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Ketamine inhibits tumor necrosis factor- and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation
Gone-Jhe Wu a,b,c , Ta-Liang Chen c,d , Yune-Fang Ueng e , Ruei-Ming Chen a,
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan b Department of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan c Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan Department of Anesthesiology, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan e National Research Institute of Chinese Medicine, Taipei, Taiwan Received 9 October 2007; revised 16 November 2007; accepted 28 November 2007 Available online 8 December 2007
a

Abstract Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor- (TNF-) and interlukin-6 (IL-6) gene expressions and its possible signaltransducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF- and IL-6 protein levels in concentrationand time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF- and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 M) significantly inhibited LPS-induced TNF- and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF- and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF- and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated sequential activations of c-Jun N-terminal kinase and activator protein-1. 2007 Elsevier Inc. All rights reserved.
Keywords: Ketamine; Macrophages; LPS; TNF-; IL-6; TLR4; JNK/AP-1

Introduction Ketamine, a widely used intravenous anesthetic agent, has more-stable hemodynamics than other anesthetic agents so it is
Corresponding author. Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, No. 250 Wu-Xing St., Taipei 110, Taiwan. Fax: +886 2 23778620. E-mail address: rmchen@tmu.edu.tw (R.-M. Chen). 0041-008X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2007.11.027

often applied as an inducer of anesthesia in critically ill patients (White et al., 1982; Bourgoin et al., 2003). Ketamine can also be a competitive N-methyl-D-aspartate-receptor antagonist and has been shown to have therapeutic potential in preventing anoxic damage from stroke in humans (Rothman et al., 1987). Clinically, induction of anesthesia with ketamine is usually associated with increases in cardiac output, arterial blood pressure, and heart rate (Reich and Silvay, 1989; Chen et al., 2005b). In addition, ketamine has also been shown to possess anti-

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inflammatory effects in both patients and experimental animals (Rofael et al., 2003; Yang et al., 2005; Suliburk and Mercer, 2007). Previous studies provided in vitro data demonstrating that ketamine can induce dysfunction of lymphocytes, natural killer cells, and neutrophils (Hofbauer et al., 1998; Weigand et al., 2000; Melamed et al., 2003). A recent study performed in our lab further showed that ketamine at a therapeutic concentration selectively suppressed macrophage functions of phagocytosis, oxidative ability, and cytokine production (Chang et al., 2005). In the innate immunity system, macrophages play pivotal roles in cellular host defense against infection and tissue injury (Calandra and Roger, 2003). In response to stimuli, macrophages can produce and release a variety of inflammatory cytokines, which induce serial inflammatory reactions (Nathan, 1987; Froidevaux et al., 2001). Tumor necrosis factor- (TNF) and interleukin-6 (IL-6), two typical and critical inflammatory cytokines predominantly produced by macrophages, are reported to have pleiotropic effects on regulating the immune response, acute-phase reaction, and hematopoiesis (Bendtzen, 1998). Previous studies showed that TNF- is involved in the progression of myocardial infarction, alcohol-induced liver disease, rheumatoid arthritis, human systemic lupus erythematosus, and macrophage-mediated tumor cytotoxicity (Jacob, 1992; Wilder and Elenkov, 1999; Frangogiannis et al., 2002; Nagy, 2004). IL-6 has been implicated as a regulator which modulates macrophage antitumor activities, myeloid cell differentiation, and endometriosis (Sachs et al., 1989; Bonta and Ben-Efraim, 1993; Sidell et al., 2002). Levels of TNF- and IL6 in macrophages can be altered by endogenous or exogenous factors, which then lead to immunomodulation (Scott and Kingsley, 2006). Toll-like receptors (TLRs) are type I transmembrane proteins with extracellular domains comprised largely of leucinerich repeats and intracellular signaling domains (Akira et al., 2006). Mammalian TLRs contain at least 12 member proteins which trigger host resistance to infection (Akira et al., 2006; Trinchieri and Sher, 2007). In macrophages, lipopolysaccharide (LPS), an important contributing factor to the pathogenesis of septic syndromes, can specifically activate TLR4, then stimulate translocation of the transcription factors, nuclear factor kappa B (NFB) and activator protein-1 (AP-1), from the cytoplasm to nuclei, ultimately inducing gene expression of inflammatory cytokines (Jones et al., 2001; Fan et al., 2004). Previous studies demonstrated that ketamine can inhibit TNF- gene expression through suppression of NFB activation (Sun et al., 2004; Yang et al., 2005). C-Jun N-terminal kinase (JNK)-mediated AP-1 activation is critical for cell differentiation, apoptosis, and tumorigenesis (Potapova et al., 2001; Paik et al., 2003). Meanwhile, the role of AP-1 in ketamine-induced immunosuppression is still unknown. Thus, this study was aimed to evaluate the effects of ketamine on the biosyntheses of TNF- and IL-6 in LPS-activated macrophages and its possible signal-transducing mechanisms from the viewpoints of cell viability, protein and RNA expressions, TLR4 knockdown, JNK phosphorylation, and AP-1 translocation and transactivation.

