Anesth Analg 2004;98:1007-1012
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000106860.27791.44
ANESTHETIC PHARMACOLOGY
Xenon and Isoflurane Differentially Modulate Lipopolysaccharide-Induced Activation of the Nuclear Transcription Factor KB and Production of Tumor Necrosis Factor-
and Interleukin-6 in Monocytes
Lothar W. de Rossi*,
Martina Brueckmann
,
Steffen Rex*,
Marco Barderschneider*,
Wolfgang Buhre*, and
Rolf Rossaint*
*Department of Anesthesiology, University Hospital, Rheinisch-Westfälische Technische Hochschule Aachen; and
Department of Cardiology, University Hospital, Mannheim, Germany
Address correspondence and reprint requests to Lothar W. de Rossi, MD, Department of Anesthesiology, University Hospital, Aachen Pauwelsstr, 30, D-52074 Aachen, Germany. Address e-mail to Lderossi{at}ukaachen.de
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Abstract
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Anesthetics are known to interfere with the production of inflammatory cytokines. In this study, we investigated the effect of xenon and isoflurane on the lipopolysaccharide (LPS)-induced activation of the nuclear transcription factor (NF)-
B and production of tumor necrosis factor (TNF)-
and interleukin (IL)-6 in vitro. Whole blood was incubated with LPS in the absence or presence of the either xenon (30 and 60 Vol%) and isoflurane (1 and 2 minimum alveolar anesthetic concentration [MAC]). After 4 h, TNF- and IL-6 were assayed in the supernatant. Involvement of NF-B was investigated using isolated monocytes from the blood samples. Whole-cell lysates were prepared, and binding of the NF-B p50 and p65 subunit to its target DNA were measured with an enzyme-linked immunosorbent assay-based NF-B kit. LPS-induced production of TNF- and IL-6 as well as activation of NF-B were significantly increased in the presence of xenon compared with controls. In contrast, isoflurane inhibited the activation of NF-B, which was associated with a decreased production of TNF- and IL-6. Our results demonstrate that xenon and isoflurane have opposite effects on the LPS-induced production of TNF- and IL-6. Furthermore, xenon increases, whereas isoflurane inhibits the activation of NF-B, providing a possible molecular mechanism for the different effects on monocyte TNF- and IL-6 production.
IMPLICATIONS: This study has shown that monocytes respond to lipopolysaccharide (LPS) in the presence of xenon with an increased activation of nuclear transcription factor (NF)-B, whereas isoflurane inhibits LPS-induced activation of NF-B. These findings suggest a possible molecular mechanism for the different effects of both anesthetics on monocyte tumor necrosis factor- and interleukin-6 production.
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Introduction
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The innate immune response is largely dependent on monocyte function. These cells phagocytose and kill invading bacteria and coordinate the following immunological response by cytokine release as well as antigen presentation. Therefore, recruitment of monocytes to an inflammatory site in response to invading bacteria followed by cytokine release is a crucial step of the acute inflammatory reaction.
Volatile anesthetics have a variety of different effects on monocyte function, including suppressive effects on chemotaxis (1,2), phagocytosis (3), respiratory burst activity (4), cytokine release (5,6), and modulation of platelet-monocyte adhesion (7). We have previously demonstrated that xenon has no suppressive effects on monocyte phagocytosis and respiratory burst activity (4) in vitro. However, further studies about the effects of xenon on monocyte function are lacking. With the present study, we determined the effects of xenon on the lipopolysaccharide (LPS)-induced production of the inflammatory cytokines tumor necrosis factor (TNF)- and interleukin (IL)-6 in vitro.
In vitro inhibition of the release of TNF- and IL-1ß by cultured mononuclear cells after exposure to isoflurane have been reported (5). The underlying mechanisms of the isoflurane-induced effects are largely unknown. The nuclear transcription factor (NF)-B plays a central role in the expression of inflammatory cytokines after activation with LPS (8). Activation of Toll-like receptor 4 (TLR4) and CD14 by LPS results in activation of NF-B, which controls the transcription of cytokines, including TNF- and IL-6. The well-known signaling pathway involves a whole cascade of scaffolding and signaling proteins, resulting in phosphorylation of the inhibitory regulator protein IB and its degradation to allow nuclear translocation of NF-B. In the nucleus, NF-B binds to specific DNA sequences, encoding various cytokines, chemokines, immunoreceptors, and cell adhesion molecules. Accordingly, we further sought to determine whether xenon and isoflurane interfere with the LPS-induced activation of NF-B.
