General anesthesia is able to modulate the inflammatory stress response characterized by peripheral leukocytosis and T-cell lymphopenia.1–6 This may result in an increased susceptibility to infectious complications, such as pneumonia or wound infection, after major surgery with concomitant anesthesia.6 Apoptosis is a genetically controlled and active form of cell death, which occurs both under physiologic as well as pathologic conditions. During apoptosis, a specific class of cysteine proteases, called caspases, are activated in an amplifying proteolytic cascade.7 Apoptosis is the predominant mechanism regulating immunological homeostasis and the termination of surgical injury-induced inflammation.2,3 However, to avoid these side affects and to improve the specificity of anesthetics, the molecular mechanisms responsible for anesthetic-mediated immunomodulation need to be identified.
The mitogen-activated protein (MAP) kinase cascades are multifunctional signaling pathways composed of three distinct subgroups: (1) extracellular signal-regulated kinases (ERKs), (2) c-Jun NH2-terminal (JNK) or stress activated protein kinases, and (3) the p38 group of protein kinases. The three principal MAP kinase pathways mediate changes in cellular gene expression in response to extracellular stimuli. The p38 MAP kinase is an important mediator of apoptosis in T-lymphocytes induced by various drugs.8–13 p38 MAP kinase is activated by an upstream MAP kinase kinase termed MKK3/MKK6. MKK3/MKK6 in turn was reported to be activated by a new MAP kinase kinase kinase, called apoptosis signal-regulating kinase1 (ASK1).14
We have demonstrated that volatile anesthetics, in particular sevoflurane, trigger apoptosis and inhibit the transcription factor activator protein-1 in primary CD3+ T-lymphocytes as well in Jurkat T-cells, which may be exerted by interfering with the p38 MAP kinase cascade.15,16 However, the precise molecular mechanism of the immunosuppressive action remains to be identified. Despite a large number of in vitro studies regarding the function of p38 MAP kinase, the role of p38 in cell death and proliferation remains controversial. Several studies have shown that p38 MAP kinase mediates apoptosis in different cell types.17–20 Other studies have described the antiapoptotic effects of the p38 MAP kinase pathway.21,22
From these and the above mentioned data, three hypotheses can be proposed: (i) sevoflurane-mediated apoptosis leads to activation of p38, (ii) induction of apoptosis is triggered by p38 MAP kinase activation, or (iii) sevoflurane-mediated p38 activation and induction of apoptosis may be unrelated. The aim of this study was to identify the induction of the p38 MAP kinase cascade and the possible association with apoptosis mediated by volatile anesthetics in Jurkat T-cells in vitro.
The following anesthetics were used: sevoflurane, isoflurane (Abbott, Wiesbaden, Germany), and desflurane (Baxter, Unterschleissheim, Germany). The remaining reagents were purchased from Sigma (Deisenhofen, Germany) unless specified otherwise.
We used a well-established cultured T-cell line the Jurkat T-cells as a model. In order to confirm the similarity between the signaling mechanisms in CD3+ T-lymphocytes and Jurkat T-cells, MAP kinase phosphorylation was evaluated under the same experimental conditions.16 Jurkat T-cells (ACC 282, DSMZ, Braunschweig, Germany) were cultured and grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% glutamine and 50 mg/mL penicillin and streptomycin (all from Gibco-BRL, Karlsruhe, Germany) in a humidified atmosphere containing 5% carbon dioxide at 37°C as previously described.16
Exposure to Volatile Anesthetics and Experimental Protocol
Jurkat T-cells were exposed according to the same experimental protocol as previously described.16 Volatile anesthetic concentrations were monitored at the chamber exit port using a ventilation monitor (PM 8050, Dräger, Lübeck, Germany). Concentrations of sevoflurane dissolved in the cell culture medium were determined by headspace gas chromatography-mass spectrometry (Agilent 5973N/6890N, Waldbronn, Germany, equipped with a CTC CombiPal Autosampler, CTC Analytics AG, Zwingen, Switzerland). Data were acquired in selected ion monitoring mode using m/z = 69 as quantifier and nitrogen (N22+, m/z = 14) as internal standard. Good linearity was achieved in the range of 6-300 μg/mL (R2 >0.99, 10-point calibration). Based on this method, a mean concentration of 1170 μmol/L sevoflurane was measured in the culture medium after 24 h of incubation with sevoflurane at a concentration corresponding to 8 Vol.% on the Vapor dial (“8” Vapor Vol.%).
