Ketamine Inhibits Maturation of Bone Marrow-Derived Dendritic Cells and Priming of the Th1-Type Immune Response

Abstract

BACKGROUND: 

Dendritic cells (DCs) play a key role as antigen-presenting cells and growing evidence suggests that DCs influence T-cell activation and regulate the polarity of the immune response. Ketamine has been reported to have immunomodulatory properties that affect immune cells, including macrophages and natural killer cells. However, the effect of ketamine on DCs has not been characterized. We examined the immunomodulation of DCs by ketamine.

METHODS: 

We used bone marrow-derived DCs induced by granulocyte–monocyte-colony stimulating factor and interleukin (IL)-4 from bone marrow and analyzed the expression of costimulatory molecules (CD40, CD80, and CD86), major histocompatibility complex class II molecules, and secretion of IL-12p40. Furthermore, we evaluated the immune response in mixed cell cultures of DCs and T cells and the contact hypersensitivity response in a whole animal.

RESULTS: 

Ketamine suppressed the expression of CD40, CD80, and major histocompatibility complex class II molecules in DCs. DCs treated with ketamine also secreted less IL-12p40 and displayed greater endocytosis. In mixed cell cultures with CD4⁺ T cells and DCs, ketamine-treated DCs showed less propensity to stimulate the proliferation of CD4⁺ T cells and the secretion of interferon from CD4⁺ T cells. Furthermore, ketamine-treated DCs impaired the induction of a cell-mediated immune response.

CONCLUSION: 

Our findings suggest that ketamine inhibits the functional maturation of DCs and interferes with DC induction of Th1 immunity in the whole animal. These novel findings provide new insight into the immunopharmacological role of ketamine.

The first step in the induction of an adaptive immune response is when professional antigen-presenting cells (APCs) present antigen to naïve T cells. This presentation of foreign antigen involves an antigen-major histocompatibility complex (MHC) and costimulatory signals to naïve T cells. Macrophages, B lymphocytes, epithelial cells, and dendritic cells (DCs) can all act as APCs, but DCs are the most potent in the initial presentation through the MHC to naïve T cells and are responsible for the subsequent T cell-specific immune response. Thus, the possibility of harnessing the power of DCs has been given prominent attention in research immunology.^1–3

In mice, DCs develop from bone marrow (BM) stem cells. Usually, DCs exist in a functionally and phenotypically immature state and in this state, because they do not express costimulatory molecules, they do not induce an immune response. Immature DCs begin to mature when they capture and process antigens in peripheral tissues. Maturing DCs stimulate naïve T cells through signaling both by Ag peptides, presented by MHC molecules, and by costimulatory molecules. DC maturation is accompanied by decreased Ag uptake, high levels of MHC class II and costimulatory molecule expression, and production of interleukin (IL)-12 on stimulation.^1,3

Anesthetics and sedatives play an essential role in the stable and safe control of critically ill patients. Furthermore, ketamine has been often used as an anesthetic for patients who are hemodynamically unstable due, for example, to sepsis or cardiac surgery. Consequently, the effects of various anesthetics on immunity have been extensively reported. Although macrophages, lymphocytes, and natural killer cells have been investigated, little attention has been paid to the effects of anesthetics and sedatives on DCs. Among many anesthetics, ketamine is an anesthetic that affects the immunoregulatory activities on macrophages,^4,5 neutrophils,⁶ mast cells,⁷ and whole blood cells.^8,9 Even so, we could find no report about how anesthetics affect DCs, the most potent of the APCs.

In this initial study, we designed experiments to illustrate the effects of ketamine on some of the functions of DCs. We found that ketamine inhibits the maturation of BM-derived DCs (BMDCs) and furthermore, impedes the ability of DCs to prime a Th1-biased immune response.

METHODS

Animals

To provide cell samples, female 4–6-week-old BALB/c and C57BL/6 mice were purchased from Japan CLEA (Tokyo, Japan). They were housed with free access to chow and water in the specific pathogen-free central animal facility of Osaka University Medical School. All experimental protocols in this study were reviewed and approved by the Institutional Animal Care and Use Committee and performed according to the National Institutes of Health guidelines.

