The modulation of immune function by trauma, surgical intervention and anaesthesia is thought to play a crucial role in the development of postoperative disorders. Anaesthetics have been suspected to impair various aspects of immune function either directly by affecting the function of immunocompetent cells or indirectly through modulation of the stress response [1,2]. A number of previous studies have investigated the effects of intravenous (i.v.) and inhalational anaesthetics on cells involved in the non-specific and specific immune response, but the results are partially conflicting. [3-6]. Since the effects of clinically used combinations of anaesthetics agents on the T-cell immune response have scarcely been investigated, the aim of the present study was to compare the concentration-dependent effects of single agents with those of clinically used agent combinations and to assess possible enhancement of compensatory effects of anaesthetic agent combinations. Based on in vivo data, we hypothesized that the use of anaesthetic agent combinations might affect the immune functions in a different manner.
Material and methods
The study was approved by the local Ethics Committee and informed consent was obtained from the participants. Eighty millilitres of peripheral venous blood was collected from 28 healthy volunteers (age 23-45 yr).
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated from heparinized peripheral blood by Biocoll/Amidotrizoate density gradient centrifugation (Biochrome, Berlin, Germany). For proliferation assays, the PBMC (2 × 106 cells mL−1) were suspended in Iscove's modified Dulbecco's medium plus 10% heat-inactivated fetal calf serum (with L-glutamine, without antibiotics; medium and fetal calf serum from GIBCO, Germany). The cells were cultured in quadruplicate for 48 h (at 37°C and 5% CO2 in NUNC 96-well, flat-bottom, microtitre plates) in the absence or presence of the mitogenic stimulus phytohaemagglutinin (PHA, 1 μg mL−1, Genzyme Virotech, Germany) and of i.v. anaesthetic agents (propofol, thiopental, fentanyl, sufentanil) and clinically used combinations (propofol plus fentanyl or sufentanil, and thiopental plus fentanyl or sufentanil). The following final agent concentrations were used: 1, 4 and 10 μg mL−1 propofol; 10, 50 and 100 μg mL−1 thiopental; 5, 10 and 20 ng mL−1 fentanyl; 0.5, 2.5 and 10 ng mL−1 sufentanil. The doses of anaesthetics were calculated by assuming a body weight of 70 kg and a plasma volume of 5 L. For exposure to volatile anaesthetics the open polystyrene containers with the mononuclear cell cultures in 10 mL Iscove's medium were placed into airtight flow chambers under sterile conditions. Air (control group) or volatile anaesthetics such as nitrous oxide/oxygen (inspired oxygen fraction (FiO2): 0.3), sevoflurane (2.0%) in oxygen/air (FiO2: 0.3) or sevoflurane (2.0%) in nitrous oxide/oxygen (FiO2: 0.3) were delivered into the chambers at a rate of 3 L min−1. Flow rates and volatile concentrations were monitored with a gas analyser (Datex-Ohmeda, Instrumentarium Corp., Finland). For the investigation of anaesthetic agent combinations, thiopental (50 μg mL−1) was added to the cell culture media. After 1 h of exposure to the volatile anaesthetics, the cell culture containers were sealed with gas-permeable tops and cultured in the absence or presence of 1 μg mL−1 PHA for 48 h. Proliferation rates were determined by 3H-thymidine incorporation (6 Ci mmol−1 added 6 h prior to harvesting) as counts per minute (cpm) using an automated well-type scintillation counter. The results were summarized from six independent experiments (six different blood donors).
The concentrations of interleukin (IL)-2 and soluble interleukin-2 (sIL-2) receptor in culture supernatants of PHA-activated PBMC (cultured for 48 h in 12-well flat-bottom plates at 37°C and 5% CO2, each substance and concentration in duplicate) were determined by using commercially available enzyme immunoassays (R&D Systems, Minneapolis, USA). The data given represent the summarized results from five independent experiments.
All data were analysed using SPSS (Statistical Package for the Social Sciences, Chicago, IL, USA). Values were tested for normal distribution with Kolmogorov-Smirnov-test. All data were normally distributed. Analysis of variance for repeated measures followed by paired, two-tailed t-test was used for intragroup analysis. Data were expressed in the tables as mean and SD. Data in the figures are represented by box plots with median and the interquartile range. A value of P < 0.05 was considered to be statistically significant.