Materials and methods

Cell culture and drug treatment. Macrophage-like Raw 264.7 cells, purchased from American Type Culture Collection (Rockville, MD, USA), were cultured in RPMI 1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal calf serum, L-glutamine, penicillin (100 IU/ml), and streptomycin (100 g/ml) in 75-cm2 flasks at 37 C in a humidified atmosphere of 5% CO2. Ketamine and LPS were dissolved in phosphate-buffered saline (PBS) (0.14 M NaCl, 2.6 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4). According to the clinical application, concentrations of ketamine at 1, 10, 100, and 1000 M, which correspond to 0.01, 0.1, 1, and 10 times the clinical plasma concentration (Domino et al., 1982; Grant et al., 1983), were chosen as the administered concentrations in this study. Prior to the addition of ketamine, macrophages were washed with PBS buffer, and nonadherent cells were removed. Control macrophages received PBS only. Assay of cell viability. Cell viability was determined by a colorimetric 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (Chen et al., 2007). Briefly, macrophages (2 104 cells per well) were seeded in 96-well tissue culture plates overnight. After drug treatment, macrophages were cultured with new medium containing 0.5 mg/ml MTT for a further 3 h. The blue formazan products in macrophages were dissolved in dimethyl sulfoxide and spectrophotometrically measured at a wavelength of 550 nm. Enzyme-linked immunosorbent assay (ELISA). Levels of TNF- and IL-6 in the culture medium of macrophages were determined according to a previously described method (Chen et al., 2005a). Briefly, macrophages (2 104 cells/well) were seeded in 96-well tissue culture plates overnight. After drug treatment, the medium was collected and centrifuged. The amounts of TNF- and IL-6 were quantified following the standard protocols of the ELISA kits purchased from Endogen (Woburn, MA, USA). Reverse-transcription polymerase chain reaction (RT-PCR) assay. Messenger RNA from macrophages exposed to LPS, ketamine, or a combination of ketamine and LPS was prepared for RT-PCR analyses of TNF-, IL-6, and actin mRNA. Oligonucleotides for the PCR analyses of TNF-, IL-6, and actin mRNA were designed and synthesized by Clontech Laboratories (Palo Alto, CA, USA). The oligonucleotide sequences of the respective upstream and downstream primers for these mRNA analyses were 5-ATGAGCACAGAAAGCATGATCCGC-3 and 3-CTCAGGCCCGTCCAGATGAAACC-5 for TNF-, 5-ATGAAGTTCCTCTCTGCAAGAGACT-3 and 3-CACTAGGTTTGCCGAGTAGATCTC-5 for IL-6, and 5-GTGGGCCGCTCTAGGCACCAA-3 and 5-CTCTTTGATGTCACGCACGATTTC-3 for -actin (Wu et al., 2003). The PCR reaction was carried out using 35 cycles of 94 C for 45 s, 60 C for 45 s, and 72 C for 2 min. The PCR products were loaded onto a 1.8% agarose gel containing 0.1 g/ml ethidium bromide and electrophoretically separated. DNA bands were visualized and photographed under UVlight exposure. The intensities of the DNA bands in the agarose gel were quantified with the aid of the UVIDOCMW ver. 99.03 digital imaging system (Uvtec, Cambridge, UK). TLR4 knockdown. Translation of TLR4 mRNA in macrophages was knocked-down using an RNA interference (RNAi) method following a small interfering (si)RNA transfection protocol provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). TLR4 siRNA was purchased from Santa Cruz Biotechnology, and is a pool of 3 target-specific 2025-nt siRNAs designed to knock-down TLR4's expression. Briefly, after culturing macrophages in antibiotic-free RPMI medium at 37 C in a humidified atmosphere of 5% CO2 for 24 h, the siRNA duplex solution, which was diluted in the siRNA transfection medium (Santa Cruz Biotechnology), was added to the macrophages. After transfecting for 24 h, the medium was replaced with normal RPMI medium, and macrophages were treated with ketamine, LPS, or a combination of ketamine and LPS. Immunoblotting analyses of JNK, phosphorylated JNK, TLR4, and -actin. Protein analyses were carried out according to a previously described method (Tai et al., 2007). After drug treatment, cell lysates were prepared in ice-

