Thiopental-induced Apoptosis in Lymphocytes Is Independent of CD95 Activation

The following reagents were obtained from commercial sources: propidium iodine (PI) and gliotoxin (Sigma Chemical Co., St. Louis, MO); Annexin-V–fluorescein isothiocyanate (FITC), terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) kit (Boehringer-Mannheim, Mannheim, Germany), pan-caspase inhibitor z-VAD-fmk16(Bachem AG, Bubendorf, Switzerland); caspase-3 fluorogenic substrate z-DEVD-afc and rhodamine 123 (Molecular Probes, Inc. Eugene, OR); agonistic anti-CD95 antibody (CH11; Immunotech, Coulter, Marseille, France); antagonistic anti-CD95 antibody (SM 1/23; Alexis, Grunberg, Germany); FITC-labeled anti-CD3 antibody (UCHT1; R&D Systems, Wiesbaden-Nordenstadt, Germany); phycoerythrin-labeled anti-CD95 antibody (33455X; Becton Dickinson, Basel, Switzerland); and thiopental (Pentothal, Lot. No. 57879TF; Abbott AG, Baar, Switzerland).

Human lymphocytes were isolated after Ficoll density centrifugation of peripheral venous blood from healthy volunteers. The interface containing monocytes and lymphocytes was washed twice and resuspended in medium RPMI-1640 (Gibco BRL, Life Technologies, Paisley, United Kingdom) supplemented with 10% fetal calf serum (Gibco BRL) and L-Glutamax (Gibco BRL). After 2 h of incubation in 24-well cell culture plates (Costar Co., Cambridge, MA) at 37°C in a humidified atmosphere (5% CO²), nonadherent cells (> 95% lymphocytes) were collected, washed, and resuspended in the same medium at a density of 1 × 10⁶/ml. Purity was measured by flow cytometry (FACS Calibur; Becton Dickinson) after staining with an FITC-CD3 antibody and was found to be greater than 95%.

Jurkat T cells (10⁶cells/ml) were maintained in RPMI-1640 supplemented with 10% fetal calf serum (Gibco BRL) and 1.5 mm L-Glutamax in 24-well cell culture plates (Costar Co.) at 37°C in a humidified atmosphere (5% CO²).

Cells were preincubated for 1 h with or without antagonistic anti-CD95 (SM 1/23) antibody (500 ng/ml), z-VAD-fmk (20 μm), or gliotoxin (0.001–0.01 μg/ml) before stimulation with or without various concentrations of thiopental (5–500 μg/ml) or agonistic anti-CD95 (CH11) antibody (20 or 100 ng/ml) for times indicated. The cell loss in Jurkat cultures was less than 5%, irrespective of the experimental design or the added proteins using trypan blue exclusion and microscopic cell counting.

Determination of apoptosis and secondary necrosis uses the high affinity of Annexin-V for phosphatidylserine, which is exposed on the cell surface of apoptotic cells, and of PI to DNA strands.28Jurkat cells (1 × 10⁶/ml) were washed with phosphate-buffered saline, resuspended in binding buffer (10 mm HEPES–NaOH, pH 7.4, 140 mm NaCl, 2.5 mm CaCl²) and incubated with 0.25 mg/ml FITC–Annexin-V and 50 μg/ml PI. The mixture was kept on ice for 5 min, and cell fluorescence was measured by two-parameter flow cytometry (FACS Calibur).28When green fluorescence (FITC) was plotted against red fluorescence (PI) in a dot plot, three distinct cell populations could be detected: viable cells (FITC negative, PI negative), apoptotic cells (FITC positive, PI negative), and secondary necrotic cells (FITC positive, PI positive). A minimum of 10,000 events (within a gate in the forward and side scatter window) was counted per sample, and data were reported as the percentage of apoptotic cells (Annexin-V–FITC positive, PI negative) and secondary necrotic cells (Annexin-V–FITC positive, PI positive).28 

For the detection of apoptosis after the experiment, cytospin preparations of freshly isolated human lymphocytes or Jurkat cells (1 × 10⁵) were treated with a commercially available kit for the TUNEL technique (Boehringer Mannheim)29according to the manufacturer’s guidelines. Briefly, air-dried cytospin preparations were fixed with 4% paraformaldehyde solution, rinsed with phosphate-buffered saline, and incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 5 min on ice. The slides were rinsed again with phosphate-buffered saline and incubated with TUNEL reaction mixture (terminal deoxynucleotidyl transferase, FITC-labeled nucleotides) for 1 h in a humidified chamber. The slides were mounted in 50% glycerol–phosphate-buffered saline and examined on a fluorescence microscope (Leica Dialux 22 EB; Leica, Wetzlar, Germany). Bright homogeneously FITC-stained nuclei were considered as typical apoptotic features.29Up to 500 cells were counted per slide. Data are reported as the percentage of apoptotic cells of all cells stained with 4′,6-diamidino-2-phenylindole following standard protocol.