Reagents and Antibodies

Recombinant mouse (rm) granulocyte–monocyte-colony stimulating factor and rmIL-4 were purchased from R&D Systems (Minneapolis, MN). Fluorescein-conjugated dextran (40,000 molecular mass) (fluorescein isothiocyanate (FITC)-dextran) and lipolysaccharide (LPS) (from Escherichia coli 055:B5) were obtained from Sigma-Aldrich (St Louis, MO). FITC- or phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) were used to detect the expression of CD11c (HL3), MHC antigen class II (I-A^d) (M5/114.15.2), CD40 (3/23), CD80 (16-10A1), and CD86 (GL1) were purchased from BD Pharmingen (San Diego, CA). For intracellular cytokine detection, we used mAbs for IL-4 and interferon (IFN) (11B11 and XMG1.2: BD Pharmingen).

Isolation and Culture of DC

DCs were generated from murine BM cells using a previously described method with minor modifications.^10,11 Briefly, BM was flushed from the tibiae and femurs of Balb/C mice and then depleted of red blood cells with ammonium chloride. BM cells were suspended in complete media (CM: RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin). Cells for each mouse were plated to two 10-cm plates and cultured for 7 days in the presence of 20 ng/mL rm granulocyte–monocyte colony-stimulating factor and rmIL-4 at 37°C in 5% CO₂. On Day 7, nonadherent cells and loosely adherent proliferating DCs were harvested and purified on 14.5% Accudenz (Accurate Chemicals, Westbury, NJ) density gradients by centrifugation at 600g for 20 min at room temperature. Purified DCs were cultured for 40 h in the absence and presence of ketamine in concentrations of 0 to 100 μM.

The viability of cultured cells was assessed by the trypan-blue exclusion test. Cell viability of more than 90% was observed in all experiments in the study.

Flow Cytometric Analysis of Surface Molecules

The expression of surface molecules on DCs was analyzed under flow cytometry. At each step of the staining, to block nonspecific binding of antibodies, 1–2 × 10⁵ cells were incubated for 15 min on ice in staining buffer containing anti-CD16/CD32 mAb. Cells were stained with specific antibodies. After mAb staining, 2% propidium iodide (PI) was applied to stain dead cells and the samples were analyzed using FACSCalibur and CellQuest software (BD Biosciences, Franklin Lakes, NJ). We used FITC or PE-labeled monoclonal Abs to stain for MHC class, CD40, CD80, CD86, and CD11c. Dead cells were flow-cytometorically gated out by staining with PI and only live cells were phenotypically assessed.

Interleukin-12 p40 Enzyme-linked Immunosorbent Assay

DCs were cultured in the presence or absence of ketamine for 40 h followed by stimulation with LPS (10 ng/mL) for 12 h. Following the manufacturer's instructions, we analyzed culture supernatants using IL-12 p40 enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN).

Intracellular Cytokine Assay

For intracellular cytokine assay, T cells were restimulated with ionomycin and phorbol myristate acetate in the presence of GolgiStop (BD Pharmingen). Following the maker's instructions, intracellular cytokines were detected using standard Cytofix/Cytoperm kits (BD Biosciences). We used PE-labeled monoclonal Abs to stain IL-4 and IFN- and FITC-labeled mAb for staining CD4.

Annexin V and Propidium Iodide Binding Assay

For analysis of DC apoptosis, using a FACSCalibur and CellQuest software (BD Biosciences), 1 × 10⁵ cells were stained with FITC-labeled anti-CD11c mAb, washed in phosphate-buffered saline, and stained with PI and allophycocyanin-labeled Annexin V (BD Pharmingen).

Quantitation of Ag Uptake

As described by Sallusto et al.,¹² endocytosis was quantitated. In brief, 2 × 10⁵ cells were equilibrated at 37°C or 4°C for 10 min and then pulsed with FITC-dextran (Sigma-Aldrich) at a concentration of 1 mg/mL. Cold staining buffer was added to stop the reaction. The cells were washed three times and stained with PE-conjugated anti-CD11c Abs and then analyzed using a FACSCalibur. Nonspecific binding of FITC-dextran to DCs was assessed by evaluating FITC-dextran uptake at 4°C. Mean fluorescence intensity at 37°C minus mean fluorescence intensity at 4°C was used as the measure of antigen uptake.

