Abstract

Ketamine, a noncompetitive N-methyl-d-aspartate receptor antagonist, is widely used as an intravenous anesthetic agent. It is known to produce increases in blood pressure and stroke volume, which implies its importance in clinical practice. Ketamine has also been shown to possess anti-inflammatory effects. Our previous studies showed that ketamine, at clinically relevant concentrations, can downregulate endotoxin-induced macrophage activation through toll-like receptor-dependent activation of mitogen-activated protein kinases and the transcription factors nuclear factor-kappa B and activator protein-1. As to the responsible mechanisms, considerable attention was devoted to ketamine-involved regulation of proinflammatory gene expression. The assessment of how ketamine regulates proinflammatory gene expressions is significant in determining the signal cascades that are influenced by this anesthetic agent and its clinical application in the tactical use of ketamine in preventing sepsis. Herein, we review the literature on the pharmacodynamics, pharmacokinetics, and possible mechanisms involved in ketamine's immunology.

Keywords

immunosuppression; immunity, innate; ketamine; mitogen-activated protein kinases; signal transduction; toll-like receptors;

1. Introduction

Ketamine, 2-(2-chlorophenyl)-2-(methylamino)-cyclohexanone, was developed by Parke Davis Laboratories in 1962 and was first described in 1965, after assessing the side effects of behavioral and psychological changes associated with the earlier product, phencyclidine.1, 2 Ketamine is a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist that can block the excitation of the NMDA receptor induced by glutamate and aspartate.² Among nonvolatile anesthetics, ketamine has more stable hemodynamics than other anesthetic agents; it is used in various clinical areas, including pediatric anesthesia, traumatic anesthesia, and obstetrics; and is quite widely serviceable in operating rooms and intensive care units.³ Clinical analyses in both human and animal studies also showed that ketamine has possible immunomodulatory and anti-inflammatory effects. In this article, we review the literature on the immunomodulatory effects of ketamine on immune cells and its possible signal-transducing mechanisms.

2. Immunosuppressive effects of ketamine on immune cells

Some immune cells protect hosts from infection by organisms in a nonspecific manner, and these cells with their mechanisms comprise the innate immune system. Inflammation is one of the first responses of the immune system to infection. Macrophages and other cells of the innate immune system initiate inflammatory responses by producing cytokines, which include tumor necrosis factor (TNF)-α, high mobility group box 1, interleukin (IL)-1, IL-6, and IL-8.⁴ Leukocytes comprising the innate immune system include natural killer (NK) cells, mast cells, eosinophils, and basophils, while phagocytes include macrophages, neutrophils, and dendritic cells. These innate leukocytes function within the immune system by identifying and eliminating pathogens that intrude into a host.⁵

Ketamine was shown, in both human and animal models, to possess anti-inflammatory effects, and several studies provided in vitro data demonstrating that this intravenous anesthetic could inhibit the function of lymphocytes, NK cells, and neutrophils.6, 7, 8 NK cells can recognize and kill the cells infected by a virus and a variety of tumor cells during the metastatic process.⁹ Melamed et al¹⁰ analyzed the count of NK cells in rat blood, and the results of flow cytometry showed a reduction of 29% in the NK-cell count after exposure to ketamine (Table 1). The NK-cell cytotoxic activity was significantly reduced by ketamine, and an injected lung tumor showed increased metastasis after ketamine treatment. Estes et al¹¹ also found that NK-cell activity and cell numbers were reduced by 28% and 33%, respectively, in rats after being anesthetized using isoflurane and ketamine.

Table 1. Effects of ketamine on immunosuppression.