Apoptosis of mitochondria was measured in Jurkat cells (1 × 10⁶/ml) stained with rhodamine 123 (0.2 μm) for 1 h at 37°C. After a washing step, fluorescence was measured in the cytometer (FACS Calibur). The mitochondrial membrane breakdown in the cells is represented by a shift of mean fluorescence to the left (decrease in channel number). Data are given as percent of shifted cells from 10,000 cells measured within a forward and side scatter gate.

Expression of CD95 on Jurkat cells was measured by flow cytometry (FACS Calibur) with a phycoerythrin–anti-human-CD95 (33455X) antibody. After 24 h incubation Jurkat cells (10⁶/ml) were stained with 0.25 mg/ml phycoerythrin-labeled anti-CD95 antibody for 1 h on ice before fluorescence was measured in FACS. A minimum of 10,000 events was counted per sample, and data were reported as the mean channel fluorescence.

Caspase-3–like activity was measured in cellular extracts from Jurkat cell samples. After incubation, cells were lysed by freeze–thaw procedure in hypotonic extraction buffer (25 mm HEPES, pH 7.5, 5 mm MgCl², 0.1% Triton-X 100, 1 mm Pefablock® with addition of pepstatin, leupeptin, and aprotinin [1 μg/ml each]) and subsequently centrifuged (15 min, 14,000g , 4°C), and the supernatant was stored at −80°C until further used. The fluorometric cleavage assay for caspase-3-like activity (DEVD-afc; Calbiochem, Nottingham, United Kingdom) was performed in microtiter plates (Greiner, Nuertingen, Germany) according to the method described by Thornberry23,30using the fluorometric plate reader Victor-2 (Wallac Instruments, Turku, Finland), with the excitation wavelength set at 385 nm and an emission wavelength of 505 nm. The protein concentrations of the respective samples were measured with a commercially available kit (Pierce Assay; Pierce, Cramlington, United Kingdom), and caspase-3–like activity was calculated as microunits per milligram protein with 1 μU/mg being equal to the cleavage of 1 pmol 7-amino-4-trifluromethylcoumarin per milligram protein and minute.

In addition, caspase-3 activation in Jurkat cells was measured by Western blot. Equal amounts of whole cell lysate (5 μl/lane, corresponding to 5 × 10⁵cells) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 10% polyacrylamide gels in a Mini Protean II chamber (BioRad, Hercules, CA). Proteins were subsequently electrotransferred onto a nylon membrane (Immobilon P; Millipore, Bedford, MA), and membranes were blocked overnight at 4°C in tris-buffered saline supplemented with 0.1% Tween-20 and 0.2% bovine serum albumin (Sigma). Caspase-3 protein was detected using a specific antibody against caspase-3 from Santa Cruz (Santa Cruz, CA) and a peroxidase coupled secondary antibody (Dako, Glostrup, Denmark). Specific binding was visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, United Kingdom) on x-ray film (Kodak, Rochester, NY) following the manufacturer’s recommendations. The molecular weight of the protein bands were determined by use of prestained low-molecular-weight markers (Sigma) on the same gel.

Data are given as mean ± SD. The dose-dependent effect of thiopental was analyzed by one-way analysis of variance and post hoc  testing (Tukey); in other experiments, mean values were compared using the Student two-tailed t  test for independent means or the Mann–Whitney U test. Differences were regarded as significant if P  was less than 0.05.

Thiopental in concentrations from 5 to 500 μg/ml dose-dependently increased apoptosis and necrosis in isolated human lymphocytes (10⁶cells/ml). The proportion of necrotic (Annexin-V–positive, PI-positive) cells was higher than that of apoptotic (Annexin-V–positive, PI-negative) cells. Apoptosis increased only marginally, whereas necrosis increased from 3.0 ± 1.3% up to 38.1 ± 16.9% after 24 h and up to 48.4 ± 19.9% after 48 h (fig. 1). Analysis of variance (P < 0.05) and post hoc  tests (Tukey) revealed significant differences of necrotic cells at thiopental concentrations above 250 μg/ml (P < 0.05) compared with medium alone.

