The In Vitro Effects of Ketamine at Large Concentrations Can Be Attributed to a Nonspecific Cytostatic Effect

Abstract

Ketamine seems to promote a favorable outcome for critically ill patients, including those with septic shock (1). It has been proposed that the nonanesthetic effects of ketamine may be attributed to antiinflammatory mechanisms. This hypothesis has been supported by in vivo findings, such as decreased levels of interleukin (IL)-6 in patients treated with a single IV small dose of ketamine during postoperative stress (2) or during abdominal hysterectomy (3). Notably, the doses that produce the antiinflammatory effects in these studies reach a plasma concentration of 0.1 μg/mL, whereas the plasma ketamine concentration required for anesthesia is 0.6–2.0 μg/mL (1,4,5). In vitro attempts to uncover the antiinflammatory mechanisms of ketamine revealed widespread inhibitory activities, but they used large ketamine concentrations. For example, inhibition of tumor necrosis factor-α and nitric oxide production in lipopolysaccharide (LPS)-stimulated mouse-activated macrophagelike cells was attained at ketamine concentrations of 7.1–142.6 μg/mL (6); inhibition of action and production of nitric oxide synthase in LPS-treated rat alveolar macrophages, at 2.38 μg/mL ketamine concentration (7); and inhibition of IL-6, IL-8, and tumor necrosis factor-α production by LPS-stimulated human whole blood, at ketamine concentrations of 100–500 μg/mL (8). We believe that the in vitro responses with large concentrations of ketamine are misleading. To verify this, we sought to clarify whether these large concentrations of ketamine exert a broad nonspecific cytostatic effect that includes both the arrest of cell proliferation and a blockade of cytokine production. We introduced varying concentrations of ketamine to both growth factor-dependent and independent human and mouse cell cultures and examined cell proliferation. Our data indicate that large doses of ketamine arrest cytokine production and cell proliferation in a strikingly similar pattern. Methods This study was approved by our institutional human investigation committee. A written informed consent was obtained from all subjects. We measured IL-6 production in human LPS-stimulated whole blood according to the protocol reported by Kawasaki et al. (8). Briefly, blood was obtained from healthy volunteers and diluted in RPMI medium containing 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. LPS (10 ng/mL) and ketamine (25–500 μg/mL; Parke-Davis, Hampshire, UK) were added to 1 mL diluted blood. After 6 h of incubation at 37°C in an atmosphere of 5% CO2 and 95% air, blood was centrifuged at 700 g for 10 min. The supernatant was examined for IL-6 levels by commercial ELISA (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Human primary tubular epithelial cells (TEC) were prepared from normal cortical tissues of hypernephrotic kidneys as previously described (9). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Sigma Israel Chemicals Ltd., Rehovot, Israel) density gradient centrifugation of heparinized peripheral blood obtained from three healthy donors. The mouse fibroblast cell line (L-cells) and the mouse T-cell line (CTLD) were obtained from the American tissue culture cell collection. Cells were seeded in a flat-bottomed 96-well plate; TEC (5 × 103 cells per well), in M199 medium; and PBMC (50 × 103 cells per well), CTLD (5 × 103 cells per well), and mouse fibroblasts (103 cells per well), in RPMI medium. Both types of medium contained 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50μM β-mercaptoethanol. PBMC proliferation was induced by phytohemagglutinin (PHA) (0.2%; Difco Laboratories, Detroit, MI), and CTLD proliferation was induced by IL-2 (50 U/mL). Cells were treated with medium or ketamine (25–500 μg/mL). At 48 h, TEC, CTLD, and mouse fibroblasts’ relative cell proliferation was measured by XTT colorimetric assay (Biological Industries, Beit Haemek, Israel), according to the manufacturer’s instructions. This is an in vitro assay for determining numbers of viable cells in proliferation and cytotoxicity studies, on the basis of the bioreduction of the tetrazolium compound XTT by viable cells. PBMC proliferation was determined by XTT assay at 72 h. The reliability of the XTT assay in the presence of ketamine was confirmed by manual cell count. Statistical analysis was performed with Pearson correlation and one-way analysis of variance. A value of P < 0.05 was considered