN GmbH, Hilden, Germany) according to the optimized protocol for Jurkat T-cells using the Cell Line Nucleofector Kit V and a Nucleofector II (Amaxa, Köln, Germany). After transfection, cells were cultured in 5 ml of prewarmed medium and six-well tissue culture plates for 18 to 20 h before treatment.

Total Cell Extracts and Electrophoretic Mobility Shift Assays. Cells were harvested by centrifugation, they were washed once in ice-cold phosphate-buffered saline, and then total cell extracts were prepared as described previously (Loop et al., 2002). Total cell extracts of CD3⁺ T lymphocytes (1 x 10⁷ cells/sample) were used for electrophoretic mobility shift assays. Inhibitors of proteinases and phosphatases were added at concentrations to the extraction and suspension buffer as described previously (Loop et al., 2002). Band-shift assays were performed using a ³²P-labeled HSF-1 oligonucleotide (Promega, Madison, WI). The kinase reaction consisted of 37 µl of purified water, 25 ng of HSF-1 oligonucleotides, 5 µl of kinase buffer, 5 µl of [-³²P]dATP (GE Healthcare), and 1.5 µl of T4 kinase (PNK buffer and PNK T4 kinase; New England Biolabs, Schwalbach, Germany), and it was incubated for 30 min at 37°C. The protein content of the cell lysates was determined using a Bradford assay system (Bio-Rad Laboratories, München, Germany), and equal amounts of protein (30 µg) were added to a 20-µl electrophoretic mobility shift assay reaction mixture containing 20 µg of bovine serum albumin, 2 µg of poly(dI-dC) (Roche Diagnostics, Mannheim, Germany), 2 µl of buffer D+ (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM dithiothreitol, and 0.1% phenylmethylsulfonyl fluoride), 4 µl of 5x Ficoll buffer (20% Ficoll 400, 100 mM HEPES, 300 mM KCl, 10 mM dithiothreitol, and 0.1% phenylmethylsulfonyl fluoride), 1 µl of 50 mM MgCl₂, 3 µl of double-distilled H₂O, and 1 µl of HSF-1 ³²P-labeled oligonucleotide. These samples were incubated at room temperature for 30 min, and then they were loaded on a 4% acrylamide gel in 0.5x Tris borate-EDTA (900 mM Tris-HCl, 900 mM boric acid, and 20 mM EDTA, pH 8.0), 400 µl of ammonium persulfate, and 40 µl of tetramethylethylenediamine. For the supershift assays, 2.5 µl of antibody HSF-1 (clone 10H8, SPA-950E; Assay Designs, Ann Arbor, MI/BIOMOL, Hamburg, Germany) or p65 (clone C20, SC-372X; Santa Cruz Biotechnology, Heidelberg, Germany) were added to the reaction simultaneously with the protein and incubated as described. Gels were vacuum dried (Gel dryer 543; Bio-Rad, Hercules, CA) for 30 min, and then they were exposed to X-ray film (Kodak, Stuttgart, Germany).