G.-J. Wu et al. / Toxicology and Applied Pharmacology 228 (2008) 105113 cold radioimmunoprecipitation assay buffer (25 mM TrisHCl (pH 7.2), 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 0.15 M NaCl, and 1 mM EDTA). Protein concentrations were quantified using a bicinchonic acid protein assay kit (Pierce, Rockford, IL, USA). The proteins (50 g/well) were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. After blocking, the phosphorylated JNKs were immunodetected using a rabbit polyclonal antibody with a synthetic phospho-peptide corresponding to residues Thr183/Tyr185 of the human JNK (Cell Signaling, Danvers, MA, USA). The JNK was detected using a mouse monoclonal antibody against the human JNK (Cell Signaling) as the internal standard. TLR4 was immunodetected using a goat polyclonal antibody against mouse TLR4 (Santa Cruz Biotechnology). Cellular -actin protein was immunodetected using a mouse monoclonal antibody against mouse -actin (Sigma, St. Louis, MO, USA) as the internal standard. These protein bands were quantified using a digital imaging system (UVtec). Extraction of nuclear proteins and immunodetection. Nuclear components were extracted, and an immunodetection was carried out following the method of Wu et al. (2007). After drug treatment, nuclear extracts of macrophages were prepared. Protein concentrations were quantified by a bicinchonic acid protein assay kit (Pierce). Nuclear proteins (50 g/well) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After blocking, nuclear c-Jun and c-Fos were immunodetected using rabbit polyclonal antibodies against mouse cJun or human c-Fos (Santa Cruz Biotechnology). Total cellular c-Jun and c-Fos were immunodetected as internal standards. Intensities of the immunoreactive bands were determined using a digital imaging system (UVtec). Electrophoretic mobility shift assay (EMSA). An EMSA was performed using a Dig gel shift kit (Roche Diagnostics, Mannheim, Germany). Briefly, AP-1 consensus oligonucleotides (Santa Cruz Biotechnology) were labeled with Dig. The nuclear extracts (10 ng) were reacted with Dig-labeled oligonucleotides at room temperature for 25 min. The complex was subjected to nondenatured polyacrylamide gel electrophoresis, and transferred to positively charged nylon membranes. After cross-linking at 120 mJ and blocking with the blocking buffer (Santa Cruz Biotechnology) at room temperature for 30 min, the membranes were immunoreacted with the anti-Dig-AP. Following washing and chemiluminescent detection, the membranes were exposed to X-ray film. Intensities of the immunoreactive bands were determined using a digital imaging system (UVtec). Statistical analysis. The statistical significance of differences among control, ketamine-, LPS-, and ketamine + LPS-treated macrophages was evaluated using nonparametric ANOVA followed by Duncan's multiple-range test, and differences were considered statistically significant at p values of b 0.05.