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Methods
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After approval of our institutional review board and informed consent, venous blood from healthy volunteers were taken who had no history of infections and had not ingested nonsteroidal antirheumatics for 1 wk before donation. Blood was collected into sterile blood collection tubes (Sarstedt, Nümbrecht, Germany) containing a 1/10 volume of 3.2% sodium citrate. Afterwards, citrated blood was further diluted 1:4 with 37°C prewarmed Roswell Park Memorial Institute 1640 medium (Sigma Chemical, St Louis, MO) in sterile polypropylene tissue culture dishes (Sarstedt). Blood samples were stimulated with 100 ng/mL of LPS from Escherichia coli O111:B4 (Sigma Chemical) 1 min before they were exposed for 4 h to the anesthetics.
To analyze the effects of the anesthetics on the spontaneous and LPS-induced cytokine response, diluted blood samples were incubated in two identical airtight boxes, as previously described (7,9). The control blood samples (control) were exposed in the first box to 21 vol% oxygen/5 vol% carbon dioxide at 37°C. In the second box, blood samples were either exposed to xenon (30 or 60 vol%) or isoflurane (1 or 2 minimum alveolar anesthetic concentration [MAC]) under the same atmospheric conditions. Xenon was delivered with a low-
P-mass flowmeter (Type F-201C-FA-22-V and E-7300-AAA, HI-TEC Bronkhorst, EC Veenendal, The Netherlands). Xenon concentrations within the box were monitored using an Ecotec 500 Euro gas analyser and measured with Masterquad V3.2MG software (both Leybold, Cologne, Germany). Isoflurane was delivered using an anesthetic machine (Sulla 909, Dräger, Lübeck, Germany). Concentrations of oxygen and carbon dioxide were continuously monitored with a multigas-analyser (Datex Compact, Helsinki, Finland). The isoflurane MAC value used in this study was 1.2 vol%.
At 4 h after the onset of anesthetic exposure, blood samples were centrifuged with 1000 g for 5 min at 4°C. The cell-free supernatant was stored at -80°C for later analysis of TNF- and IL-6.
TNF- and IL-6 levels in cell-free supernatants were assayed in duplicate and quantified by enzyme-linked immunosorbent assay (ELISA) (OptEIA®, Pharmingen, San Diego, CA) according to the manufacturer’s instructions. The lower detection limits of these assays were 4.7 pg/mL (IL-6) and 7.8 pg/mL (TNF- ), respectively.
Unstimulated and LPS-stimulated (E. coli O111:E4; 100 ng/mL) blood samples were incubated with air (control), 60 vol% xenon, or 1 MAC of isoflurane, as described above. Immediately after the end of the incubation, peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque density gradient centrifugation (Lymphoprep®, Nycomed, Norway). PBMC at the interface were harvested, washed twice with ice-cold PBS + 10% fetal calf serum and resuspended to a concentration of 1 x 107/mL. Isolation of monocytes from the PBMC suspension was performed with an immunomagnetic cell isolation technique, as recommended by the manufactures. In brief, PBMC (1 x 107/mL) were incubated for 30 min on ice with anti-CD14 monoclonal antibodies conjugated with uniform, superparamagnetic polystyrene beads (Dynabeads® M-450 CD14, Dynal Biotech ASA, Norway). Separation of monocytes was performed using a magnetic particle concentrator (Dynal MPC-L). Purity of the monocyte suspension, assessed by morphology, exceeded 98%. The cell suspension and used buffers were kept cold (2°C) during incubation and separation procedures to prevent artificial monocyte activation.
Whole-cell extracts from the monocyte suspension were prepared, as previously described (10), using a commercial kit (Nuclear Extract Kit, Actif Motif, Carlsbad, CA). After isolation, monocytes were washed with ice-cold PBS/phosphatase inhibitor and lysed with complete lysis buffer for 20 min on ice. Cell debris was isolated at 4°C by centrifugation (14,000 g) for 20 min. Supernatants were transferred to prechilled aliquots and stored at -80°C for NF-B analysis. Protein concentration in each sample was determined using a standard Bradford protein assay (Bio-Rad Laboratories, Milan, Italy). A quantity of 5 µg of protein from each whole-cell extract sample was used for the NF-B binding assay.