Spontaneous apoptosis (Annexin-V positive, PI negative) of cultured Jurkat cells (10⁶cells/ml) was 4.4 ± 1.9% after 24 h and 6.1 ± 2.0% after 48 h (fig. 2), whereas 3.0 ± 1.0 and 4.0 ± 1.0% necrotic (Annexin-V–positive, PI-positive) cells were detected after 24 and 48 h, respectively (fig. 2). Incubation with 5–500 μg/ml thiopental dose-dependently increased apoptosis up to 29.7 ± 2.8% after 24 h and 39.7 ± 3.2% after 48 h and secondary necrosis up to 21 and 29%, respectively (fig. 2). In contrast to lymphocytes, Jurkat cells exhibited a lower proportion of secondary necrotic (Annexin-V–positive, PI-positive) cells compared with apoptotic (Annexin-V–positive, PI-negative) cells. The amount of pure necrotic (Annexin-V–negative, PI-positive) cells was 1.7 ± 2.3% after 24 h and 2.0 ± 2.0% after 48 h and was independent of thiopental treatment.

Thiopental-induced apoptosis was also seen in cytospin slides stained by the TUNEL method. Here, the percent of TUNEL-positive cells after 24 h in freshly isolated lymphocytes and Jurkat cells (3.2 ± 2.6 and 5.5 ± 3.2%, respectively) increased dose dependently after incubation with thiopental up to 17.7 ± 4.9 and 20.1 ± 8.2%, respectively (table 1).

To investigate whether thiopental-induced apoptosis was mediated through CD95 expression and subsequent autocrine and/or paracrine induction of cell death, expression of CD95 on Jurkat cells was measured in FACS after incubation with thiopental (5–500 μg/ml). However, expression of CD95 on the cell surface of Jurkat cells (6.9 ± 0.6 mean fluorescence) was not altered significantly 4 h after incubation with 250 μg/ml thiopental (8.6 ± 0.9 mean fluorescence) (table 2). To further test whether the CD95 system is involved in thiopental-induced apoptosis, Jurkat cells were preincubated with an antagonistic anti-CD95 (SM 1/23) antibody (500 ng/ml) before stimulation with thiopental (250 μg/ml) or an agonistic anti-CD95 (CH11) antibody (100 ng/ml). Anti-CD95 induced apoptosis (16.7 ± 0.4%) could be completely blocked (9.7 ± 2.8%) similar to necrosis (60.0 ± 7.0 vs.  5.1 ± 3.3%, respectively) by preincubation with antagonistic anti-CD95 antibody (SM 1/23), whereas no effect was observed in thiopental-induced apoptosis (15.3 ± 0.7 vs.  16.0 ± 0.6%, respectively) or necrosis (37.8 ± 13.1 vs.  38.2 ± 10.0%) (fig. 3). This indicates that thiopental-induced apoptosis and necrosis is independent of the CD95 system.

To test whether caspase-3 was involved in thiopental-induced apoptosis, caspase-3–like activity was measured in Jurkat cells after incubation with increasing doses of thiopental (5–500 μg/ml). Thiopental dose-dependently and in parallel to apoptosis significantly (P < 0.05) increased caspase-3–like activity from basal level (88.0 ± 14.4 μU) to 364.0 ± 95.1 μU after incubation with 500 μg/ml thiopental for 24 h (fig. 4). The activation of caspase-3 was confirmed in Western blot. Whole cell lysates from Jurkat cells incubated for 24 h with medium or thiopental (5–500 μg/ml) were separated by electrophoresis and Western blot stained with caspase-3–specific antibodies. Activation of caspase-3 is characterized by a reduction of the pro–caspase-3 band. As depicted in figure 5, thiopental dose-dependently reduces pro–caspase-3 band, indicating activation of caspase-3 by thiopental (fig. 5). Densitometric analysis of Western blots where the medium control was set at 100% yielded values for incubation with 5, 50, 100, 250, and 500 μg/ml thiopental of 97.5 ± 9.6, 94.6 ± 11.7, 81.2 ± 18.6, 64.1 ± 24.3, and 65.1 ± 21.5%, respectively. Values were derived from five separate Western blots.

The involvement of caspase-3 was further studied by preincubating Jurkat cells (10⁶cells/ml) and human lymphocytes (10⁶cells/ml) with the pan-caspase inhibitor z-VAD-fmk (20 μm), and apoptosis was measured after 24 and 48 h. Thiopental (250 μg/ml)–induced apoptosis after 24 h of Jurkat cells (20.2 ± 2.5%) was reduced only insignificantly by z-VAD-fmk (17.2 ± 2.5%), whereas secondary necrosis (17.0 ± 3.0%) was significantly reduced (6.6 ± 1.2%) (fig. 6). In contrast, CD95-induced apoptosis (27.0 ± 2.0%) was completely (P < 0.05) blocked (8.1 ± 1.8%). These result are also supported by TUNEL-stained cells (table 1) showing a reduction of thiopental-stimulated TUNEL-positive cells (15.1 ± 3.7%) after preincubation with z-VAD-fmk (6.8 ± 2.0%).