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Fig. 1. Effects of ketamine (KTM) and lipopolysaccharide (LPS) on macrophage viability. Macrophages were exposed to 1, 10, 100, and 1000 M KTM (A), or to 100 ng/ml LPS or a combination of LPS and KTM at 1, 10, 100, and 1000 M (B) for 1, 6, and 24 h. Cell viability was determined using a colorimetric method. Each value represents the mean SEM for n = 6. The asterisk () indicates that the value significantly differs from the respective control, p b 0.05. O.D., optical density.

Results To evaluate the toxic effects of ketamine and LPS on macrophages, cell viability was assayed (Fig. 1). Treatment with 1, 10, and 100 M ketamine for 1, 6, and 24 h was not cytotoxic to macrophages (Fig. 1A). In 1- and 6-h-treated macrophages, ketamine at 1000 M did not affect the cell viability. However, after exposure to 1000 M ketamine for 24 h, the cell viability was significantly reduced by 48%. Administration of 100 ng/ml LPS to macrophages for 1, 6, and 24 h did not influence cell viability (Fig. 1B). The combination of LPS and ketamine at 1, 10, and 100 M for 1, 6, and 24 h was not toxic to macrophages. When the administered time interval reached 24 h, exposure to the combination of LPS and 1000 M ketamine caused 50% of macrophages to die (Fig. 1B). Amounts of TNF- and IL-6 were detected to determine the effects of ketamine on the syntheses of these two cytokines in LPS-activated macrophages (Fig. 2). Exposure of macrophages to LPS for 24 h caused a 7-fold increase in the levels of cellular

TNF- (Fig. 2A). Ketamine at 1 M did not change LPSinduced TNF- synthesis. Treatment with 10, 100, and 1000 M ketamine significantly decreased cellular TNF- by 23%, 53%, and 74%, respectively. Administration of 100 ng/ml LPS to macrophages for 1, 6, and 24 h caused significant increases of 2.3-, 3.8-, and 7.3-fold, respectively, in cellular TNF- levels (Fig. 2B). Treatment with ketamine at a therapeutic concentration of 100 M for 1, 6, and 24 h did not affect TNF- production. Exposure to 100 M ketamine for 1 h did not influence LPS-induced TNF- synthesis. After being administered for 6 and 24 h, a clinically relevant concentration of ketamine significantly decreased LPS-induced augmentation in the levels of TNF- by 42% and 58%, respectively (Fig. 2B). Administration of 100 ng/ml LPS to macrophages for 24 h increased cellular IL-6 amounts by 30-fold (Fig. 2C). Ketamine at 1 M did not change the LPS-induced IL-6 production. Meanwhile, when the concentrations reached 10, 100, and 1000 M, ketamine significantly reduced LPS-induced IL-6 synthesis by 33%, 61%, and 83%, respectively. Exposure to 100 ng/ml LPS for 1, 6, and 24 h augmented cellular IL-6 levels by 8-, 20-, and 34-fold, respectively (Fig. 2D). Treatment of macrophages with 100 M ketamine for 1, 6, and 24 h did not change the cellular IL-6 amounts. In 1-h-treated macrophages, ketamine did not influence LPS-induced IL-6 production. After

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Fig. 2. Concentration- and time-dependent effects of ketamine (KTM) on lipopolysaccharide (LPS)-induced TNF- and IL-6 productions. Macrophages were exposed to either 100 ng/ml LPS or a combination of LPS and 1, 10, 100, and 1000 M KTM for 24 h (A and C), or to 100 ng/ml LPS, 100 M KTM, or a combination of KTM and LPS for 1, 6, and 24 h (B and D). The levels of TNF- and IL-6 in the culture medium of macrophages were quantified using enzyme-linked immunosorbent assays. Each value represents the mean SEM for n = 6. The symbols, and #, indicate that a value significantly (p b 0.05) differed from the control or LPS-treated groups, respectively.