Allogeneic Mixed Cell Culture Reaction

To enrich CD3⁺ T cells in the sample, splenocytes, isolated from female 5–8-week-old C57BL/6 mice (haplotype: I–A^b), were purified in a magnetic cell sorting and separation of biomolecules (MACS) column (Miltenyi Biotec, Bergisch Gladbach, Germany). DCs induced from Balb/C mice (haplotype: I–A^d) were incubated in the presence or absence of ketamine. Subsequently, DCs were stimulated by LPS (20 ng/mL) for 12 h and treated with mitomycin C (50 μg/mL). Enriched CD3⁺ T cells were cocultured with mitomycin C-treated DCs. In 96-well round-bottom plates, these mixed samples were cultured for 4 days in RPMI 1640 supplemented with 10% fetal bovine serum at 37°C in 5% CO₂ in air. Cell proliferation was estimated based on uptake of [³H]-thymidine. For this purpose, the cells were pulsed with [³H]-thymidine for the final 18 h of mixed cell culture. Radioactivity was then measured using a liquid scintillation counter (PerkinElmer, Waltham, MA). Intracellular cytokines were detected using standard Cytofix/Cytoperm kits following the manufacturer's instructions (BD Bioscience).

Establishing Contact Hypersensitivity by Adoptive Transfer of DCs

DCs were prepared as described earlier. After being cultured in the presence or absence of ketamine, 5 × 10⁵ DCs (in 100 μL of saline) were pulsed with 100 μg/mL 2,4-dinitrobenzene sulfonic acid (DNBS) and injected subcutaneously on Day 0. After 5 days, mice were challenged by the application on both sides of the right ear of 10 μL of 0.2% 2,4-dinitro-1-fluorobenzene (DNFB) in 4:1 acetone/olive oil solution. Negative control animals were injected with only 100 μL of saline at the time of initial immunization and exposed to DNFB 5 days later. After 24 h, using an engineer's spring-loaded micrometer (Mitsutoyo, Kawasaki, Japan), we measured the right (challenged) and the left (unchallenged) ear thickness. The increase in the ear thickness was evaluated by simple subtraction: thickness of the right (challenged) ear—thickness of the left (unchallenged) ear.

Statistics

Data for samples were compared using the Student's t-test. When P < 0.05 the difference was considered statistically significant. All statistical analysis was performed using JMP software (SAS Institute, Cary, NC).

RESULTS

Ketamine Inhibits Maturation of Murine DCs

To examine whether ketamine influences the maturation of DCs, we cultured immature DCs induced after 7 days of culture for another 40 h in the presence or absence of different concentrations of ketamine. The expression of surface molecules (CD11c, MHC class II) on DCs was assessed using flow cytometry. As DC maturation progressed, we observed a higher presence of MHC class II molecules. Figure 1 shows CD11c cell populations. Ketamine had no effect on the numbers of BM cells found in the wells of culture dishes after BM cells were cultured in the presence or absence of ketamine in various concentrations of up to 200 μM (data not shown). Similarly, DCs cultured in the presence or absence of ketamine, at least in concentrations of up to 100 μM, showed no statistically significant differences in the total number of CD11c⁺ DCs including mature MHC class II^high and immature MHC class II^lo DCs (Fig. 1A). To further exclude the possibility that ketamine has a toxic action on cultured DCs, using an Annexin V-PI binding assay, we assessed the effect of ketamine, in concentrations of up to 100 μM, on apoptosis (Fig. 1B and C). Our findings showed that ketamine has no harmful effect on DCs in concentrations of up to 100 μM. In subsequent protocols, we used ketamine in concentrations of 40 μM.

Figure 1.: Effect of ketamine on CD11c⁺ DC. In concentrations of up to 100 μM, ketamine has no influence and no cytotoxicity on CD11c⁺ DC. DCs were generated as described in Methods section (without or in the indicated concentration of ketamine) and analyzed at Day 9 by flow cytometry. DCs were stained for CD11c (A), and Annexin V and PI (B and C). A single typical result of Annexin V and PI staining is shown from samples for each set of three independent experiments. The percentage within each histogram represents the incidence of CD11c⁺ cells (A) and Annexin-V⁺PI⁺ cells (C). The cytotoxic effect of ketamine was evaluated from the fraction of necrotic cells that were detected as AnnexinV⁺PI⁺ cells. Results are means ± sds (n = 6). DC = dendritic cell; PI = propidium iodide.