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Neutrophils play a major role in host defense. They rapidly migrate through vascular endothelial cell monolayers to the site of infection and destroy the invading bacteria by changing their morphology from rounded and relatively smooth cells to elongated and ruffled cells with pseudopodia.³² Adhesion of leukocytes to the endothelium is mediated by distinct families of adhesion molecules.³³ Schmidt et al determined the rates of leukocyte adherence and changes in microhemodynamics in rat mesenteric venules after exposure to ketamine, using in vivo video microscopy. A 6.3-fold increase in the number of adherent leukocytes was observed after administration of endotoxins, while in ketamine-pretreated rats, only a 2.6-fold increase in leukocyte adherence occurred after endotoxin exposure, suggesting a reduced expression of adhesion molecules.³⁴ In lipopolysaccharide (LPS)-stimulated leukocytes, ketamine was reported to attenuate cell adherence and migration of human neutrophils through human umbilical endothelial cell monolayers to 52%.³⁵ Weigand et al¹² used adhesion molecules as markers and found that ketamine inhibited upregulation of CD18 and CD62L in activated human neutrophils (Table 1). CD11b and CD16 can bind to complement- or immunoglobulin-opsonized particles and microorganisms, and enhance their phagocytosis.¹³ Welters et al¹⁴ studied the effects of racemic ketamine on expressions of CD11b and CD16 by neutrophils after stimulation of whole blood with LPS. They found that LPS-stimulated increases in both CD11b and CD16 expressions by human neutrophils were significantly inhibited by ketamine.

3. Ketamine-involved downregulation of chemotactic activity and oxidant production

Chemotactic activity and oxidant production by neutrophils were also demonstrated to be suppressed by ketamine.12, 36 Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a critical regulator of the production of superoxide anions by neutrophils; p47^phox is one subunit of NADPH oxidase, and its phosphorylation upregulates superoxide anion production.³⁷ Sakamoto et al³⁸ found that the p38 mitogen-activated protein kinase (MAPK) pathway is important for chemotactic activity, and plays a crucial role in NADPH oxidase activation and superoxide anion generation in neutrophils. p38 MAPK is a subfamily of MAPKs. Compared to an inhibitor specific for p38 MAPK, Lu et al²⁸ found that ketamine has a similar effect to SB203580 of inhibiting the phosphorylation of p47phox and generating superoxide anions by neutrophils by suppressing p38 MAPK activation. Both ketamine and SB203580 inhibit superoxide generation, and phosphorylation of the NADPH oxidase p47^phox subunit and p38 MAPK. Similar inhibitory effects between ketamine and SB203580 strongly suggest that ketamine is likely to inhibit p38 MAPK activation and then downregulate phosphorylation of p47^phox and generation of superoxide anions by neutrophils (Table 1).²⁸

4. Ketamine-involved inhibition of TNF-α, IL-1β, and IL-6 gene expressions

As a type of neutrophil, macrophages are also important in cellular host defense against infection and tissue injury. Macrophages can destroy invading microorganisms and tumor cells during inflammation by adopting reactions such as chemotaxis, phagocytosis, oxidant production, and inflammatory cytokine release.³⁹ In LPS-activated macrophages, ketamine was reported to reduce TNF-α and nitric oxide production, both of which play critical roles during inflammation, and inhibition of these activities may affect macrophage-mediated immunity (Table 1).15, 40 To evaluate the possible mechanism of ketamine-induced immunosuppression in macrophages, Chang et al¹⁶ studied TNF-α, IL-1β, and IL-6 messenger RNA (mRNA) syntheses in murine macrophages and found that ketamine inhibited mRNA syntheses by LPS-activated macrophages. Therefore, those studies also provide in vitro data to support the theory that ketamine exerts suppressive effects on macrophage functions via inhibition of phagocytic activities, oxidative ability, and TNF-α, IL-1β, and IL-6 mRNA syntheses; ketamine-induced suppression of TNF-α and IL-6 syntheses occurs at the transcriptional level (Table 1). Chang et al also found that ketamine can reduce the mitochondrial membrane potential. Because depolarization of the mitochondrial membrane can lead to mitochondrial dysfunction or even cell insult,⁴¹ they proposed that ketamine suppresses macrophage function by reducing the mitochondrial membrane potential but not through a reduction in cell viability, which could possibly be one of the mechanisms explaining the suppression of macrophage functions.¹⁶