Fig. 3. Effects of ketamine (KTM) on lipopolysaccharide (LPS)-induced TNF- and IL-6 mRNA syntheses. Macrophages were exposed to either 100 ng/ml LPS, 100 M KTM, or a combination of KTM and LPS for 6 h. Messenger RNA from control and drug-treated macrophages was prepared for RT-PCR analyses of TNF- and IL-6 mRNA (A and C, top panels). -Actin mRNA was detected as an internal standard (bottom panels). These DNA bands were quantified and analyzed (B and D). Each value represents the mean SEM for n = 4. The symbols, and #, indicate that a value significantly (p b 0.05) differed from the control or LPS-treated groups, respectively.

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Fig. 4. Effects of TLR4 small interfering (si)RNA on TNF- and IL-6 productions. TLR4 siRNA was applied to macrophages for 24 and 48 h. The amounts of TLR4 were immunodetected using a goat polyclonal antibody against mouse TLR4 (A, top panel). -Actin was quantified as an internal standard (bottom panel). These immunoreactive protein bands were quantified and analyzed (B). After application of TLR4 siRNA for 48 h, macrophages were exposed to either 100 ng/ml lipopolysaccharide (LPS), 100 M ketamine (KTM), or a combination of KTM and LPS for another 24 h. Enzyme-linked immunosorbent assays were performed to determine the levels of TNF- and IL-6 in the culture medium of macrophages. Each value represents the mean SEM for n = 6. The symbols, , #, and indicate that a value significantly (p b 0.05) differed from the control, LPS-, or KTM + LPS-treated groups, respectively.

Fig. 5. Effects of ketamine (KTM) on lipopolysaccharide (LPS)-induced phosphorylation of JNK and the translocation of c-Jun and c-Fos. Macrophages were exposed to either 100 ng/ml LPS, 100 M KTM, or a combination of KTM and LPS for 1 h. Phosphorylated JNK (P-JNK) was immunodetected using a rabbit polyclonal antibody with a synthetic phospho-peptide corresponding to residues Thr183/Tyr185 of human JNK (A, top panel). JNK was analyzed using a mouse monoclonal antibody against human JNK as an internal standard (bottom panel). Nuclear c-Jun (Nc-Jun) and c-Fos (Nc-Fos) were immunodetected using rabbit polyclonal antibodies against mouse c-Jun or human c-Fos (C). Total c-Jun (Tc-Jun) and c-Fos (Tc-Fos) were detected as internal standards. These protein bands were quantified and analyzed (B and D). Each value represents the mean SEM for n = 4. The symbols, and #, indicate that a value significantly (p b 0.05) differed from the control or LPS-treated groups, respectively.