In this study, the effects of single anaesthetic agents as well as combinations of agents on immune cell functions were assessed.
Thiopental inhibited PHA-induced proliferation of isolated PBMC from healthy donors in a concentration-dependent manner. The proliferation inhibition caused by thiopental was much more pronounced than that of propofol, which tended to be significant only at higher concentrations. Fentanyl was found to have no effect on 3H-thymidine uptake, whereas sufentanil in concentrations of 2.5 and 10 ng mL−1 induced a significant reduction of PHA-induced lymphocyte proliferation (Fig. 1).
The proliferative response of isolated PBMC in the presence of thiopental (50 μg mL−1) plus fentanyl (10 ng mL−1) or sufentanil (2.5 ng mL−1) was reduced to the same degree (inhibition by 40%) as compared to thiopental alone. Likewise, fentanyl and sufentanil had no additional effects on the propofol-induced alterations of PBMC proliferation (Fig. 2).
Fentanyl and sufentanil alone were found to have no effects on the PHA-induced IL-2 or sIL-2R release of treated cells. The release of IL-2 into the culture supernatant tended to be higher at concentrations of 100 μg mL−1 thiopental (from 1760 to 3180 pg mL−1; P = 0.09). Thiopental and propofol caused a significant reduction of sIL-2 receptor shedding at higher concentrations (100 μg mL−1 for thiopental and 10 μg mL−1 for propofol; Table 1).
Exposure of freshly isolated PBMC to nitrous oxide induced a slight, but significant depression of PHA-induced proliferation (P < 0.05), whereas exposure to sevoflurane did not change the PBMC proliferation rate. The combination of sevoflurane and nitrous oxide slightly increased proliferation. However, this effect was not statistically significant (Fig. 3). The PHA-induced release of the T-cell growth factor IL-2 or the shedding of the IL-2 receptor was not affected by sevoflurane and/or nitrous oxide (Table 2).
The anaesthetic agent combination commonly used in a balanced anaesthetic technique consisting of thiopental (50 μg mL−1) plus sevoflurane (2.0%) in nitrous oxide/oxygen (FiO2: 0.3) did not induce a significant reduction of the PHA-induced PBMC proliferation rate or release of T-cell growth factor IL-2 and soluble IL-2 receptor (Fig. 3).
The main result of the present study is that the previously described strong immunosuppressive effects of thiopental are not enhanced by other anaesthetic agents. Moreover, for certain substance combinations, we observed compensatory effects, which might be beneficial for immune-compromised patients.
Immune suppression in patients with trauma, shock or sepsis is associated with high morbidity and mortality . In patients undergoing major surgery, the depressed postoperative immune response is mainly attributed to surgery. Previous reports had demonstrated that anaesthetics can influence various aspects of lymphocyte function in vitro and in vivo, but the results of these were often contradictory [3,8]. Barbiturates, such as thiopental, have already been shown to reduce the growth of PHA-stimulated PBMC or isolated T-lymphocytes [6, 8-10]. The depressed proliferative T-cell response makes patients susceptible to postoperative infections . Consequently, it has been suggested that barbiturates administered over long periods may cause iatrogenic immunosuppression. Indeed, a higher incidence of infections has been described in head injured patients receiving prolonged infusions of thiopental to control increased intracranial pressure [12,13].
In our hands, thiopental alone caused a significant decrease of up to 50% of mitogen-induced PBMC proliferation even in concentrations which are typically attained after induction of anaesthesia. Thiopental has an elimination half-life of approximately 11 h and may accumulate during long-term administration. Thus, the in vivo effects of thiopental on the proliferative capacity of lymphocytes may be of particular relevance. The reduction of mitogen-induced PBMC proliferation is comparable to that induced by 10−7 mol L−1 methylprednisolone .
The activation of T- and B-lymphocytes is characterized by the release of the autocrine growth factor IL-2, the upregulation of the high-affinity IL-2 receptor trimer and the simultaneous release of a soluble form of the IL-2 receptor α-subunit (shedding). Dysregulation of IL-2-production and IL-2-receptor expression in the accessory signalling pathways have been shown to result in immune defects or autoimmune diseases . The immunosuppressive effects of thiopental are associated with a marked reduction in the density of IL-2 receptors on the cell membrane and the numbers of cells expressing IL-2 receptors . In the present study, a significant decrease of the concentration of soluble IL-2 receptors in culture supernatants of thiopental-treated cells was observed, which confirms the previous reports .