RNA Isolation and Northern Blot Analysis. Total RNA was extracted from approximately 2 x 10⁷ CD3⁺ T lymphocytes/sample according to the manufacturer's recommendation (a monophasic one-step solution of phenol and guanidine isothiocyanate; Invitrogen). Aliquots of total RNA (10 µg/lane) were size-fractionated on a denaturating 1% agarose gel, they were transferred to a nylon membrane (Hybond-N; GE Healthcare) by capillary blotting in 20x sodium saline citrate (3 M NaCl and 0.3 M sodium citrate), and then they were cross-linked to the membrane by UV irradiation. The membrane was preincubated for 30 min in hybridization solution (ExpressHyb; Clontech, Palo Alto, CA), and then they were incubated overnight at 68°C with a ³²P-labeled (Prime-It II labeling kit; Stratagene, La Jolla, CA) probe. The probes consisted of hsp27, hsp32, hsp70, and hsp90 cDNA fragment. All blots were stripped and reprobed with an 18S ribosomal RNA cDNA to confirm equal loading. The following specific primers were used: hsp27, 5'-ACGTCAACCACTTCGCTCCTGAGG-3' as upper primer and 5'-CTTGGCTCCAGACTGTTCCGAACTC-3' as lower primer; hsp70, 5'-CAG CGG CAG GCC ACC AAG GAC-3' as upper primer and 5'-TGC ACC GCC GCC CCG TAG G-3' as lower primer; hsp90, 5-GCG AGT CGG ACG TGG TCC-3 as upper primer and 5-CTG AGG GTT GGG GAT GAT GTC-3 as lower primer; and 18S, 5'-CGC CGC GCT CTA CCT TAC CTA CCT-3' as upper primer and 5'-GAC CGC CCG CCC GCT CCC AAG AT-3' as lower primer.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. Total cell extracts of 1 x 10⁷ cells/sample were boiled in Laemmli sample buffer, and then they were subjected to 10% SDS-polyacrylamide gel electrophoresis as described previously (Loop et al., 2002). Proteins were transferred to Immobilon P membranes (Millipore Corporation, Eschborn, Germany). Equal loading was confirmed by stripping membranes and incubating them with specific antibodies at the end of the procedure. Primary antibodies used for Western blotting were hsp70 (clone C92F3A-5, SPA-810; Assay Designs/BIOMOL; dilution 1:1000), heat shock cognate protein (Hsc)70 (polyclonal, SPA-816; Assay Designs/BIOMOL; dilution 1:1000), HSF-1 (polyclonal, SPA-901; Assay Designs/BIOMOL; dilution 1:1000), and β-actin (polyclonal, 4967; Cell Signaling Technology Inc., Beverly; dilution 1:1000). Nonspecific binding sites were blocked by immersing the membrane into blocking solution [20 mM Tris-HCl, pH 7.6, 0.1% Tween 20 with 5% (w/v) nonfat dry milk powder (Fluka, Buchs, Switzerland)]. Membranes were washed in 20 mM Tris-HCl, pH 7.6, plus 0.1% Tween 20, and then they were incubated in a recommended dilution of specific antibodies. Bound antibody was detected by goat anti-rabbit/horseradish peroxidase-conjugated secondary antibody (7074; Cell Signaling Technology Inc.; dilution 1:2000). The immunocomplexes were detected using enhanced chemiluminescence Western blotting reagents (GE Healthcare) according to the manufacturer's instructions. Exposure to enhanced chemiluminescence Western blot films (GE Healthcare) was performed for 15 s to 1 min.

Fluorogenic Caspase Activity Assay. Total protein cell extracts of 1 x 10⁷ cells per sample (10 µl) were mixed with 90 µl of assay buffer (100 mM HEPES, pH 7.5, 2 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride). The respective fluorogenic substrate for caspase-3 and -7, 7-amino-4-methylcoumarin (1 µl, 60 µM; Alexis Corporation, Gruenberg, Germany), was added and the fluorescence was measured at 30°C for 30 min in a Microplate Spectra Max Gemini XS reader (Molecular Devices, Sunnyvale, CA) at 380/460 nm.

Flow Cytometric Analysis of CD3⁺ T Lymphocytes. After the experimental treatment, CD3⁺ T lymphocytes (1 x 10⁵ cells/sample) were harvested by centrifugation, they were washed in phosphate-buffered saline, and then they were stained in annexin binding buffer containing annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (all obtained from BD Biosciences, Heidelberg, Germany) or with a monoclonal FITC-conjugated hsp70 antibody (clone C92F3A-5, SPA-810F; Assay Designs/BIOMOL) using a 1:40 dilution for 30 min according to the manufacturer's recommendations. Subsequently, the percentage of early apoptotic lymphocytes was measured using a flow cytometer (FACSCalibur; BD Biosciences). Lymphocytes were gated using a forward/side scatter, and fluorescence intensity was measured in 1 x 10⁴ lymphocytes/sample in fluorescence channel (FL)1 for annexin V and FL2 for propidium iodide.