Similar results were seen after 48 h in Jurkat cells (fig. 6). In isolated lymphocytes, no effect of z-VAD-fmk was seen on thiopental-induced apoptosis and necrosis after 24 and 48 h (fig. 7).

CD95-induced caspase-3–like activity (551.0 ± 56.9 μU), as well as thiopental (250 μg/ml)–induced activity (232.0 ± 33.6 μU) in Jurkat cells after 24 h was completely blocked by z-VAD-fmk (7.0 ± 3.0 and 8.5 ± 3.6 μU, respectively) (fig. 8), confirming the effectiveness of z-VAD-fmk.

It has been shown that thiopental leads to activation of mitochondria.31Therefore, Jurkat cells (10⁶cells/ml) were incubated with increasing doses of thiopental (5–500 μg/ml) for 24 and 48 h, and cells were stained afterward with rhodamine 123 for detection of mitochondrial membrane breakdown. The mitochondrial membrane breakdown is represented by a shift of mean fluorescence (decrease in channel number) in the cells. Parallel to apoptosis, thiopental dose-dependently increased the amount of rhodamine-shifted cells (13.9 ± 3.9 to 60.2 ± 10.9%) after 24 and 48 h (82.6 ± 13.4%) of all cells counted (fig. 9). Incubation with agonistic CD95 antibody increased rhodamine-negative cells to 94.1 ± 2.7% after 24 h and 99.0 ± 0.4% after 48 h (data not shown).

Recently, activation of NF-κB by thiopental has been shown in Jurkat cells. Therefore, gliotoxin, a specific inhibitor of NF-κB, was used as a pharmacologic tool to investigate the involvement of NF-κB in the regulation of thiopental-induced apoptosis. Because of the high rate of cell death after incubation with agonistic CD95 antibody, after 24 h, the concentration of agonistic CD95 antibody was decreased from 100 ng/ml to 20 ng/ml, and the incubation time was reduced to 16 h in this experiment. Jurkat cells (10⁶cells/ml) were incubated for 16 h with increasing doses of gliotoxin (0.001–0.01 μg/ml) in presence or absence of thiopental (250 μg/μl) or agonistic CD95 antibody (20 ng/ml). Gliotoxin dose-dependently increased the proapoptotic effect of thiopental from 29.9 ± 3.6 to 72.7 ± 6.5% cell death (apoptosis and secondary necrosis; fig. 10) and in the highest concentration (0.01 μg/ml) induced cell death (54.9 ± 12.2 vs.  13.1 ± 2.7%) by itself, indicating that NF-κB activity is necessary for survival of Jurkat cells. Even CD95-induced cell death after 16 h (65.9 ± 6.7%) was further increased by 0.01 μg/ml gliotoxin (86.8 ± 6.2%), indicating that NF-κB activation is also necessary in CD95-induced apoptosis (fig. 10).

In patients with severe traumatic brain injury, the barbiturate coma can be the final resort in the therapy of life-threatening increases of the ICP, whenever other therapeutic regimens do not reduce ICP.7However, this therapy is often complicated by disturbed immunocompetence with leukopenia and an increased infection rate.10–12It could be speculated that thiopental would have proapoptotic activity on immune cells. This suggestion is supported by the observation that intravenous administration of barbiturate depresses lymphocyte activation.14 

The presented results demonstrate the proapoptotic activity of thiopental on Jurkat T cells and human lymphocytes. In previous studies, drug-induced apoptosis of immune cells was focused on cancer therapy.21,32,33Villunger et al.  21showed that drug-induced apoptosis is associated with enhanced CD95 ligand expression but occurs independently of CD95 signaling in human acute lymphatic leukemia T cells. In contrast, Krammer and coworkers postulated CD95/CD95 ligand signaling as the central effector pathway of drug-induced apoptosis.34,35Although thiopental enhanced the CD95-induced apoptosis in Jurkat cells, the facts that thiopental did not alter CD95 expression on Jurkat cells and thiopental-induced Jurkat cell apoptosis could not be prevented by pretreatment with antagonistic anti-CD95 antibody points to a CD95-independent pathway of thiopental-induced lymphocyte apoptosis.