Total Protein Cell Extracts
Total protein cell extracts were prepared using a high-salt detergent buffer “Totex” (20 mM HEPES, pH 7.9, 350 mM NaCl, 20% (v/v) glycerol, 1% (w/v) NP-40, 1 mM MgCl2, 0.5 mM ethylene diamine tetra acetic acid, 0.1 mM EGTA). Inhibitors of proteinases and phosphatases were added at the following concentrations to the extraction and suspension buffer: 10 μg/mL aprotinin, 25 μM leupeptin, 2 mM phenyl-methylsulfonyl-fluoride, 2 mM iodoacetamide, 10 mM sodium fluoride, 0.2 mM sodium orthovanadate, 0.1 mM β-glycerophosphate, and 0.1 mM sodium pyrophosphate. Cells were harvested by centrifugation, washed once in ice-cold phosphate-buffered saline and resuspended in four cell volumes of the detergent buffer. The cell lysate was incubated for 30 min on ice, and then centrifuged for 5 min at 13000g at 4°C. The supernatant containing the cell protein lysate was stored at −80°C.
Sodium-Dodecyl-Sulfate-Polyacrylamide Gel Electrophoresis and Western Blotting
Total cell extracts of Jurkat T-cells (30 μg) were boiled in Laemmli sample buffer and subjected to 10% sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis. Western blotting was performed according to the protocol previously described.15,16 Membranes were incubated in a recommended dilution of specific antibodies (phospho-p38 #9211, p38 #9212, phospho-ERK #9101, ERK #9102, phospho-JNK #9251, JNK #9258, phospho-ASK (Ser83) #3761, phospho-ASK (Ser967) #3764, ASK #3762, phospho-MKK3/MKK6 #9231, MKK3/MKK6 #9232, phospho-activating transcription factor-2 (ATF-2) #9221, ATF-2 #9226, caspase-3 #9662, cleaved caspase-3 #9661, all 1:1000, all Cell Signaling Technology, Danvers) in 20 mM Tris-HCl (pH 7.6) plus 0.1% Tween-20 plus 5% bovine serum albumine overnight at 4°C. After washing 4 times (5 min each), the immunocomplexes were detected using ECL Western blotting reagents (Amersham-Pharmacia, Freiburg, Germany) according to the manufacturers instructions. Exposure to ECL Western blot films (Amersham-Pharmacia, Freiburg, Germany) was performed for 15 s to 1 min.
p38 MAP Kinase and Fluorogenic Caspase Activity Assay
To investigate the activity of p38 MAP kinase, a nonradioactive p38 MAP kinase activity assay kit (Cell Signaling Technology, Danvers) was used and performed according to the manufacturer’s instructions. In brief, immobilized Phosphop38 MAPK (Thr180/Tyr182) antibody was used to immunoprecipitate p38 MAP kinase before performing an in vitro kinase assay using ATF-2 as a substrate. ATF-2 phosphorylation was then detected by Western blotting using Phospho-ATF-2 (Thr71) Antibody.
Fluorogenic caspase activity assay was performed as previously described.15
After exposure to the volatile anesthetics, Jurkat T-cells were washed in phosphate-buffered saline and resuspended in 50 μL binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 140 mM NaCl, 2.5 mM CaCl2, pH 7.9) containing 12.27 μg/mL green fluorescent protein (GFP)-annexin-V und 0.5 μg/mL propidiumiodide. Samples were incubated in the dark at room temperature for 15 min. Subsequently, 450 μL binding buffer was added, and the percentage of early apoptotic lymphocytes was measured using a flow cytometer (FACSR-CaliburFCM, Becton Dickinson, Heidelberg, Germany). Jurkat T-cells were gated using forward scatter and side scatter, and fluorescence intensity was measured in 2 × 105 cells. The fluorescence intensity of GFP-annexin-V was measured in the fluorescence 1 (FL1, 515-545 nm) channel, and the fluorescence intensity of propidiumiodide was measured in the fluorescence 2 (FL2, 564-606 nm) channel.
Quantitative and Statistical Analysis
The fold induction in immunoblot analysis for the phosphorylation status was calculated by performing densitometry of the individual Western blot results using the Chemi-Smart 5000 image acquisition system (Vilber Lourmat, France). The density values of the phosphorylation read-outs were corrected for the density values of the total protein read-outs for each individual intervention to exclude the influence of difference in total amount of protein. The value of the negative control was then set to 1 in order to be able to compare between the different interventions.