5. Roles of nuclear factor-κB and AP-1 in ketamine-induced gene inhibition

Studies showed that ketamine can inhibit TNF-α gene expression through suppression of nuclear factor (NF)-κB activation (Table 1).7, 29 During inflammation, LPS first binds to the LPS-binding protein (LBP) in the bloodstream.⁴² Then, the LPS–LBP complex induces certain gene expressions via toll-like receptor (TLR)-dependent mechanisms. TLRs, which are type-I transmembrane proteins with extracellular domains, consist largely of leucine-rich repeats and intracellular signaling domains; at least 12 members of TLRs have been found in mammalian cells.⁴³ TLRs recognize essential structures expressed by pathogens and also some endogenous mediators released by injured tissue.⁴⁴ During inflammation, LPS first specifically activates TLR4 on macrophages, and then stimulates translocation of the transcription factors, NF-κB and activator protein (AP)-1, from the cytoplasm to nuclei, eventually inducing gene expression of inflammatory cytokines such as TNF-α and IL-6.⁴⁵ To determine the signal-transducing mechanisms of ketamine-caused inhibition of TNF-α and IL-6 gene expressions in LPS-activated macrophages, Wu et al³⁰ evaluated phosphorylation of c-Jun N-terminal kinase (JNK)1/2 and translocations of c-Jun and c-Fos from the cytoplasm to nuclei in macrophages (Table 1). They found that treatment with ketamine not only decreased LPS-induced JNK1/2 phosphorylation but also inhibited LPS-caused increases in nuclear c-Jun and c-Fos. Wu et al³⁰ suggested that ketamine reduces the biosynthesis of TNF-α and IL-6 in LPS-activated macrophages through suppression of TLR4-dependent JNK activation and AP-1 translocation and transactivation. In neutrophils and mononuclear cells, inhibition of LPS-induced NF-κB/AP-1 activity by ketamine was also observed (Table 1).14, 31

6. Cascade downregulation of MAPK phosphorylation

Recently, a study showed that transduction of TLR signaling to various biological functions, such as cell growth and differentiation, might be mediated by the Ras protein.⁴⁶ After being phosphorylated by the Ras protein, the activated Raf kinase sequentially triggers MAPKs, (MEK)1/2, and extracellular signal-regulated kinases, (ERK)1/2.47, 48 They trigger ERK1/2 and then transactivate the inhibitor of κB kinase (IKK) by stimulating the translocation and transactivation of NF-κB, which in turn induces expressions of certain inflammatory genes.49, 50 Previous studies showed that ketamine could alleviate LPS-induced NF-κB activation in the rat jejunum and lung.7, 29 Chen et al¹⁷ tried to evaluate the role of the Ras/Raf/MEK/ERK/IKK cascade in ketamine-induced regulation of these inflammatory gene expressions in the RAW 264.7 murine macrophage cell line (Table 1). A therapeutic concentration of ketamine not only interfered with LPS binding to the LBP, but also downregulated LPS-induced increases in Ras activity and Raf phosphorylation, and then disrupted LPS-induced phosphorylations of MEK1/2, ERK1/2, and IKK. Consequentially, the translocation and transactivation of NF-κB induced by LPS were significantly depressed after exposure to ketamine, which subsequently decreased the production of TNF-α, IL-1β, and IL-6.¹⁷ Recently, the increasing prevalence of sepsis from Gram-positive bacterial pathogens prompted another examination of the molecular pathogenesis of septic shock. Lipoteichoic acid (LTA) is a major component of the outer membranes of Gram-positive bacteria and was shown to be one of the key factors participating in the pathogenesis of sepsis by Gram-positive bacteria.⁵¹ The TLR2 receptor was reported to be responsible for LTA activation in macrophages.⁵² A previous study reported that ketamine could decrease syntheses of TNF-α and IL-6 in Gram-negative endotoxin LPS-activated macrophages. Chang et al¹⁸ showed that ketamine also suppressed TNF-α and IL-6 productions in Gram-positive endotoxin LTA-activated macrophages by inhibiting TNF-α and IL-6 gene expressions (Table 1). This suppression of TNF-α and IL-6 gene expressions occurs through NF-κB-involved transcriptional regulation due to decreased phosphorylation of ERK1/2. TLR2-mediated signal-transducing activation of ERK1/2 may be one of the multiple pathways that regulate ketamine-involved downregulation of NF-κB activation and TNF-α and IL-6 gene expressions. These data indicate that ketamine has an anti-inflammatory effect, which reveals its possible role in treating septic patients.³⁴