In a recent study, it was shown that opioids, commonly used as potent analgesics, induced CD95 expression on human lymphocytes and promoted CD95-mediated apoptosis.36,37This effect was blocked by naloxone, an antagonist of the opioid receptors, which are also expressed on lymphocytes.32In the same line, Yin et al.  33showed that stress modulates the immune system with an increase of T-cell apoptosis through CD95-mediated apoptosis dependent on endogenous opioids. These data were in accord with previous results from Oka et al.  38who demonstrated that circulating lymphocytes in the early perioperative period are susceptible to CD95-mediated apoptosis, which may cause depletions of circulating lymphocytes after surgery. However, in the current study, no influence of naloxone was observed on the thiopental-induced apoptosis (data not shown). Therefore, in contrast to opioids, thiopental acts via  a different mechanism.

The observation that caspase-3 activity increased in parallel to apoptotic rate after incubation with thiopental suggested a pivotal role of caspase-3 in thiopental-induced apoptosis. However, the pretreatment with the pan-caspase-inhibitor z-VAD-fmk did not completely inhibit the apoptotic effect by thiopental, whereas the anti-CD95–induced apoptosis was completely blocked by z-VAD-fmk. The current dogma that caspase activity is essential for execution of apoptosis has been under scrutiny recently,24,30because certain apoptotic characteristics such as cell shrinkage, membrane blebbing, and nuclear condensation were found to be insensitive toward caspase inhibitors.39This is confirmed through the results of this study that effective inhibition of the complete caspase cascade through the pan-caspase inhibitor z-VAD-fmk did not completely reduce thiopental-induced Jurkat cell apoptosis and lymphocyte necrosis. This caspase independence and the differences seen between Jurkat cells and freshly isolated lymphocytes in the mode of cell death and induction of necrosis in our experiments have been described before.40Therefore, our results indicate that further factors must be involved in the regulation and execution of thiopental-induced cell death, i.e. , apoptosis and necrosis. Today, apoptosis and necrosis are seen as the two extremes of a wide spectrum of cell demise including caspase-independent cell death, with a multitude of factors involved.24 

Recently other proteases, namely calpains,13,41cathepsins,42,43or the proteasome44have been discussed as alternative factors of cellular apoptosis. As further alternative concept of apoptotic execution, a loss in mitochondrial membrane barrier function is discussed in the literature.15This is supported by recent findings showing an exaggerated generation of oxygen radicals after incubation with 20 mmol/ml thiopental.31This increased production of oxygen radicals by thiopental could enhance lymphocyte commitment to apoptosis and is also in accord with our observation of decreased rhodamine staining after thiopental incubation. However, further investigations are necessary to clarify this mechanism.

Recently, it has been shown that thiopental inhibits activation of NF-κB in lymphocytes and Jurkat cells25,via  inhibition of the calmodulin–calcineurin complex pathway.45We could confirm these findings by gliotoxin inhibition of NF-κB, which enhanced the spontaneous apoptosis and the proapoptotic effect of thiopental in Jurkat cells. This shows that NF-κB plays a central role not only in regulation of drug-induced apoptosis but also in the survival of lymphocytes during homeostasis, possibly by transcription of survival genes. It has been shown in another study that NF-κB controls the transcriptional activity of a gene, which induces the synthesis of survival proteins.46Furthermore, in a number of immune cells, NF-κB activation has been shown to play a pivotal role in regulating the genes for inflammatory cytokines, which inhibit the apoptotic process, probably by activation of NF-κB in a positive-feedback loop.46Recently, Humar et al.  47showed that thiopental and other barbiturates indirectly suppress the activation of the transcription factor activator protein 1. However, in contrast to other closely related barbiturates, thiopental does not act through activation of mitogen-activated protein kinases and small G proteins, but through other mechanisms.47 

In conclusion, we could show that thiopental acts as an apoptosis-inducing agent for lymphocytes and, through this, might contribute to the severe immunosuppression during thiopental coma in patients with traumatic brain injury. We are aware of the preliminary nature of our observations, and the exact mechanisms of thiopental-induced cell death in lymphocytes need further investigation. Elucidating the mechanism of thiopental-induced cell death might help to improve thiopental therapy and prevent lymphocyte apoptosis and risk of nosocomial infections in thiopental-treated patients.

The authors thank Ursula Steckholzer (Technician, Division of Trauma Surgery, Department of Surgery, University Hospital Zurich, Zurich, Switzerland) for perfect technical assistance and Silke Ludwig (Nurse, Division of Surgical Intensive Care, University Hospital Zurich) for help in the intensive care unit.