Cell Staining and Multiparameter Flow Cytometry of Jurkat T Cells. Cells were harvested by centrifugation, they were washed in phosphate-buffered saline, and then they were permeabilized using the Cytofix/Cytoperm kit (BD Biosciences). The permeabilized cells were stained with a monoclonal FITC-conjugated hsp70 antibody (BD Biosciences) using a 1:40 dilution for 30 min. Transfection efficiency of the used siRNAs estimated by Alexa Fluor 488-labeled hspA1A-siRNA was >90% (data not shown).

For the detection of apoptosis and necrosis, cells were harvested by centrifugation, they were washed in phosphate-buffered saline, and then they were stained in annexin binding buffer containing annexin V-phycoerythrin (PE) and 7-amino-actinomycin D (7-AAD) (all obtained from BD Biosciences) at a 1:100 dilution according to the manufacturer's recommendation. Flow cytometry of 3 x 10⁴/sample was performed with a Cyan cytometer (Dako Denmark A/S, Glostrup, Denmark). Cells were gated using a forward/side scatter, and fluorescence intensity was measured in FL2 for annexin V and FL4 for 7-AAD.

Quantitative and Statistical Analysis. Differences in measured variables between the experimental conditions were assessed using a one-way analysis of variance followed by a Student-Newman-Keuls post-hoc test for multiple comparisons (normality test passed) for caspase-3 activity data and a one-way analysis of variance on ranks followed by a nonparametric Student-Newman-Keuls test for multiple comparisons or Student's t test for flow cytometric data. Results were considered statistically significant at p < 0.05. The tests were performed using the SigmaStat software package (SPSS Inc., Chicago, IL).

	Results

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Thiopental Induces the Expression of Various Heat Shock Proteins at the Transcriptional and Translational Level. To investigate whether thiopental induces hsp expression, we performed Northern blot experiments to examine the effects of thiopental treatment on the mRNA levels of hsp27, hsp32, hsp70, and hsp90 in T lymphocytes. Thiopental strongly increased the mRNA levels of hsp27 as well as that of hsp70 and hsp90 in CD3⁺ T lymphocytes (Fig. 1A, blots 1, 3, and 4). In contrast, thiopental did not alter hsp32 mRNA levels (Fig. 1A, blot 2). Western blot analyses similarly revealed hsp70 protein accumulation in response to thiopental treatment (Fig. 1B). Flow cytometric analysis with a monoclonal FITC-conjugated hsp70 antibody revealed hsp70 expression in >50% CD3⁺ T cells after thiopental treatment (*, p < 0.05).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Effects of thiopental on the expression of various heat shock proteins in human CD3+ T lymphocytes. A, effect of thiopental treatment for 2 h at different concentrations on the mRNA levels of heat shock proteins hsp27, hsp32, hsp70, and hsp90 (blots 1–4). CD3+ T lymphocytes (2 x 107) were used per sample. mRNA levels were analyzed by Northern blotting. Accuracy of loading and transfer was confirmed by reprobing using a cDNA probe for 18s rRNA (blot 5). In blot 2, rat liver after hemorrhagic shock served as positive control. B, hsp70 protein accumulation in response to thiopental treatment. Protein levels were analyzed by Western blotting. CD3+ T lymphocytes (1 x 107) were used per sample. Accuracy of loading and transfer was confirmed by reprobing using an antibody recognizing constitutively expressed Hsc70. The results are representative of four separate experiments. C, flow cytometric determination of hsp70 protein accumulation in CD3+ T lymphocytes after thiopental treatment. CD3+ T lymphocytes (1 x 105) were used per sample (*, p < 0.05 versus negative control, ANOVA, normality test passed; n = 8).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Effects of thiopental on the DNA binding activity of HSF-1 in human CD3+ T lymphocytes. CD3+ T lymphocytes (1 x 107/sample) were treated with thiopental for 2 h at the concentrations indicated (lanes 3–5). Cells exposed to 42°C for 1 h served as positive control (lane 2). Binding activity was assessed by electrophoretic mobility shift assay. Supershift analyses using a specific antibody recognizing an HSF-1 subunit and competition analyses using unlabeled HSE oligonucleotides confirmed the specificity of the protein/DNA interaction (lanes 6–9). Position of the HSF-1/DNA complexes (). A nonspecific activity binding to the probe (); unbound oligonucleotide (). The results are representative of four separate experiments.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Effects of various other anesthetics on the DNA binding activity of HSF-1 in human CD3+ T lymphocytes. CD3+ T lymphocytes (1 x 107/sample) were treated for 2 h with either midazolam (A, lanes 2–4), propofol (B, lanes 1–3), etomidate (C, lanes 2–4), or s-ketamine (D, lanes 1–3) at the concentrations indicated. Cells exposed to 42°C for 1 h served as positive controls (A, lane 5; B, lane 4; C, lane 5; and D, lane 4). Binding activity was assessed by electrophoretic mobility shift assay. Position of the HSF-1/DNA complexes (). A nonspecific activity binding to the probe (); unbound oligonucleotide (). The results are representative of four separate experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Effects of structurally diverse barbiturate analogs on the DNA binding activity of HSF-1 in human CD3+ T lymphocytes. CD3+ T lymphocytes (1 x 107/sample) were treated for 2 h with either thiopental (lanes 2–4) or pentobarbital (lanes 7–9) at equimolar concentrations indicated, corresponding to approximately 100, 400, and 1000 µg/ml thiopental. Cells exposed to 42°C for 1 h served as positive controls (lanes 5 and 10). Binding activity was assessed by electrophoretic mobility shift assay. Position of the HSF-1/DNA complexes (). A nonspecific activity binding to the probe (); unbound oligonucleotide (). The results are representative of four separate experiments.