Differences in measured variables and densitometric analysis between the experimental conditions were assessed using a one-way analysis of variance on ranks followed by a nonparametric Student-Newman-Keuls test for multiple comparisons. Results were considered statistically significant if P < 0.05. The tests were performed using the Sigma Stat software package (Jandel Scientific, San Rafael, CA).
Sevoflurane Exposure Induces p38 Phosphorylation and Activity in Jurkat T-Cells
In order to determine whether volatile anesthetics may induce phosphorylation of p38 MAP-kinase, Jurkat T-cells were exposed to different concentrations of sevoflurane (2.5, 5, 8 Vol.%). Phosphorylation was studied by Western blot analysis using a phospho-specific antibody against p38. While exposure of the cells to sevoflurane 2.5 Vol.% had no effect, treatment of the cells with higher concentrations of sevoflurane (5, 8 Vol.%) induced a comparable level of increased phosphorylation of p38 MAP kinase (Fig. 1A, upper panel, lanes 3 and 4). Time kinetic evaluation demonstrated p38 MAP kinase phosphorylation after 12 hours of sevoflurane exposure (Fig. 1B, upper panel, lanes 4–7). p38 MAP kinase activity, measured by phosphorylation of its physiological target ATF-2, was substantially increased after exposure to 8 Vol.% sevoflurane (Fig. 1C, upper panel, lane 2). Sevoflurane-induced ATF-2 phosphorylation was completely inhibited by the specific p38 inhibitor SB 202190 (10 μM; Fig. 1C, upper panel, lane 3). The total amount of p38 MAP kinase was constant throughout each experiment (Fig. 1A, B, and C, lower panels, all lanes).
Isoflurane, but not Desflurane, also Induces p38 Phosphorylation in Jurkat T-Cells
While exposure of the cells to desflurane had no effect on the phosphorylation level of p38 MAP-kinase (Fig. 2B, upper panel, lanes 2-4), treatment of the cells with isoflurane (“2.5” and “5” Vapor Vol.%) induced a comparable level of increased phosphorylation of p38 MAP kinase (Fig. 2A, upper panel, lane 4). Stimulation of cells with phorbol-myristate-acetate (PMA, 15 ng/mL) and ionomycin (I, 700 ng/mL) served as positive controls to validate the negative result.
Sevoflurane Exposure does not Induce Phosphorylation of c-Jun-NH2-Terminal Kinase (JNK) and p44 (ERK) MAP Kinase in Jurkat T-Cells
In order to assess whether sevoflurane specifically induces p38 phosphorylation or whether other MAP kinases such as ERK and JNK are also activated, we performed additional Western blots using phosphospecific antibodies for ERK and JNK. Treatment of cells with sevoflurane (2.5, 5, 8 Vol.%) did not detectably affect phosphorylation of ERK or JNK kinase (Fig. 3A and B, upper left panels, lanes 2-4). Neither was the phosphorylation of these kinases affected by isoflurane or desflurane (Fig. 3A and B, upper middle and right panels, lanes 2-4). Stimulation of cells with PMA (15 ng/mL) and ionomycin (I, 700 ng/mL) served as positive controls. The total amount of these MAP kinases was constant throughout each experiment (Fig. 3A and B, lower left, middle, and right panels, lanes 1-5).
Sevoflurane Exposure Induces Phosphorylation of ASK1, MAP Kinase Kinase 3 and 6 (MKK3/MKK6), and ATF-2 in Jurkat T-Cells
In order to determine whether other members of the p38 MAP kinase signaling pathway are also affected by sevoflurane, we analyzed the phosphorylation of the upstream kinases ASK1 and MKK3/MKK6, as well as ATF-2, a target substrate of p38, in Jurkat T-cells after treatment with sevoflurane. Sevoflurane (8 Vol.%) induced phosphorylation of ASK1 at Serine residues 83 and 967 (Fig. 4A, upper and third panel, lane 2). Furthermore, phosphorylation of MKK3/MKK6, which is activated by ASK1, was also induced by sevoflurane (8 Vol.%; Fig. 4B, upper panel, lane 2). In addition, phosphorylation of ATF-2, a substrate of p38, was also induced by sevoflurane (8 Vol.%; Fig. 4C, upper panel, lane 3). Preincubation of cells with SB202190 before sevoflurane exposure reduced sevoflurane-mediated ATF-2 phosphorylation (Fig. 4C, upper panel, lane 4). (Note that this effect, which is seen in whole cell extracts, is weaker than in the in vitro kinase assay using ATF-2 in excess as a substrate.) Stimulation of cells with PMA (15 ng/mL) and ionomycin (700 ng/mL) served as a positive control (Fig. 4A, B, upper panels, lane 3, and C, upper panel, lane 5). The total amount of ASK, MKK3/MKK6, and ATF-2 was constant throughout each experiment (Fig. 4A, second and lower panel, lanes 1-3, B, lower panel, lanes 1-3, and C, lower panel, lanes 1-5).