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exposure for 6 and 24 h, ketamine at a therapeutic concentration (100 M) significantly decreased LPS-induced IL-6 synthesis by 40% and 53%, respectively (Fig. 2D). To determine the mechanism of ketamine-caused suppression of TNF- and IL-6 syntheses in LPS-activated macrophages, the RNA levels of these two cytokines were quantified (Fig. 3). In untreated macrophages, low levels of TNF- and IL6 were detected (Figs. 3A, C, top panels, lane 1). After exposure to 100 ng/ml LPS for 6 h, TNF- and IL-6 mRNA were obviously induced (Figs. 3A, C, top panels, lane 2). Treatment with ketamine alone did not affect the synthesis of TNF- or IL6 mRNA (Figs. 3A, C, top panels, lane 3). Meanwhile, ketamine at a clinically relevant concentration (100 M) inhibited LPSinduced TNF- and IL-6 mRNA productions (Figs. 3A, C, top panels, lane 4). The amounts of -actin mRNA were detected as internal controls (Figs. 3A, C, bottom panels). These DNA bands were quantified and analyzed (Figs. 3B, D). Administration of LPS caused significant 63- and 20-fold increases in cellular TNF- and IL-6 mRNA levels, respectively. A therapeutic concentration (100 M) of ketamine significantly inhibited LPS-induced TNF- and IL-6 mRNA levels by 73% and 79%, respectively (Figs. 3B, D). TLR4 siRNA was applied to macrophages to determine the roles of this membrane receptor in ketamine-induced suppression of TNF- and IL-6 syntheses in LPS-activated macrophages (Fig. 4). After treatment with TLR4 siRNA for 24 and 48 h, levels of TLR4 protein in macrophages were obviously downregulated (Fig. 4A, top panel). The amounts of -actin in macrophages were detected as internal standards (bottom panel). These protein bands were quantified and analyzed (Fig. 4B). Application of TLR4 siRNA to macrophages for 24 and 48 h significantly decreased cellular TLR4 levels by 47% and 87%, respectively. Administration of TLR4 siRNA for 48 h slightly affected the basal levels of TNF- or IL-6 (Figs. 4C, D). After application of TLR4 siRNA in macrophages for 48 h, the LPS-induced increases in the amounts of TNF- and IL-6 decreased by 66% and 70%, respectively. Administration of ketamine at a therapeutic concentration (100 M) significantly decreased LPS-induced syntheses of TNF- and IL-6 by 70% and 72%, respectively. By comparison with alone administration of TLR4 siRNA, co-treatment of macrophages with ketamine and TLR4 siRNA caused more suppression in the LPS-induced TNF- and IL-6 syntheses (Figs. 4C, D). To determine the signal-transducing mechanisms of ketamine-caused inhibition of TNF- and IL-6 gene expressions in LPS-activated macrophages, phosphorylation of JNK1/2 and the translocations of c-Jun and c-Fos from the cytoplasm to nuclei were evaluated (Fig. 5). Exposure of macrophages to LPS obviously increased the phosphorylation of JNK1/2 (Fig. 5A, top panel, lane 2). Ketamine did not influence JNK1/2 phosphorylation (lane 3). However, treatment with ketamine decreased LPS-induced JNK1/2 phosphorylation (lane 4). Nonphosphorylated JNK was immunodetected as the internal standard (Fig. 5A, bottom panel). These protein bands were quantified and analyzed (Fig. 5B). Administration of LPS increased phosphorylation of JNK1 and JNK2 by 2.5- and 2.2fold, respectively. After exposure to a therapeutic concentration

of ketamine, LPS-induced phosphorylation of JNK1 and JNK2 was significantly ameliorated by 37% and 46%, respectively (Fig. 5B). Exposure to LPS increased nuclear levels of c-Jun and c-Fos (Fig. 5C, top 1 and 3 panels, lane 2). Treatment with ketamine did not affect nuclear c-Jun or c-Fos levels (lane 3). After exposure to a clinically relevant concentration of ketamine, the LPS-caused increases in nuclear c-Jun and c-Fos were significantly reduced (lane 4). Total levels of c-Jun and c-Fos in macrophages were immunodetected as internal standards (Fig. 5C, top 2 and bottom panels). These immunoreactive protein bands were quantified and analyzed (Fig. 5D). Administration of LPS caused significant 2.8- and 2.2-fold enhancements in the nuclear c-Jun and c-Fos levels, respectively. After treatment with ketamine, the LPS-induced increases in the amounts of nuclear c-Jun and c-Fos significantly suppressed (Fig. 5D). Analysis by EMSA was performed to evaluate the downstream events of ketamine-caused suppression of the phosphorylation of JNK and the sequential translocation of c-Jun and c-Fos (Fig. 6). Administration of LPS to macrophages increased the DNA binding activity of nuclear extracts to AP-1 consensus oligonucleotides (Fig. 6A, top panel, lane 2). Treatment with ketamine alone did not influence the binding activity of AP-1 (lane 3). However, the LPS-induced enhancement in the DNA