We ascertained that the total number of CD11c⁺ DCs, including mature MHC class II^high and immature MHC class II^lo DCs, was not affected by exposure to ketamine in concentrations of up to 100 μM. As immature DCs matured, we observed higher expression of MHC class II, CD40, CD80, and CD86 molecules. We found that ketamine specifically decreased the number of MHC class II^high DCs. Figure 2 shows data for the expression of MHC class II, CD40, CD80, and CD86. The expression of CD40 and CD80 molecules, important for costimulating T-cell activation, was also suppressed when ketamine was present. This finding indicates that the phenotypic maturation of DCs is at least partially retarded by the presence of ketamine. Conversely, no differences in the expression of CD86 molecules were detected between samples that had been exposed to ketamine and control samples.

Figure 2.: Effect of ketamine on expression of costimulatory molecules on DCs. Ketamine suppresses DC maturation. DCs were treated with ketamine (40 μM). A, Flow cytometry was used to assess the expression rate for CD40, CD80, CD86, and major histocompatibility complex (MHC) class II molecules in CD11c-gated cells. Ketamine inhibits the expression on DCs of CD40, CD80, and MHC class II molecules. B, Representative histogram of the expression of each surface molecule on DC. The number in each histogram shows the percentage of DCs (CD11c⁺ cells) expressing highly each molecule. Results are means ± sds (n = 4). DC = dendritic cell.

Ketamine Inhibits Secretion of Interleukin-12 p40 from DC

Mature DCs are important for the synthesis and secretion of cytokines that affect T-cell differentiation and are largely responsible for the quality of immune response. DCs produce proinflammatory cytokines: IL-12 production is a particular marker of DC maturation and can be used as method of selecting the Th1-dominant adjuvant. Here, we tested the expression of IL-12p40 from LPS-stimulated DCs. As Figure 3A shows, ketamine inhibited the secretion of IL-12p40.

Figure 3.: Effect of ketamine on production of interleukin-12 p40 from DCs (A) and on endocytotic activity of DCs (B). Ketamine inhibits the production of interleukin-12 p40 (IL-12p40) from DCs. A, DCs were treated with ketamine (40 μM) for 40 h and thereafter exposed to 10 ng/mL of lipopolysaccharide (LPS) for 12 h. The expression of IL-12p40 was measured by cytokine-specific ELISA in culture supernatant. B, Ketamine-treated DCs exhibit enhanced endocytotic capacity. DCs were generated as described in Methods section and harvested on Day 7. FITC-dextran was analyzed on CD11c-PE–positive cells by flow cytometry. The uptake of FITC-dextran is shown as the product of mean fluorescence intensity (MFI) at 37°C minus MFI at 4°C. Results are means ± sds (n = 6). DC = dendritic cell; PE = phycoerythrin; FITC = fluorescein isothiocyanate; ELISA = enzyme-linked immunosorbent assay.

Ketamine Induces Immature State DCs with High Endocytotic Capacity

Evaluation of the expression of surface molecules and IL-12p40 secretions indicated that exposure to ketamine significantly suppressed the phenotypic and functional maturation of DC generated in vitro. These results did not exclude the possibility that ketamine might, however, cause a general inhibition of DC functions. Consequently, we assessed whether exposure of DCs to ketamine alters the ability of DCs to capture Ag through the uptake of FITC-conjugated dextran. As Figure 3B shows, after 40 h exposure to ketamine, DCs displayed increased endocytotic capacity for FITC-dextran. All our findings, including the expression of surface molecules and IL-12p40 and endocytotic activity, strongly suggest that ketamine prevents the maturation of DCs.

Ketamine inhibits the ability of DCs to stimulate T-cell proliferation and to prime the Th1-type immune response in allogeneic mixed cell culture reaction.