Thiopental Leads to HSF-1 Phosphorylation. Because inducible HSF-1 phosphorylation on serine residues is important for HSF-1 activity, we examined the phosphorylation state of thiopental-induced HSF-1 by Western blot. HSF-1 hyperphosphorylation was detected after treatment of CD3⁺ T cells with thiopental (400 µg/ml) after 1 and 2 h (Fig. 5, lanes 3 and 5).

View larger version (38K): [in this window] [in a new window]   Fig. 5. Effects of thiopental on the phosphorylation of HSF-1 in human CD3+ T lymphocytes. CD3+ T lymphocytes (1 x 107/sample) were treated with thiopental for the durations and at the concentrations indicated (lanes 2–7). Cells exposed to 42°C for 1 h served as positive control (lane 2). Protein levels were analyzed by Western blotting. Accuracy of loading and transfer was confirmed by reprobing using an antibody recognizing β-actin. The results are representative of four separate experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Effects of thiopental on caspase-3-like activity (A) and externalization of phosphatidylserine (B and C) after staurosporine-induced apoptosis in human CD3+ T lymphocytes. CD3+ T lymphocytes were pretreated with thiopental for 4 h at the concentrations indicated before the induction of apoptosis adding 2 µM staurosporine for an additional 4 h (with thiopental still present). A, caspase-3-like activity was assessed using fluorogenic caspase activity assay. CD3+ T lymphocytes (1 x 107) were used per sample. Relative fluorescent units (RFU) denotes relative fluorescent units (***, p < 0.001 versus staurosporine, ANOVA, normality test passed; n = 8). B, representative flow cytometric analysis after annexin V-FITC staining. CD3+ T lymphocytes (1 x 104) were analyzed per sample. C, statistical analysis of six independent experiments (*, p < 0.05 versus staurosporine, ANOVA on ranks, normality failed; n = 6). PI, propidium iodide.

