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.
1. Salo M. Effects of anaesthesia and surgery on the immune response. Acta Anaesthesiol Scand
2. McBride WT, Armstrong MA, McBride SJ. Immunomodulation: an important concept in modern anaesthesia. Anaesthesia
3. Correa-Sales C, Tosta CE, Rizzo LV. The effects of anesthesia with thiopental
on T lymphocyte responses to antigen and mitogens in vivo
and in vitro
. Int J Immunopharmacol
4. Hamra JG, Yaksh TL. Halothane inhibits T cell proliferation
and interleukin-2 receptor expression in rats. Immunopharmacol Immunotoxicol
5. Mikawa K, Akamatsu H, Nishina K et al. Propofol
inhibits human neutrophil functions. Anesth Analg
6. Galley HF, Webster NR. Effects of propofol
and thiopentone on the immune response. Anaesthesia
7. Angele MK, Faist E. Clinical review: immunodepression in the surgical patient and increased susceptibility to infections. Crit Care
8. Devlin EG, Clarke RSJ, Mirakhur RK, McNeil TA. Effects of four i.v. induction agents on T-lymphocyte proliferations to PHA in vitro
. Br J Anaesth
9. Spiers EM, Potts RC, Simpson JR, MacConnachie A, Beck JS. Mechanisms by which barbiturates suppress lymphocyte responses to phytohaemagglutinin stimulation. Int J Pharmacol
10. Devlin EG, Clarke RSJ, Mirakhur RK, McNeill TA. The effects of thiopentone and propofol
on delayed hypersensitivity reactions. Anaesthesia
11. Hensler T, Hecker H, Heeg K et al.
Distinct mechanisms of immunosuppression as a consequence of major surgery. Infect Immun
12. Stover JF, Stocker R. Barbiturate coma may promote reversible bone marrow suppression in patients with severe isolated traumatic brain injury. Eur J Clin Pharmacol
13. Eberhardt KE, Thimm BM, Spring A, Maskos WR. Dose-dependent rate of nosocomial pulmonary infection in mechanically ventilated patients with brain oedema receiving barbiturates: a prospective case study. Infection
14. Briggs WA, Eustace J, Gimenez LF, Choi MJ, Scheel Jr PJ, Burdick JF. Lymphocyte suppression by glucocorticoids with cyclosporine, tacrolimus, pentoxifylline, and mycophenolic acid. J Clin Pharmacol
15. Hanisch UK, Quirion R. Interleukin-2 as a neuroregulatory cytokine. Br Res Rev
16. Yang KD, Liou WY, Lee CS, Chu ML, Shaio MF. Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation. J Leukoc Biol
17. Loop T, Liu Z, Humar M et al. Thiopental
inhibits the activation of nuclear factor kappaB. Anesthesiology
18. Le Grue SJ, Munn CG. Comparison of the immunosuppressive effects of cyclosporine, lipid-soluble anesthetics, and calmodulin antagonists. Response to exogenous interleukin 2. Transplantation
19. Salo M, Pirttikangas CO, Pulkki K. Effects of propofol
emulsion and thiopentone on T helper cell type-1/type-2 balance in vitro
20. Pirttikangas CO, Perttila J, Salo M. Propofol
emulsion reduces proliferative responses of lymphocytes from intensive care patients. Intens Care Med
21. Galley HF, Nelson LR, Webster NR. Anaesthetic agents decrease the activity of nitric oxidase synthase from human polymorphonuclear leukocytes. Br J Anaesth
22. Humar M, Pischke SE, Loop T et al.
Barbiturates directly inhibit the calmodulin/calcineurin complex: a novel mechanism of inhibition of nuclear factor of activated T cells. Mol Pharmacol
23. Tian J, Chau C, Hales TG, Kaufmann DL. GABA(A) receptors mediate inhibition of T cell responses. J Neuro-immunol
24. McCarthy L, Wetzel M, Sliker JK, Eisenstein TK, Rogers TJ. Opioids, opioid receptors, and the immune response. Drug Alcohol Depend
25. Welters ID, Menzebach A, Goumon Y et al.
Morphine inhibits NF-κB nuclear binding in human neutrophils and monocytes by a nitric oxide dependent mechanism. Anesthesiology
26. Bidlack JM. Detection and function of opioid receptors on cells from the immune system. Clin Diagn Lab Immunol
27. Yeager MP, Procopio MA, DeLeo JA, Arruda JL, Hildebrandt L, Howell AL. Intravenous fentanyl
increases natural killer cell cytotoxicity and circulating CD16(+) lymphocytes in humans. Anesth Analg
28. Jacobs R, Karst M, Scheinichen D et al.
Effects of fentanyl
on cellular immune functions in man. Int J Immunopharmacol
29. Bruce DL. Halothane inhibition of RNA and protein synthesis of PHA-induced human lymphocytes. Anesthesiology
30. Mitsuhata H, Shimizu R, Yokoyama MM. Suppressive effects of volatile anaesthetics on cytokine release in human peripheral blood mononuclear cells. Int J Immunopharmacol
31. De Hert SG, Ten Broecke PW, Mertens E et al. Sevoflurane
but not propofol
preserves myocardial function in coronary surgery patients. Anesthesiology
32. Horn NA, de Rossi L, Robitzsch T, Hecker KE, Hutschenreuter G, Rossiant R. The effects of sevoflurane
and desflurane in vitro
on platelet-leukocyte adhesion in whole blood. Anaesthesia
33. Matsuoka H, Kurosawa S, Horinouchi T, Kato M, Hashimoto Y. Inhalation anesthetics induce apoptosis in normal peripheral lymphocytes in vitro
34. Loop T, Scheiermann P, Doviakue D et al. Sevoflurane
inhibits phorbol-myristate-acetate-induced activator protein-1 activation in human T lymphocytes in vitro
: potential role of the p38-stress kinase pathway. Anesthesiology
35. Stevenson GW, Hall SC, Rudnick S, Seleny FL, Stevenson HC. The effect of anaesthetic agents on the human immune response. Anesthesiology
36. Ferrero E, Ferrero ME, Marni A et al.In vitro
effects of halothane on lymphocytes. Eur J Anaesthesiol
37. Tschaikowsky K, Ritter J, Schröppel K, Kühn M. Volatile anesthetics differentially affect immunostimulated expression of inducible nitric oxide synthase: role of intracellular calcium. Anesthesiology
38. Nakamura K, Terasako K, Toda H et al.
Mechanisms of inhibition of endothelium-dependent relaxation by halothane, isoflurane, and sevoflurane
. Can J Anaesth
Keywords:© 2005 European Society of Anaesthesiology
ANAESTHETICS INTRAVENOUS; propofol; thiopental; ANALGESICS OPIOID; fentanyl; sufentanil; ANAESTHETICS INHALATIONAL; sevoflurane; nitrous oxide; CELL PROLIFERATION; human mononuclear; LYMPHOCYTE ACTIVATION; CYTOKINES