To clarify the relevance to immune responses of the ketamine-mediated alteration of DC functions that we had so far observed, we analyzed the effect of ketamine on the mixed cell culture reaction of lymphocytes and DCs. Using samples derived from Balb/C mice (haplotype: I–A^d), DCs that had been incubated in the presence or absence of ketamine were tested for their capacity to stimulate allogeneic T cells derived from C57BL/6 (haplotype: I–A^b). From Day 7 cultures that had been incubated with ketamine for 40 h, DC samples were tested for their capacity to stimulate allogeneic T cells.

Although coculture with LPS-stimulated DCs effectively enhanced proliferative responses, when LPS-stimulated DCs were pretreated with ketamine, there was less [³H] uptake by allogeneic T cells (Fig. 4A). Another determinant of DCs potency is how well they are able to adhere to T cells to form clusters (Fig. 4B). Compared with control cells, we found that ketamine-treated DCs formed fewer clusters on T cells.

Figure 4.: In the presence and absence of ketamine: in vitro dendritic cell (DC)-induced proliferation of allogeneic T cells and Th1 response. Ketamine impeded the proliferation of allogeneic T cells and Th1 response. DCs were incubated with and without ketamine (40 μM) for 48 h with the administration of lipolysaccharide (LPS) for final 12 h. The DCs were washed and cocultured with T cells derived from Balb/C mice as described in Methods section. A, Mixed cell culture was performed for 4 days. For the final 18 h of mixed cell culture, [³H]-TdR was added to the culture. Cell proliferation was estimated based on uptake of [³H] thymidine, which was pulsed during the final 18 h of culture. The radioactivity of the harvested cells was measured using a liquid scintillation counter (PerkinElmer). B, After 64 h of mixed cell culture, clustering was counted under microscopic analysis. Results are means ± sds (n = 6).

We also evaluated the effects on IFN- and IL-4 synthesis when CD4⁺ T cells were cocultured with ketamine-treated DCs. Intracellular cytokine analysis revealed a lower density of IFN-producing CD4⁺ cells in cocultures that contained ketamine-treated BMDCs (Fig. 5). No differences in the secretion of IL-4 were detected in ketamine-treated and control DC samples.

Figure 5.: Ketamine inhibits the production of interferon (IFN) from CD4⁺ T cells stimulated by allogeneic dendritic cell (DC) culture in the presence or absence of ketamine. In the presence or absence of ketamine: production of IFN-γ from CD4⁺ T cells stimulated by allogeneic DC culture. Ketamine inhibited the production of IFN-γ. Mixed cell culture was performed with bone marrow-derived DCs (BMDCs) from Balb/C mice (I–A^d) and CD3⁺ T cells from C57BL/6 mice (I–A^b) for 4 days. At Day 4, T cells were harvested and restimulated with ionomycin and phorbol myristate acetate (PMA) in the presence of GolgiStop (BD Pharmingen). The production of IFN-γ and interleukin (IL)-4 from CD4⁺ T cells was assessed by flow cytometry with intracellular staining of cytokine using Cytofix/Cytoperm kits (BD Pharmingen). Single, typical results are shown from each set of three independent experiments. Results are means ± sds (n = 6).

Ketamine-Treated DCs Fail to Elicit Contact Hypersensitivity Response

Contact hypersensitivity (CHS) is a typical cell-mediated immune response that is induced mainly by Th1-type T cells. CHS is originally induced by epicutaneous immunization and subsequent challenge with haptens, such as DNFB. CHS can be also induced by a single subcutaneous injection at Day 0 of 5 × 10⁵ DCs that have been pulsed with DNBS (the water-soluble derivative of DNFB) and subsequent epicutaneous challenge with DNFB at Day 5.^13,14 We used this model to assess the in vivo effect of ketamine on DCs and DC-mediated Th1-type immune response in the whole animal. Immunization with ketamine-treated DCs elicited less CHS response than immunization with phosphate-buffered saline-treated DCs (Fig. 6).

Figure 6.: Ketamine inhibits Th1-type immune response in DC-transfer model of contact hypersensitivity (CHS). Ketamine-treated DCs fail to induce a normal cell-mediated immune response. As described in Methods section, after being cultured in the presence or absence of ketamine, 5 × 10⁵ DCs were pulsed with 100 μg/mL DNBS and injected subcutaneously on Day 0. After 5 days, mice were challenged by the application on both sides of the right ear of 10 μL 0.2% DNFB in 4:1 acetone/olive oil solution. Right (challenged) and left (unchallenged) ear thicknesses were measured after 24 h and the results are shown as simple subtraction. Results are means ± sds (n = 6). DC = dendritic cell; DNBS = 2,4-dinitrobenzene sulfonic acid; DNFB = 2,4-dinitro-1-fluorobenzene.