Fig. 6. Effects of ketamine (KTM) on lipopolysaccharide (LPS)-induced DNA binding activity of the AP-1 transcriptional factor. Macrophages were exposed to either 100 ng/ml LPS, 100 M KTM, or a combination of KTM and LPS for 1 h. AP-1 consensus oligonucleotides were labeled with digoxigenin, and then reacted with 10 g of nuclear extracts. The complex was electrophoretically separated and blotted onto nylon membranes. After cross-linking and blocking, the membranes were immunoreacted with the anti-digoxigenin-AP. Following washing and chemiluminescent detection, the membranes were exposed to X-ray films, and the amounts of free probes were detected as an internal standard (A). These DNAprotein binding bands were quantified and analyzed (B). Each value represents the mean SEM for n = 4. The symbols, and #, indicate that a value significantly (p b 0.05) differed from the control or LPS-treated groups, respectively.

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binding activity of nuclear extracts to AP-1 consensus oligonucleotides was obviously ameliorated following administration of a clinically relevant concentration of ketamine (100 M; lane 4). The free probes were detected as internal standards (Fig. 6A, bottom panel). These protein-DNA bands were quantified and analyzed (Fig. 6B). Exposure of macrophages to LPS increased the DNA-binding activity of nuclear extracts to AP-1 consensus oligonucleotides by 12.6-fold. Meanwhile, treatment with ketamine significantly decreased LPS-induced increases in the association of nuclear extracts with AP-1 DNA oligonucleotides (Fig. 6B). Discussion Ketamine at a therapeutic concentration (100 M) can downregulate TNF- and IL-6 syntheses. This study showed that exposure of macrophages to LPS significantly increased cellular TNF- and IL-6 protein levels. Administration of ketamine caused concentration- and time-dependent decreases in LPS-induced TNF- and IL-6 productions. Ketamine is an intravenous anesthetic agent usually used for critically ill patients (White et al., 1982; Bourgoin et al., 2003). Ketamine at 100 M is within the range of clinically relevant concentrations (Domino et al., 1982; Grant et al., 1983). After exposure to a therapeutic concentration of 100 M, ketamine was not cytotoxic to macrophages but significantly ameliorated biosyntheses of TNF- and IL-6 induced by LPS. A previous study reported that ketamine suppressed cytokine production in rats which were suffering acute lung injury, but only at a large dose (Yang et al., 2005). Thus, this study further showed that ketamine at a clinically relevant concentration could inhibit TNF- and IL-6 productions in LPS-activated macrophages. LPS has been implicated as a critical factor in the process of septic syndrome (Raetz et al., 1994). TNF- and IL-6 are two major inflammatory cytokines produced by macrophages, and have been reported to participate in regulating the immune response, acute-phase reaction, and hematopoiesis (Bendtzen, 1998). Decreases in the levels of these two cytokines can lead to immunosuppression. Our previous study showed that ketamine can specifically reduce phagocytosis and the oxidative ability of macrophages (Chang et al., 2005). In this study, we found that the ketamine-involved suppression of TNF- and IL-6 biosyntheses contributed to its anti-inflammatory effects in LPSactivated macrophages. The mechanism of ketamine-induced suppression of TNF- and IL-6 syntheses occurs at the transcriptional level. Exposure of macrophages to LPS increased the amounts of TNF- and IL6 proteins in concentration- and time-dependent manners. After administration of ketamine, the LPS-induced enhancements of TNF- and IL-6 proteins were significantly ameliorated. In rats injected with endotoxin, ketamine was shown to attenuate septic syndrome through downregulating TNF- and IL-6 biosyntheses at the protein level (Taniguchi et al., 2001). Meanwhile, RNA levels of TNF- and IL-6 in macrophages were augmented following LPS administration. Administration of ketamine at a therapeutic concentration inhibited LPS-induced TNF- and IL6 mRNA productions. Analysis of transcription factors further