significant. Results are presented as mean ± sem. Results Introduction of LPS to human whole blood induced IL-6 secretion that was inhibited by ketamine at a 50% inhibitory concentration (IC50) of approximately 100 μg/mL (Fig. 1). The proliferation of PHA-stimulated human PBMC and the primary culture of human TEC was also inhibited by ketamine (IC50 50 μg/mL and IC50 200 μg/mL, respectively) (Fig. 2). This pattern of inhibition was also observed while introducing ketamine to a mouse T-cell line (IC50 200 μg/mL) and a mouse fibroblast cell line (IC50 350 μg/mL) (data not shown). The inhibition of IL-6 secretion correlated with the inhibition of proliferation of PBMC (r = 0.81, P = 0.0055) (Fig. 3) and to the inhibition of proliferation of CTLD (r = 0.92, P < 0.005).Figure 1: The effect of ketamine on interleukin (IL)-6 production. Whole blood from healthy volunteers was diluted fivefold in RPMI medium. Lipopolysaccharide (LPS; 10 ng/mL) and the indicated ketamine concentrations were added to 1 mL diluted blood. After 6 h of incubation, the supernatant was examined for IL-6 levels by specific enzyme-linked immunoassay. The histogram indicates the level of IL-6 secretion from unstimulated blood. This experiment is representative of three independent experiments. Error bars indicate sem. ** P < 0.005 (one-way analysis of variance).Figure 2: The effect of ketamine on cell proliferation. A, Primary cultures of human renal tubular epithelial cells (TEC); B, human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin were seeded in a 96-well plate in medium containing increasing concentrations of ketamine. At 48 h (TEC) or 72 h (PBMC), cell number was evaluated by XTT assay. Relative cell number is represented by percentage of ketamine-untreated cells. The histogram in graph A represents the level of proliferation of TEC in the presence of cycloheximide (CHX), a potent protein synthesis inhibitor. Ketamine 50% inhibitory concentration is &sim;200 μg/mL for TEC and &sim;50 μg/mL for PBMC. This experiment is representative of three independent experiments performed on PBMC from three healthy volunteers. *** P < 0.001 (one-way analysis of variance).Figure 3: Correlation between ketamine inhibition of interleukin (IL)-6 production and human peripheral blood mononuclear cells (PBMC) number. The data presented in Figures 1 and 2A were analyzed by linear regression to demonstrate the correlation between the inhibition of increasing doses of ketamine on IL-6 levels and the relative number of phytohemagglutinin-stimulated PBMCs.Discussion Studies involving the administration of ketamine to patients and animal models have demonstrated an antiinflammatory activity at a plasma concentration of 0.1 μg/mL (2,3). At this concentration, ketamine has no anesthetic effect (1,4,5). Despite these findings, various in vitro studies have examined the antiinflammatory activity of ketamine at concentrations 10- to 1000-fold larger. These studies, without exception, show inhibitory activities to ketamine (6–8,10,11). In this study, we sought to establish whether previously described inhibitory effects of ketamine at large concentrations could be attributed to nonspecific cytostatic effects. We have demonstrated that large concentrations of ketamine exert a broad, nonspecific cytostatic effect. This effect includes failure to proliferate, as well as the previously reported failure to produce cytokines (8). This was established by observing the inhibition of proliferation of human PTEC and of human PHA-stimulated PBMC. Additionally, a mouse T-cell line and a mouse fibroblast cell line exhibited the same pattern of inhibition under ketamine treatment (data not shown). Several reports have attempted to exclude the presence of cytotoxic effects by performing trypan-blue staining within the first few hours of ketamine treatment (8). However, trypan-blue staining is indicative of cell death associated with membrane leakage and does not reflect cytostatic events in intact, viable cells. Although this assay excludes a cytotoxic activity, it cannot eliminate a cytostatic effect. We conclude that reported in vitro inhibitory effects of ketamine at large concentrations are misleading. Because of the cytostatic activity exerted by large ketamine concentrations on a variety of cell types and cell functions, we recommend that the valuable antiinflammatory effects of ketamine be examined by using concentrations that do not exceed the upper anesthetic plasma concentrations of 2.0 μg/mL.