Sevoflurane-Induced p38 Phosphorylation is Caspase-Independent
Based on the above findings, we evaluated whether sevoflurane-induced p38 phosphorylation was dependent on caspases, or could also be executed in a caspase-independent manner. For that purpose, we performed Western blots using a p38 phosphospecific antibody in the presence (pretreatment 1 h before start of sevoflurane exposure) or absence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk). Z-VAD.fmk did not prevent the phosphorylation of p38 induced by sevoflurane (Fig. 5, upper panel, lane 4 vs 3). Z-VAD.fmk alone had no effect on the phosphorylation of p38 (Fig. 5, upper panel lane 2). The total amount of p38 MAP kinase was constant throughout the experiment (Fig. 5, lower panel, lanes 1-4).
Sevoflurane-Induced Apoptosis is not p38 MAP-Kinase-Dependent
To determine whether primary CD3+, as well as Jurkat T-cells, caspase-3 processing of sevoflurane15 is a result of the activation of p38 MAP kinase, we exposed Jurkat T-cells to sevoflurane (8 Vol.%, Fig. 6A) in the presence or absence of specific p38 inhibitors (SB2020190 or SB203580), and studied the processing of p32 procaspase-3 to its active p19/17 forms by immunoblotting. These experiments demonstrated that sevoflurane-induced caspase-3 processing could not be (completely) abolished by pretreatment with the specific p38 inhibitors (Fig. 6A). To confirm and quantify caspase-3 activation, we performed a caspase-3 activity assay of the cytosols of these T-cells by using a specific fluorogenic caspase-3 substrate (DEVD-AMC). Jurkat T-cells exposed to sevoflurane showed a significant increase in caspase-3 activity (Fig. 6B; P < 0.05 vs untreated cells, n = 12 each), while induced caspase-3 processing could not be abolished by pretreatment with the specific p38 inhibitors (Fig. 6B). In addition, GFP-annexin-V/PI staining, an indicator of apoptosis, was not reduced by SB203580 (Fig. 6C, plot 3 vs 2). Statistical analysis of the apoptotic cells of independent experiments revealed no statistically significant effect of pretreatment using SB203580 (Fig. 6D, table: comparison sevoflurane-treated versus sevoflurane plus inhibitor, n = 7; *P < 0.05). The p38 inhibitor SB203580 (5 μM) alone had no influence on the GFP-annexin-V/PI staining (data not shown).
Volatile anesthetics are widely used for the maintenance of anesthesia during major surgery. In addition to their anesthetic effects, volatile anesthetics have been shown to modulate the inflammatory stress response.1,6 This is of particular importance because anesthetics are exclusively administered to patients undergoing surgical procedures, already exposing them to a particular risk of developing infections. A compromised immune status could affect the postoperative infection rate, healing reactions, and the rate and size of tumor metastases disseminated during surgery.6,23–25 Numerous immunomodulatory and immunosuppressive effects of volatile anesthetics on leukocyte and lymphocyte function have been described in vitro.26–29
Here, we present evidence that sevoflurane and isoflurane induce the activation of p38 MAP kinase in Jurkat T-cells. This effect appears to be selective and specific, since (i) sevoflurane and isoflurane act in the same dose-dependent manner, whereas desflurane did not exert an effect, (ii) sevoflurane did not affect the phosphorylation of other MAP kinases such as ERK and JNK. The induction of p38 kinase activity could be completely abolished by the specific p38 inhibitor SB 202190. From our results we conclude that sevoflurane-mediated p38 activation and induction of apoptosis may be unrelated, because blocking of caspase processing by the general caspase inhibitor Z-VAD.fmk was not able to prevent phosphorylation of p38 by sevoflurane. Neither did inhibition of sevoflurane-mediated p38 phosphorylation by specific inhibitors attenuate caspase-3 processing and apoptosis in Jurkat T-cells.