DISCUSSION

As far as we know, this is the first report detailing the effects of ketamine on the phenotypic and functional properties of murine DCs. As such, it is also the first study to show that the presence of ketamine in cultures makes DCs less able to elicit T-cell Th1-type immune response.

Several previous studies have reported that ketamine affects various types of immune cells, including macrophages.^4–9 Although these studies mentioned the direct effects of ketamine on particular types of cells, they did not investigate the effect of ketamine on immune responses that arise from the interaction of different immune cells.

During functional maturation, DCs show increased expression of MHC and costimulatory molecules (CD40, CD80, CD86, etc), increased IL-12-secreting activity, and reduced endocytosis of antigens.^1–3 In this study, the DC phenotype changes induced by ketamine included reduced expression of MHC class II and costimulatory molecules, less secretion of IL-12, and increased capacity to internalize antigens. These results suggest that ketamine suppresses the functional maturation of DCs. As they mature, DCs become increasingly able to stimulate T cells to proliferate and differentiate into effector T cells. We found that DCs matured by LPS acquired the ability to stimulate the proliferation of allogeneic T cells. In contrast, LPS-stimulated DCs treated with ketamine did not manifest the same ability. This reversal suggests that ketamine affects the ability of DCs to elicit T-cell response.

There is accumulating evidence that the cytokine production of DCs depends on DC subsets or on stimuli received by DCs.¹⁵ IL-12 has multiple immunoregulatory functions, including the activation of the Th1 T-cell subset, which plays a pivotal role in the induction of inflammation and host-defense response. Many lines of evidence indicate that the development of the Th1-type immune response is regulated by DC-derived IL-12.^16–18 Our current finding that ketamine inhibits DC production of IL-12 suggests that ketamine strongly affects the differentiation of T cells. In fact, culturing CD4⁺ T cells with ketamine-treated DCs suppressed the differentiation of CD4⁺ T cells into IFN-producing Th1 T cells. Conversely, culturing CD4⁺ T cells with ketamine-treated DCs did not induce the differentiation of IL-4–producing Th2 T cells. Overall, our data demonstrate that ketamine affects, at least in part, the ability of DCs to tilt the immune response toward an increased Th1 response rather than Th2 response. With practical development in the prospect of inducing Th1 responses, such as ex vivo manipulation of DCs for cell transfer,^19–21 our findings indicate a candidate means of counter-regulatory DC therapy. Conversely, the effect of ketamine on DCs and the Th1-type immune response suggest immunosuppression by ketamine, which possibly compromises the crucial initial protective Th1-type immune response against invading pathogens.

Several studies of postoperative patients have reported that ketamine suppresses the immune response.^22–24 It seems that just a single preoperative administration of ketamine (0.15 mg/kg) is enough to attenuate the production of proinflammatory cytokines from peripheral blood mononuclear cells and the proliferative response of mononuclear cells.²³ Meanwhile, the impairment of IL-12 production from monocytes is reported to be a predictor of lethal outcome in postoperative sepsis.^25,26 Moreover, a rat model of tumor metastasis has shown that ketamine promotes tumor metastasis.²⁷ We suggest that, in view of the central role of DCs in the induction and regulation of the immune response,^1–3 the suppressive effects of ketamine on various immune responses observed in these several reports can be mediated by the effect of ketamine on DCs.^22–24,27 Thus, in certain clinical settings, the immunosuppressive action of ketamine on the functions of DCs, including the suppression of IL-12 production may increase risk, so cautious use of ketamine is advised.