Thiopental-Mediated Protection from Staurosporine-Induced Apoptosis in Jurkat T Cells Is Mediated by the Expression of hsp70. To evaluate the role of hsp70 in the thiopental-mediated protection from staurosporine-induced apoptosis, we assessed whether the protective properties of thiopental would be altered after modifying the expression level of this protein in response to thiopental. For this purpose, Jurkat T cells were chosen as cell model and transfected with siRNA directed against hsp70-mRNA before thiopental treatment. Flow cytometry experiments after staining of cells with FITC-labeled hsp70 antibody revealed that the percentage of cells positive for hsp70 significantly increased after thiopental treatment (Fig. 7, column 2 versus column 1; **, p < 0.001). However, the rate of hsp70-positive cells was significantly reduced in cells initially transfected with siRNA directed against hsp70-mRNA before thiopental treatment (Fig. 7, column 3 versus column 2; ***, p < 0.001). In contrast, initial transfection of cells with nonsilencing RNA not targeting any known eukaryotic gene product left the percentage of cells positive for hsp70 unaltered after thiopental treatment (Fig. 7, column 4 versus column 2).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effects of the transfection with hsp70 siRNA on the thiopental-induced expression of hsp70 protein in Jurkat T lymphocytes. Jurkat T lymphocytes were transfected with siRNA directed against hsp70-mRNA (hsp70 siRNA) or nonsilencing siRNA not targeting any gene product. After transfection, cells were treated with thiopental for 4 h at the concentration indicated. Expression of hsp70 was assessed by flow cytometry after staining with a FITC-labeled antibody directed against hsp70 protein (HSP70-FITC-ab). Data represent the median, 10th, 25th, 75th, and 90th percentile (***, p < 0.001 versus control, ANOVA on ranks; **, p < 0.01 versus thiopental, Mann-Whitney rank sum test; n = 9).

Subsequent flow cytometric analysis using annexin V-PE and 7-AAD staining was performed for the detection and quantification of apoptosis and necrosis. As shown in a representative experiment in Fig. 8A, staurosporine (2 µM; 4 h) induced a strong increase in the rate of annexin V-positive cells [Fig. 8A; histogram I; 62.5% (white-filled curve) versus 21.6% control (gray-filled curve)]. Preincubation of the T cells with thiopental (100 µg/ml; 4 h) before staurosporine-treatment (2 µM; 4 h) lead to a substantial decrease in the rate of annexin V-positive cells [Fig. 8A; histogram II; 39.6% (white-filled curve) versus 62.5% (gray-filled curve)]. Compared with cells treated with thiopental before the addition of staurosporine, initial transfection of cells with siRNA directed against hsp70-mRNA before thiopental and subsequent staurosporine treatment lead to a significant increase in the rate of annexin V-positive cells [Fig. 8A; histogram III; 54.4% (white-filled curve) versus 39.6% (gray-filled curve)], whereas transfection of nonsilencing siRNA did not [Fig. 8A; histogram IV; 29.2% (white-filled curve) versus 39.6% (gray-filled curve)]. Statistical analysis of five independent experiments revealed that the changes were significant [Fig. 8B; 35.4 ± 7.2% (thiopental + staurosporine, column 3) versus 62.4 ± 9.2% (staurosporine, column 2), **, p < 0.01; and 51.0 ± 9.9% (hsp70 siRNA + thiopental + staurosporine, column 4) versus 35.4 ± 7.2% (thiopental + staurosporine, column 3), *, p < 0.05].

View larger version (23K): [in this window] [in a new window]   Fig. 8. Effects of the transfection with hsp70 siRNA on the thiopental-mediated decrease from staurosporine-induced externalization of phosphatidylserine in Jurkat T lymphocytes. Jurkat T lymphocytes were transfected with siRNA directed against hsp70-mRNA (hsp70 siRNA) or nonsilencing siRNA not targeting any gene product. After transfection, cells were treated with thiopental for 4 h at the concentrations indicated before induction of apoptosis using staurosporine (2 µM; 4 h). A, representative flow cytometric analysis after annexin V-PE staining. Depicted are four histogram overlays (I–IV), each demonstrating the course of the number of cells positive for annexin V in response to the interventions stated below each overlay in normal (gray-filled curve) and underlined (white-filled curve) writing. Accordingly, the percentage stated above the marker line in each overlay corresponds to cells of the gray-filled curve, whereas the underlined percentage stated below the marker line corresponds to cells of the white-filled curve, fulfilling the criterion of the marker. Jurkat T lymphocytes (3 x 104) were analyzed per sample. ST, staurosporine; T100, thiopental, 100 µg/ml. B, statistical analysis of five independent experiments (mean + S.D.; **, p < 0.01 versus staurosporine; *, p < 0.05 versus staurosporine + thiopental; t test).