Interestingly, we found an increased IL-2 production in the presence of higher thiopental concentrations. Since diminished lymphocyte proliferation is often associated with decreased IL-2 levels [3,16,17], this unexpected finding was validated by additional experiments. All further experiments, even additionally performed quantitative polymerase chain reaction (PCR) measurement of IL-2 transcripts (data not shown), confirmed the initial data. However, these results are in contradiction to a previous report of Loop and colleagues, who described a decrease in the production of the IL-2, -6 and -8, as well as interferon-γ by CD3(+) lymphocytes. This group provided data suggesting that thiopental interferes with activation of nuclear transcription factor κB in T-lymphocytes, which is probably mediated via the suppression of IκB kinase .
Based on the differences in experimental design, it cannot be ruled out that in our experiments using PBMC preparations, other leucocyte sub-types such as B-cells or natural killer (NK) cells contributed mainly to the increased IL-2 levels. Direct effects of thiopental on these cells have not been studied in detail as yet.
In previous studies, Le Grue, Spiers and colleagues reported that the immunosuppressive effects of barbiturates could not be restored by the addition of exogenous IL-2, which indicates that the inhibitory effects of barbiturates on proliferation are not directly IL-2 dependent. Furthermore, they provided first evidence for cytotoxic effects of thiopental [8,17]. Salo and colleagues described the decreased release of IFN-γ and IL-4 with unaffected IL-2 release . Taken together these data suggest that the reduction of PHA-induced proliferation in the presence of thiopental is due to a down-modulation of the high-affinity IL-2 receptor expression rather than to reduced levels of the main lymphocyte growth factor IL-2.
Propofol caused a mild inhibition of PHA-induced PBMC proliferation at high concentrations in this study. These data are in accordance with results published by Pirttikangas and colleagues, who observed a proliferation suppressing effect of propofol in vitro only in PBMC obtained from critically ill patients, who were primarily immunosuppressed . Devlin and colleagues investigated the effects of thiopental and propofol on lymphocyte proliferation after PHA stimulation. In their studies, neither propofol nor its solvent intralipid caused T-lymphocyte depression. The authors concluded that propofol may be the safest drug for patients receiving prolonged surgery or for sedation in the intensive care unit .
While the release of sIL-2R was depressed in the presence of higher propofol concentrations, IL-2 production by PBMC was found to increase with increasing propofol concentrations. Salo and colleagues did not find any effect of propofol on IL-2 production, but described an increased production of the TH1 cytokine interferon-γ by isolated T-cells with an increase in the IFN-γ/IL-4 ratio at propofol concentrations up to 10 μg mL−1 . In addition, barbiturates, but not propofol, suppressed the activation of transcription factor nuclear κB in human T-cells . These findings suggested that the NF-κB pathway is a target for the immunosuppressive effect of thiopental. Galley and colleagues studied the inhibitory aspect of thiopental on the activity of nitric oxide synthetase from human polymorphonuclear leucocytes . Humar and colleagues provide evidence that the nuclear factor of activated T-cells is a target of barbiturate-mediated immunosuppression in human T-cells . Thus, the barbiturate effect is probably due to the cytoplasmatic transduction cascade rather than the previously described effects mediated through GABA-receptors on T-lymphocytes .
Opioids, such as fentanyl and sufentanil may affect the immune function directly or indirectly, but data on specific immune cell functions are scarce. In the present study, fentanyl did not influence PHA-induced lymphocyte proliferation in clinically relevant concentrations. Sufentanil in higher concentrations suppressed the mitogen-induced T-cell response, while the release of IL-2 and sIL-2 receptor was unaffected. Likewise, neither substance had an influence on the immunosuppressive effects of thiopental or propofol.
There are well-documented, dose-dependent, immunosuppressive effects of morphine, which is known to impair monocyte and neutrophil function, NK cell-mediated cytotoxicity, lymphocyte proliferation and cytokine release. Morphine promotes apoptosis in lymphocytes and macrophages by activating enzymes involved in apoptotic cell death. Furthermore, it affects nitric oxide release and inhibits cell adhesion. Opioids are known to exert their effects via specific opioid receptors expressed on immunocompetent cells. Recent studies estimating the effects of synthetic opioids used in general anaesthesia showed no more transient immunomodulatory changes [24-26].