Binding of the NF-B subunit p50 and p65 to the NF-B binding sequence 5'-GGGACTTTCC-3'was measured with the ELISA-based Trans-Am NF-B kit (Actif Motif) using the whole-cell lysates prepared from the isolated monocytes. The Trans-Am NF-B ELISA kit consists of 96-well microtiter plates precoated with an oligonucleotide containing the appropriate NF-B binding consensus sequence. The active form of the p50 and p65 subunit is detected using antibodies specific for an epitope that is accessible only when the appropriate subunit has bound to its target DNA. Addition of a secondary antibody conjugated to horseradish peroxidase provided a colorimetric readout that was quantified by spectrophotometry. Optical density was read at 450 nm, and the results are expressed after subtraction of the blank values (use of lysis buffer instead of cell lysates). The assay was performed exactly according to the manufacturer’s instructions. A wild-type consensus oligonucleotide, provided as a competitor for NF-kB binding, and a mutated consensus oligonucleotide with no effect on NF-kB binding were used to monitor the specificity of the assay. Previously, the Trans-Am kit showed a good correlation and better sensitivity compared with an electrophoretic mobility shift assay in detecting the DNA binding activity of NF-kB (11).
The Kolmogorov-Smirnov test showed that the cytokine data were not normally distributed. Thus, results are expressed as median (25–75 percentile) unless otherwise indicated. Differences between the anesthetic exposed samples and control samples were tested by the Wilcoxon test. P < 0.05 was considered significant.
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Results
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After 4 h of incubation, spontaneous cytokine production of TNF- and IL-6 barely exceeded the lower detection limit of the ELISA assays in all samples. Both anesthetics did not alter the spontaneous cytokine production in comparison with unexposed control samples (data not shown).
In contrast to the lacking effect on the spontaneous cytokine release, xenon affected substantially the LPS-induced cytokine production. The TNF- and IL-6 concentrations in the supernatant of the untreated control samples and the xenon exposed samples after 4 h of incubation are shown in Figure 1. The LPS-induced TNF- production was increased by 128% (P < 0.05) in the 30 vol% xenon group and by 50% (P < 0.05) in the 60 vol% xenon group, as compared with controls. LPS-induced production of IL-6 was increased in the presence of xenon by 104% (P < 0.05) and 83% (P < 0.01), respectively.
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Figure 1. Effect of xenon on the lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)- and interleukin (IL)-6 in cultured human whole blood. Data represent median and range of eight independent experiments for each concentration of xenon. *P < 0.05 and #P < 0.01 compared with the control in the absence of xenon.
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Isoflurane significantly inhibited the LPS-induced release of TNF- by 48% at 1 MAC and by 50% (P < 0.05) at 2 MAC of isoflurane in comparison with unexposed control values (Fig. 2). Corresponding values for IL-6 were 42% and 55% (P < 0.05), respectively.
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Figure 2. Effect of isoflurane on the lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)- and interleukin (IL)-6 in cultured human whole blood. Data represent median and range of eight independent experiments for each concentration of isoflurane. *P < 0.05 and #P < 0.01 compared with the control in the absence of isoflurane.
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We explored a possible mechanism for the different effects of both anesthetics by examining the effect on NF-kB, the main transcription factor involved in the LPS-induced production of TNF- and IL-6. In the unstimulated blood samples, xenon did not alter nuclear translocation of both NF-kB subunits, but isoflurane significantly reduced binding of the p50 subunit to its target DNA by 20% (Fig. 3 and 4). LPS-induced activation of NF-kB strongly increased in the presence of 60 vol% xenon in monocytes compared with controls (Fig. 3 and 4). Binding of the NF-kB p50 and p65 subunit to its target DNA was significantly increased in the presence of xenon. In contrast, isoflurane partially reduced LPS-induced activation of NF-kB. After 4 h of incubation with 1 MAC of isoflurane, binding of both NF-kB subunits was reduced after stimulation with LPS as compared with control values (Fig. 3 and 4; P < 0.05).
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Discussion
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The main findings of our study are that xenon and isoflurane differentially modulated the LPS-induced production of TNF- and IL-6 in human whole blood. Xenon increased the LPS-induced secretion of both cytokines, in association with an increased activation of the transcription factor NF-B. In contrast, isoflurane reduced the secretion of TNF- and IL-6, which was not reversed within one hour after the end of the isoflurane exposure. Furthermore, the ability of isoflurane to inhibit the LPS-induced activation of the transcription factor NF-B may identify a possible underlying molecular mechanism.
Volatile anesthetics are known to modify leukocyte cellular metabolism and function. These effects may either be beneficial during inflammation (12) or be harmful in patients with preexisting immunological disorders (13). Recently, we have demonstrated that xenon, which is currently under intensive clinical investigation as an anesthetic (14), does not suppress neutrophil and monocyte phagocytosis and oxidative killing capacity in vitro (4). With the present study, we have determined the effects of xenon on the basal and LPS-induced production of the two important inflammatory cytokines TNF- and IL-6 in an established whole-blood model.