The activation of the p38-MAP kinase pathway mediated by sevoflurane was associated with an activation of the upstream kinases ASK1 and MKK3/MKK6 and accompanied by the phosphorylation of a p38 target, the transcription factor ATF-2. Recent data provide evidence that the ATF/CREB family of transcription factors plays a functional role in ischemia and reperfusion injury. Very few physiologic activators of ATF-2 have been identified. These include reperfusion of ischemic organs and viral infection.30–33
While many stimulants may induce several MAP kinase pathways simultaneously, oxidative stress has been shown to selectively activate p38.34,35 Compared with propofol, volatile anesthetics augment both systemic and local (i.e., pulmonary) oxidative stress.36–39 Thus, it would be possible, and in agreement with our findings, that sevoflurane may activate the p38 MAP kinase pathway by inducing oxidative stress.
The MAP kinase p38 is involved in the molecular mechanisms of adaptive cellular functions after tissue injury due to ischemia and reperfusion.40 For example, activation of p38 MAP kinase by isoflurane before ischemia induces neuroprotection. This effect can be blocked using a p38 inhibitor (SB203580). Zheng et al.41 used a rat model wherein p38 MAP kinase was induced by isoflurane in the cerebral neocortex for at least 24 h. Subsequently, brain ischemic tolerance was measured. Animals, pretreated with isoflurane showed significantly smaller infarct sizes with less necrosis and apoptosis.41 Isoflurane similarly has a preconditioning effect against renal ischemia/reperfusion injury when administered before ischemia. In addition to p38 activation, an inhibition of JNK and ERK might be involved in this mechanism.42
However, little is known about the role of the MAP kinase cascade in apoptosis induced by volatile anesthetics. In the past, the role of p38 MAPK in the context of cell death was mostly considered to be sensitizing to or enhancing apoptosis. For example, it was shown that apoptosis induced in human fibroblasts by the nonsteroidal anti-inflammatory drug sodium salicylate was suppressed by a p38 MAP kinase inhibitor.18 Likewise, p38 MAP kinase inhibition protected PC12 cells from apoptosis after nerve growth factor withdrawal.43 Previous data in our model demonstrated that sevoflurane and isoflurane induce apoptosis in CD3+ T as well as in Jurkat T-cells via increased mitochondrial membrane permeability and caspase-3 activation, but independent of death receptor signaling.15 By contrast, desflurane did not exert any proapoptotic effects.15 Here we demonstrate that sevoflurane mediates the phosphorylation of ASK1 in Jurkat T-cells. ASK1 is a ubiquitously expressed MAP3-kinase, which is able to activate the JNK- and p38-MAP kinase signaling cascades. ASK1 appears to act as a proapoptotic intermediate and multifunctional stress-sensing kinase through mitochondria-dependent caspase activation and is required for apoptosis induced by oxidative stress, tumor necrosis factor, and endoplasmic reticulum stress.44–49
The phosphorylation and induction of p38 activity mediated by sevoflurane was observed in a dose-dependent manner and was also observed at in vitro concentrations, which are higher than those attained in the plasma of patients during administration of sevoflurane for general anesthesia, allowing for only careful conclusions regarding clinical practice.50,51 In addition, conclusions due to the use of Jurkat T-cells instead of primary CD3+ T-cells could be of limited value. However, Jurkat T-cells originate from a T-leukemia cell line and are widely used as a well established T-cell model.52 Besides, sevoflurane-mediated phosphorylation of p38 MAP kinase has been shown for primary CD3+ T-cells.16
We had originally developed three alternative hypotheses regarding the role of p38 in sevoflurane-induced apoptosis. We therefore conclude that the third hypothesis, stating that sevoflurane-mediated p38 activation and induction of apoptosis may be unrelated to one another is the most plausible. Induction of apoptosis leading to activation of p38, our first hypothesis, seems to be less likely, because inactivation of caspase-3 did not inhibit induction of p38 phosphorylation by sevoflurane. Neither could our second hypothesis be confirmed, stating a direct role for p38 in mediating apoptosis by sevoflurane, because treatment of cells with p38 inhibitors before sevoflurane exposure could not attenuate caspase-3 processing and apoptosis in Jurkat T-cells. In contrast, ERK and JNK have been shown to become activated by caspases.53
In summary, our data suggest that sevoflurane (and isoflurane) are specific inducers of the ASK1-, MKK3/MKK6-p38 MAP kinase cascade in Jurkat T-cells as illustrated in Figure 7. We further conclude that sevoflurane-induced p38 activation and sevoflurane-mediated apoptosis are two uncorrelated phenomena.
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