7. Roles of TLRs in ketamine-caused gene inhibition

Despite a decrease in mortality over the last decade, sepsis remains one of the most common causes of death in Western countries. A study in 2009 estimated that 1.21% of patients developed postoperative sepsis after elective surgery in the USA.⁵³ In spite of progress in better recognizing and improving standards of care, mortality still ranges from 30% to 50% in patients with septic shock.⁵⁴ Roles of proinflammatory cytokines and TLR signaling in the pathogenesis and development of sepsis were reviewed recently.55, 56 The discovery of the TLR explained the missing link between endotoxin recognition by LBP and CD14, and how this intracellular signaling pathway activates and translocates NF-κB to nuclei and subsequently transactivates the production of proinflammatory cytokines. To prevent overactivation of the TLR and its likely side effects, hosts develop strategies to control TLR expression and signaling. For instance, RP105, a TLR-like homolog mainly expressed by B cells, directly interacts with the TLR4 signaling complex, reduces its ability to bind to LPS, and prevents TLR4 from overstimulation.⁵⁷ Since LPS is a specific ligand for the TLR4 signaling pathway and TLR4 expression by monocytes is enhanced in septic patients, TLR4 and its signaling pathway have garnered more attention.¹⁹ Although the concept of anti-inflammatory effects of ketamine is well accepted and documented in many in vitro animal and human models, the effects on TLR expression were only recently explored. In a rat model, both intravenous LPS-induced TLR4 expression and NF-κB activation in the intestines and lungs were shown to be reduced by ketamine (Table 1).20, 21 In another model, rats had polymicrobial sepsis after cecal ligation and puncture, but treatment with ketamine after the procedure decreased proinflammatory cytokines such as TNF-α and IL-6, as well as NF-κB activation and TLR4 and TLR2 mRNA expressions in the intestines and lungs. Ketamine also improved the survival of rats after cecal ligation and puncture.22, 23 Chen et al¹⁷ studied the mechanisms of action of ketamine in the cultured murine RAW 264.7 macrophage cell line. They found that ketamine interfered with LPS binding to LBP and also decreased phosphorylation of various kinases involved in TLR4 intracellular signaling, thereby inhibiting the signal pathway. When stimulated with the TLR2 agonist LTA, ketamine decreased TNF-α and IL-6 production by macrophages. This results from decreased phosphorylation of ERK1/2, an upstream protein kinase for activating the IKK, leading to decreased NF-κB translocation to nuclei.¹⁸ The clinical relevance of those results has to be assessed for patients with sepsis and those sedated with ketamine. However, those studies provide useful information for future effective and targeted treatment for patients with sepsis.

8. Ketamine-induced postoperative immunoresponses

The immune system of a patient undergoing surgery is affected by tissue damage, anesthesia, postoperative pain, and psychological stress, which may have adverse influences on the postoperative outcome. Surgical trauma can induce a complex system of cytokine cascades with different effects on the host patient; for example, some proinflammatory cytokines in the immune system are excessively stimulated, while some cell-mediated immunity is suppressed.²⁴ Some undesirable effects, such as hypotension, shock, and multiple organ failure, may occur and put the patients in grave danger if the proinflammatory response is exaggerated.²⁵ It was suggested that there are interactions and regulation between nociceptive and proinflammatory cytokines.⁵⁸ Therefore, it is important to attenuate postoperative pain, then reduce suppression of the lymphocyte-mediated immune response, and attenuate the proinflammatory cytokine reaction to surgery by undertaking effective pain management to reduce surgical stress responses during the postoperative period.⁵⁹ In a study of patients after coronary artery bypass grafting, ketamine inhibited leukocyte reactivity and suppressed superoxide anion produced by neutrophils, thus demonstrating an anti-inflammatory effect.⁶⁰ Roytblat et al⁶¹ also reported that giving small doses of ketamine to patients undergoing cardiopulmonary bypass during induction of anesthesia can reduce serum levels of IL-6 significantly during the course of 7 postoperative days (Table 1). Takahashi et al⁶² used an animal model to demonstrate that ketamine anesthesia can effectively improve mouse survival after LPS and Escherichia coli challenge compared to sevoflurane anesthesia. In that study, mice were anesthetized with ketamine or sevoflurane during a laparotomy, and then mouse survival rates and cytokine secretions were examined after challenging mice with E. coli or LPS. Compared with sevoflurane anesthesia, ketamine anesthesia increased the mouse survival rate from the LPS challenge after laparotomy, whereas such an effect of ketamine was not observed after E. coli challenge. However, if bacterial growth was controlled using an antibiotic, ketamine anesthesia produced higher mice survival than sevoflurane anesthesia.⁶² Neutralization of TNF-α with an antibody also improved survival in mice challenged with sevoflurane anesthesia, indicating that better survival rate with ketamine may be due to suppression of TNF-α. TNF-α is the cytokine that stimulates the production of IL-6 and IL-8 by macrophages. TNF-α and IL-6 are involved in both acute and chronic inflammation, and in the case of polymicrobial sepsis in animal studies, production of both TNF-α and IL-6 was inhibited by ketamine.⁶² These data suggest that suppression of IL-6 and TNF-α production by ketamine may have favorable influences on a patient's recovery.63, 64 The timing of ketamine treatment is also important in boosting the survival rate of infected rats. A trend toward better survival and lower IL-6 production with ketamine administered close to the insult, either burn injury or E. coli challenge, was obtained compared to ketamine treatment 24 hours before and 2 hours after the insult (Table 1).65, 66, 67