Xenon did not modify the basal concentration of TNF- and IL-6 in unstimulated whole blood but strongly increased the LPS-induced production of both cytokines. In contrast, isoflurane has been reported to attenuate the LPS-induced increase in TNF- (6), whereas sevoflurane had no effects (15). In addition, IV anesthetics also show mixed results concerning their effects on the LPS-induced production of TNF- (16). Comparable to xenon, propofol augmented the LPS-stimulated TNF- response, even in clinically used concentrations.
Most of our findings about the effect of isoflurane on monocyte cytokine secretion are in line with previous in vitro (5,6) and in vivo studies (12,13,17,18). Sato et al. (6) showed that isoflurane inhibits the LPS-induced secretion of TNF- and IL-1ß by peripheral blood mononuclear cells. In the current study, isoflurane additionally inhibited the LPS-induced secretion of IL-6, but we did not observe any dose-dependent effect of isoflurane on monocyte cytokine secretion. Interestingly, our results are also consistent with in vivo studies. As shown by Plachinta et al. (12), pretreatment with 1.4% isoflurane for 30 min inhibits the LPS-induced inflammation in rats, including an attenuated increase in TNF-. In humans, clinical investigations showed no alterations of the basal cytokine levels of TNF- and IL-6 under isoflurane anesthesia alone before surgery (13,17,18). In our study, the basal concentrations of both cytokines were not modified by isoflurane.
The transcription factor NF-B is a key factor in the immune response triggered by LPS and therefore a potential molecular target for the effects of xenon and isoflurane in monocytes. Upon engagement of LPS with TLR4 and CD14, several intracellular signal transduction pathways are activated, leading to NF-B activation (19). The signal transduction pathway includes the myeloid differentiation protein MyD88, IL-1-receptor associated kinase, tumor necrosis factor receptor-associated kinase TRAF 6, tumor growth factor-ß-activated kinase 1, and IB kinase. NF-B exists in the cytoplasm in an inactive form associated to the regulatory inhibitory protein IB. The most prevalent activated form of NF-B is a heterodimer consisting of a p50 and p65 subunit and the p50 homodimer. Upon phosphorylation of IB by IB kinase, NF-B subunits are released from its cytoplasmic anchor and translocate into the nucleus to initiate gene expression after binding to target DNA elements (20). The p50 subunit lacks a transactivation domain and is believed to serve as a regulatory subunit modulating the DNA-binding activity of p65. The p65 subunit, which contains transactivation domains required for gene induction, stimulates transcription of various inflammatory cytokines, such as IL-1, -2, -6, -8, and TNF-.
In the present study, we determined the activation of NF-B by measuring the active form of the p50 and p65 subunit, using antibodies specific for an epitome that is accessible only when the appropriate subunit has bound to its target DNA. We determined that xenon does not activate NF-B by itself in unstimulated blood samples. In contrast, LPS-induced binding of both NF-B subunits in monocytes is increased in the presence of xenon. Because binding of the NF-B p65 subunit to its target DNA stimulates transcription, this finding might provide a possible mechanism for the augmented monocyte cytokine response in the presence of xenon.
Interestingly, isoflurane inhibited nuclear translocation of the NF-B p50 subunit in the unstimulated blood samples but not the activation of the p65 subunit. This inhibition of the p50 subunit was not associated with an effect of isoflurane on spontaneous cytokine release, which might be explained by the missing transactivation domain of p50. However, our observation suggests that isoflurane suppresses the LPS-induced TNF- and IL-6 production by inhibiting the activation of NF-B. However, the molecular mechanisms by which xenon and isoflurane modify the activation process of NF-B remain to be determined.
At present, it is unclear whether the augmented cytokine response by xenon is significant for the clinical use of xenon as an anesthetic. However, propofol also increased the LPS-stimulated TNF- response in clinically used concentrations, but reports about complications because of an overwhelming proinflammatory reaction after the application of propofol are missing.
In conclusion, our study demonstrated that xenon and isoflurane differentially modify the LPS-induced activation of the transcription factor NF-B in monocytes. These findings might also provide a possible molecular mechanism for the augmented LPS-induced release of TNF- and IL-6 under xenon as well as for the inhibitory effects of isoflurane.
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Acknowledgments
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Supported, in part, by START, a research grant of the Rheinisch-Westfälische Technische Hochschule Aachen, Germany.
This study was supported in parts by START, a research grant of the Rheinisch-Westfälische Technische Hochschule Aachen, and the Department of Anaesthesiology, University Hospital, Rheinisch-Westfälische Technische Hochschule, Aachen, Germany. Xenon was donated by Messer GmbH, Griesheim, Germany.
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Accepted for publication October 28, 2003.
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Abstract
Introduction