The percentage of cells positive for 7-amino-actinomycin D, which represent necrotic and end stage apoptotic cells, did not differ significantly between the treatments or transfections of the cells (data not shown).

	Discussion

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Barbiturates may be beneficial in patients with severe head injury and refractory intracranial hypertension. This conclusion is based on a series of clinical studies showing that administration of barbiturates can reduce intracranial pressure and increase cerebral perfusion pressure after brain trauma (Ghajar et al., 1995). However, little is known about whether these beneficial effects are specific for barbiturates and whether they are reflected by changes at the cellular level.

The data presented here support the hypothesis that thiopental induces a heat shock response and that it mediates cytoprotection at clinically relevant tissue concentrations by several lines of evidence: 1) Thiopental-mediated heat shock protein gene expression seems to be selective: whereas hsp32 was not induced at all, hsp27 was activated only weakly, and hsp70 and hsp90 were strongly induced. 2) Thiopental-induced hsp expression was dose-dependent. 3) The induction of a HSR seems to be a specific characteristic of thiopental, because other anesthetics tested over a broad range of clinically relevant concentrations did not exert any effect on HSF-1 activation. 4) Induction of HSF-1 DNA binding activity by thiopental is linked to inducible serine phosphorylation. 5) Thiopental exerts cytoprotective properties, because induction of caspase-3-like activity and apoptosis by staurosporine, a nonselective protein kinase inhibitor commonly used to elicit apoptotic cell death, were attenuated after pretreatment with thiopental. 6) hsp70 seems to be pivotal for this protection, because the knockdown of its expression suspends the thiopental-mediated cytoprotection.

Other i.v. and volatile anesthetics vary in their ability of inducing one or several hsps. Xenon and isoflurane preconditioning has been reported to induce cardiac hsp27, and pentobarbital reduced the levels of hsp27 mRNA in aortic smooth muscle cells, whereas propofol had no effect (Kozawa et al., 2000). Exposure of rats to isoflurane has been shown to induce mRNA, protein, and activity of hepatic hsp32 (Hoetzel et al., 2002). However, neither hsp27 nor hsp70 mRNA could be detected after 6 h of anesthesia with desflurane, sevoflurane, or isoflurane (Hoetzel et al., 2002).

In eukaryotic cells, HSF-1 has been identified as the primary stress-inducible molecule sensing environmental changes and mediating heat shock gene expression (Westerheide and Morimoto, 2005). The data presented here provide evidence that thiopental is able to induce a multistep process involving binding of HSF-1 and subsequent transactivation of heat shock genes. It is noteworthy that this mechanism is similar to the events that occur during hyperthermia, and it is quite different from the activation by other drugs and stressors. For example, despite its ability to bind to the promoter of the endogenous hsp70 gene, indomethacin-induced HSF-1 is inert, and transcription is not induced (Lee et al., 1995). In our study, thiopental was the only agent tested to induce the activation of HSF-1, which is responsible for mediating the expression of several but not all heat shock proteins. This observation, which is in accordance with our study, has become evident when overexpression of HSF-1 followed by heat shock experiments revealed that HSF-1 repressed heme oxygenase-1 (hsp32) gene expression by directly binding to the HSE of the heme oxygenase-1 gene promoter (Chou et al., 2005).