9. Summary and conclusions

Nowadays, ketamine is used as an anti-inflammatory drug, which emphasizes the importance of research on its interactions with the immune system. Major surgery or sepsis causes the release of proinflammatory cytokines, which, in excessive amounts, can cause some undesirable effects, such as hypotension, shock, and multiple organ failure. Ketamine was proved to be a modulator of proinflammatory cytokines such as TNF-α, IL-1, and IL-6 in in vivo and animal models. Although limited data are available from human studies, it was revealed that a single dose of ketamine administered prior to anesthesia induction reduced the postoperative serum level of IL-6. The results imply the importance of future studies with more cases and exploration of relationships between IL-6 and mortality and morbidity.

The postulated effects of ketamine on proinflammatory gene expressions are summarized in Fig. 1. Administration of ketamine can result in TLR4 pathway dysfunction via downregulation of LPS-induced increases in Ras activity and Raf phosphorylation, followed by suppression of LPS-induced signal transduction of MEK1/2, ERK1/2, and IKK. Inhibition of the binding between LPS and LBP is also another way to inhibit TLR4 activation by ketamine. Through inhibition of the Ras/Raf/MEK/ERK/IKK cascade, the translocation and transactivation of NF-κB induced by LPS were depressed after exposure to ketamine. As a result, ketamine suppressed the production of TNF-α, IL-1β, and IL-6.

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Fig. 1. Molecular mechanisms of ketamine-induced downregulation of proinflammatory cytokine gene expressions. Administration of ketamine can result in the dysfunction of TLR 4 pathway via interference with LPS–LBP binding and reduction of LPS-induced increases in Ras activity, and then in the suppression of the LPS-induced signal transduction of MEK1/2, ERK1/2, and IKK. Through the inhibition of the Ras/Raf/MEK/ERK/IKK cascade, the translocation and transactivation of nuclear factor NF-κB induced by LPS were depressed after exposure to ketamine, which then suppressed the production of TNF-α, IL-1β, and IL-6. AP = activator protein; ERK = extracellular signal-regulated kinase; IKK = inhibitor of kappa B kinase; IL = interleukin; JNK = c-Jun N-terminal kinase; LPB = lipopolysaccharide-binding protein; LPS = lipopolysaccharide; NADPH = nicotinamide adenine dinucleotide phosphate; NF-κB = nuclear factor-kappa B; TLR = toll-like receptor; TNF = tumor necrosis factor.

Recently, anticytokine therapy was applied to control sepsis. Huang et al⁶⁸ used an extracorporeal hemoperfusion device that specifically absorbs mediators such as cytokines by a neutral microporous resin, to treat sepsis. They found that the hemoperfusion group showed significantly lower levels of plasma IL-6 and IL-8; significant increases in the cardiac index, systemic vascular resistant index, and withdrawal of vasoactive agents; and decreases in the heart rate compared to controls. Although a small dose of ketamine was observed to reduce postoperative serum levels of TNF-α and IL-6 in humans (Table 1),27, 28 unfortunately there were no multicenter evaluations or randomized controlled trials. In addition, NK cytotoxic activity was reduced significantly by ketamine, and the injected lung tumor showed increased metastasis after ketamine treatment in rats.¹⁰ These findings, if confirmed with more studies, may impact the choice of anesthetics for surgical patients under tumor excision, because any intervention that may lead to an increased risk of tumor metastasis in patients with suppressed NK cell activity should be withheld. However, it reveals the tactical use of ketamine to prevent sepsis. There is still a need for more efforts to elucidate detailed mechanisms of ketamine-caused suppression of the proinflammatory cascade and its influence in the future.