For anesthesia in which ketamine is used as the main anesthetic reagent for major surgery, it has been reported that the plasma concentration of ketamine is about 3 μg/mL.^28,29 Furthermore, when administrated in combination with midazolam for sedation in emergency trauma care, the concentration of ketamine has been reported to be 2.6 ± 2.2 μg/mL.³⁰ For pharmacological research, the concentration of ketamine used in our in vitro experiment (40 μM = 10.9 μg/mL) was two to three times the reported concentration of ketamine in anesthesia or sedation. For an in vitro experiment, the different concentration of ketamine was reasonable. Two studies found that the administration of a single small dose of ketamine (0.15–0.50 mg/kg) in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB)²² or major abdominal surgery²³ reduced postoperative cytokine secretion, such as IL-6 and tumor necrosis factor-α. Just one dose of ketamine resulted in a plasma concentration of 400 ng/mL. In in vivo settings, such as postoperative periods after major abdominal surgery or cardiac surgery on CPB in these studies, immunomodulatory activities induced by major surgery or the use of CPB could have a synergistic immunosuppressive effect on immune response together with ketamine. Thus, ketamine may have effects at lower concentrations than in an in vitro study.

Other commentators have suggested that ketamine might have nonspecific inhibitory effects.³¹ One report suggested that the use of ketamine in high concentrations could be nonspecifically cytostatic to various biological activities, and the authors cautioned about the use of high concentrations in in vitro experiments. To clarify the situation, by demonstrating that the expression of CD86 was not altered in the presence of ketamine, we confirmed that ketamine did not generally suppress all DC functions. We further investigated the capacity of DCs to internalize FITC-dextran. Our assay showed that, far from being suppressed, endocytosis had increased. These findings exclude the possibility that the action of ketamine is generally and nonspecifically suppressive. The specific actions are distinctively characteristic of immature DCs.⁶ Furthermore, through the use of Annexin V and PI staining, we confirmed that ketamine in the range of the concentration used in this experiment was not cytotoxic.

Based on our in vitro observations, we conjecture that treatment with ketamine may impair the ability of DCs to initiate a cell-mediated immune response. It has been shown that, in recipient mice transferred with DNBS-DCs, 5 × 10⁵ DNBS-pulsed DCs (DNBS-DC) could induce strong CHS.^13,14 We checked the ability of DCs to induce the T-cell–mediated immune response by sensitizing recipient mice for CHS to DNBS-DCs. Subcutaneous immunization with 5 × 10⁵ DNBS-DC made the mice susceptible to CHS, but when similarly exposed to DNBS-DCs pretreated with ketamine they showed no hypersensitivity. This result is evidence that ketamine suppresses the DC-mediated induction of the Th1-type immune response in the whole animal. Furthermore, these results show that the impaired ability of ketamine-pretreated DCs to stimulate the T-cell response cannot be reversed in vivo after withdrawal of ketamine.

Mostly based on in vitro experiments, it is questionable how far the findings of the current study are relevant to the in vivo conditions found in whole animals. The BMDC culture system used in most of the experiments reported here has, however, been widely used as an established means for investigating the effects of various agents on DCs.^10,11 Furthermore, to assess whether the effect of ketamine found in in vitro experiments can be extrapolated into in vivo conditions, we showed the suppressive effect of ketamine on the DC-induced immune response in a whole animal by the use of adoptive transfer model of CHS. Although we treated DCs with ketamine for 40 h and assessed the change of phenotype, we also assessed the effect of ketamine on DCs for shorter periods of exposure, including 10 h, which more closely matches ketamine administration in clinical applications, such as anesthesia. The results after 10 h incubation with ketamine show the same tendencies as results after 40 h incubation (Supplementary Figure). The findings obtained by this system are generally assumed to closely resemble what happens in whole animals.

Supplemental Figure.: Effect of duration of dendritic cell (DC) exposure to ketamine on expression of costimulatory molecules. Ketamine suppresses DC maturation. DCs were treated with ketamine (40 μM) for indicated duration (10 hr and 40 hr). A-D, flow cytometry was used to assess the expression rate for CD80 and CD86 in CD11c gated cells. Results are means ± sds (n = 4) DC, dendritic cell.

In conclusion, we have characterized a variety of effects exerted by ketamine on DCs. Resulting in a significant inhibition of Th1 development, ketamine inhibited phenotypic maturation and modulated cytokine production in these murine DCs. Exposure to ketamine may provide a nontoxic means of modulating of the immunostimulatory capacity of DCs. During sedation and analgesia in critical care, the use of ketamine can suppress the DC-mediated immune response.