Several studies have documented in vitro and in vivo interactions between the induction of an HSR and the activation of NF-B when both pathways are activated sequentially (DeMeester et al., 2001; Malhotra and Wong, 2002). NF-Bis activated only after its inhibitory subunit IB has been phosphorylated by IB kinase. Own previous results suggest that thiopental applied before an inflammatory stimulus is an inhibitor of NF-B, its trans-acting potency, and its downstream effects on immune cell function by altering the activity of IB kinase, confirming previous in vitro studies (Wong et al., 1997a,1997b; Loop et al., 2002, 2003; Malhotra and Wong, 2002). It is noteworthy that oxybarbiturates failed to inhibit NF-B in equimolar amounts in these investigations (Loop et al., 2002, 2003). Because our previous data revealed that thiopental prevented the proteolysis of immunoreactive IB, these findings suggest that the suppression of NF-B activation and HSR induction by thiopental may involve the stabilization of IB, making it a point of interaction and a putative novel hsp (DeMeester et al., 1997; Wong et al., 1997a; Loop et al., 2002, 2003).

Our finding that thiopental induces an HSR and protects from apoptotic cell death via hsp70 in this T cell model is supported by reports about various heat shock proteins as key determinants in the regulation of apoptosis (Xanthoudakis and Nicholson, 2000): hsp70, which functions as molecular chaperone and confers protection against various stressful stimuli in vitro (Wong et al., 1996, 1998) and in vivo, such as ischemia/reperfusion injury (Marber et al., 1995), has also been identified to inhibit cell death by preventing mitochondrial cytochrome c release and activation of procaspases into caspases (Xanthoudakis and Nicholson, 2000). This hsp also acts downstream of cytochrome c release and upstream of the activation of caspase-3 (Xanthoudakis and Nicholson, 2000). In addition, hsp70 might be involved in preventing a proposed caspase-independent cell death by suppressing c-Jun NH₂-terminal kinase activity (Gabai et al., 1997). Both hsp70 and/or hsp90 can bind Apaf-1, a component of the so-called apoptosome, thereby inhibiting its formation and the activation of caspase-9 (Xanthoudakis and Nicholson, 2000). Whether hsps other than hsp70 are also involved in the thiopental-mediated cytoprotection remains to be identified in future studies. Thiopental has recently been shown to induce apoptosis in lymphocytes and Jurkat cells by a CD95-independent mechanism and caspase-3 activation, which seems to be in contrast to our results (Keel et al., 2005). Keel and coworkers had also used freshly isolated human lymphocytes but incubated the cells with thiopental for 24 and 48 h, considerably longer than treatment times in our model, which were 4 h for the pretreatment and an additional 4 h during which apoptosis was induced.

Although some possible explanations exist, the exact mechanism by which thiopental treatment leads to an HSR still remains to be identified. That other thiol compounds have been shown to act as nonthermal inducers of the HSR by destabilizing or directly denaturating proteins suggests that thiopental may elicit the HSR by a similar mechanism (Senisterra et al., 1997). The relevance of the thiol group at position C2 within the thiopental molecule is further supported by the fact that pentobarbital, the structural oxyanalog of thiopental, failed to induce HSF-1 in equimolar amounts.

In conclusion, the present study demonstrates that thiopental induces a differential HSR mediated by the activation and phosphorylation of HSF-1 and that thiopental exerts cytoprotective effects via the expression of hsp70. Thus, the results of the present study provide a molecular rationale for future investigations that systematically examine the organ-specific induction of the HSR by thiopental.

	Footnotes

 
M.R. and D.S. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.133108.

ABBREVIATIONS: NF-B, nuclear factor-B; HSR, heat shock response; HSF, heat shock factor; HSE, heat shock element; hsp, heat shock protein; hsp, heat 70-kDa shock protein; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; FL, fluorescence channel; PE, phycoerythrin; 7-AAD, 7-amino-actinomycin D; IB, inhibitor of nuclear factor-B; ANOVA, analysis of variance; Hsc, heat shock cognate protein.

Address correspondence to: Dr. Torsten Loop, Anaesthesiologische Universitaetsklinik, Hugstetterstrasse 55, D-79106 Freiburg, Germany. E-mail: torsten.loop{at}uniklinik-freiburg.de

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