Yeager and colleagues found enhanced NK-cell cytotoxicity and increased relative number of CD16(+) and CD8(+) after an i.v. bolus dose (3 μg kg−1) and subsequent infusion of fentanyl (1.2 μg kg−1 h−1 for 2 h) in healthy volunteers . In a similar study, fentanyl increased the NK-cell (CD16(+)/CD56(+)) number, but superoxide production of polymorphonuclear cells and the number of circulating B- and T-lymphocytes remained unchanged . These results suggest a centrally mediated rather than a direct effect of fentanyl on NK-cells.
Previous in vitro studies have provided evidence that volatile anaesthetics might alter the immune response. In cultured human leucocytes, halothane inhibited mitogen-induced RNA and protein synthesis, and depressed the secretion of IFN-γ . In rats exposed to halothane for up to 5 h, lymphocytes from the spleen revealed reduced mitogen-induced proliferation and IL-2 receptor expression . Extended exposure suppressed the mitogen-induced lymphocyte proliferation and the expression of the IL-2 receptor. Mitsuhata and colleagues investigated the effects of volatile anaesthetics (sevoflurane, isoflurane, enflurane) in clinically relevant concentrations on the cytokine release of human PBMC-stimulated NK sensitive tumour cells. None of the anaesthetics reduced the levels of IL-2 .
The volatile anaesthetics tested in the present study had different effects on PBMC functions: exposing the cells to nitrous oxide decreased the proliferative capacity of PHA-activated PBMC, while exposure to sevoflurane had no effect. Surprisingly, the combination of both volatile anaesthetics induced a slight increase in the proliferation rate. This might suggest a protective effect of sevoflurane. Interestingly, a comparable compensatory effect was achieved by treating PBMC with thiopental plus sevoflurane in nitrous oxide. The depressed mitogen-induced lymphocyte proliferation in the experimental setting using thiopental, sevoflurane and nitrous oxide seems to be mediated by thiopental rather than sevoflurane.
Sevoflurane was found to be beneficial in coronary surgery patients . The mechanism of these different cellular effects remains to be elucidated, but it has been hypothesized that it involves reducing oxidative stress.
Horn and colleagues recently reported a promoting effect of sevoflurane on the binding of platelets to the surface of lymphocytes, neutrophils and monocytes. Sevoflurane increased the expression of P-selectin, a transcription factor AP-1 regulated mediator of platelet-leucocyte adhesion . In contrast, other authors described the induction of lymphocyte apoptosis by sevoflurane, which may be due to the increased caspase 3-like activity seen at high concentrations of volatile anaesthetics. The lymphocytotoxicity of isoflurane was greater than that of sevoflurane . Recently, Loop and colleagues demonstrated that sevoflurane is a specific inhibitor of the activator protein-1 (AP-1) in isolated T-lymphocytes, while isoflurane did not exert any inhibition. AP-1 controlled the production of IL-2, which could explain the anaesthetic-induced apoptosis in lymphocytes . The authors postulated that variations in the total number and subsets of circulating T-lymphocytes as well as the decreased neutrophil respiratory burst observed during inhalational anaesthesia would be consistent with the inhibition of AP-1.
Possible molecular mechanisms involved in the depression of the T-cell response by volatile anaesthetics may include an impairment of calcium ion influx, modulation of the adenylate cyclase phosphodiesterase enzymatic balance, altered signal transduction and gene transcription, inhibition of nitric oxide production and expression of the inducible nitric oxidase synthetase [35-38].
In summary, our data showed that among the tested anaesthetic agents, only thiopental and nitrous oxide had a significant inhibitory effect on the activation of freshly isolated human PBMC. The combination of these immunosuppressive substances with other anaesthetic agents may have different effects.
Opioids such as fentanyl and sufentanil do not influence the immunosuppressive effects of thiopental or propofol. The immunosuppressive effect of thiopental was associated with a marked reduction of IL-2 receptor expression. Neither nitrous oxide nor sevoflurane reduced IL-2 or IL-2 receptor expression.
The authors would like to thank Ines Meinert and Nicole Seliger for the excellent technical assistance, Anke Lux and Uwe Schmidt for the helpful advice with the statistical methods and the colleagues from the Department of Anaesthesiology of the University, Magdeburg for their support and helpful discussions.
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