revealed that exposure to LPS increased the translocation and transactivation of c-Jun and c-Fos, but ketamine significantly alleviated the augmentation. Jun and Fos are two critical components for constructing the heterodimers of the transcription factor, AP-1 (Hess et al., 2004). AP-1 consensus DNA motifs have been found in the promoter regions of both TNF- and IL-6 genes (Lu et al., 2005). Therefore, ketamine inhibits the biosyntheses of TNF- and IL-6 through a transcriptional mechanism. The ketamine-involved suppression of TNF- and IL-6 productions may be TLR4-dependent. Mammalian TLRs mediate a host's resistance to infection (Akira et al., 2006; Trinchieri and Sher, 2007). This study showed that application of TLR4 siRNA into macrophages decreased cellular TLR4 protein expression. In parallel with TLR4 knockdown, the LPS-induced enhancements of TNF- and IL-6 syntheses were simultaneously reduced. A previous study used targeted disruption of genomic TLR4 in mice to also confirm that the TLR4 receptor is necessary for sensitive responses to LPS (Hoshino et al., 1999).Thus, the LPScaused enhancements of TNF- and IL-6 syntheses are TLR4dependent. Co-treatment of macrophages with ketamine and TLR4 siRNA caused more suppression of the LPS-induced TNF- and IL-6 expressions than alone treatment with TLR4 siRNA. Therefore, TLR4 could be used as an effector involved in the ketamine-caused suppression of TNF- and IL-6 productions in LPS-activated macrophages. Ketamine can decrease JNK phosphorylation to inhibit TNF- and IL-6 gene expressions and macrophage activation. Exposure of macrophages to LPS increased JNK phosphorylation. Meanwhile, administration of ketamine at a clinically relevant concentration (100 M) significantly decreased LPS-induced JNK phosphorylation. In response to LPS stimulation, protein kinase JNK is activated via a TLR4-dependent mechanism (Jones et al., 2001; Fan et al., 2004). Our results revealed that ketamine can attenuate TLR4-mediated signals in LPSactivated macrophages. Thus, the ketamine-induced reduction of JNK phosphorylation may be due to suppression of TLR4involved signal transduction by this intravenous anesthetic. JNK is an upstream protein kinase for activating AP-1 (Potapova et al., 2001). Phosphorylation of JNK can activate AP-1 and induce certain gene expression. For example, a previous study showed that the JNK pathway participates in upregulating LPS-induced inducible nitric oxide (NO) synthase expression and NO production (Zhang et al., 2006). In the rat hippocampus, pretreatment with ketamine was shown to decrease JNK phosphorylation induced by cerebral ischemiareperfusion (Lahti et al., 2003). In this study, we further demonstrated that ketamine can decrease LPS-induced JNK phosphorylation. In addition, stress-induced JNK activation has been reported to be important in cell differentiation, apoptosis, and tumorigenesis (Potapova et al., 2001; Paik et al., 2003). Therefore, ketamine can decrease JNK phosphorylation to inhibit TNF- and IL-6 gene expressions, which leads to suppression of LPS-induced macrophage activation. Ketamine ameliorates LPS-induced translocation of c-Jun and c-Fos from the cytoplasm to nuclei via suppression of JNK activation. Exposure of macrophages to LPS significantly

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increased the nuclear levels of c-Jun and c-Fos. After administration of a therapeutic concentration of ketamine, the LPScaused enhancements significantly decreased. JNK activation has been reported to phosphorylate c-Jun and c-Fos, which triggers the translocation of these two DNA-binding proteins from the cytoplasm to nuclei (Potapova et al., 2001). Treatment with ketamine can reduce LPS-induced JNK phosphorylation. The total protein levels of c-Jun and c-Fos were not influenced by the administration of ketamine, LPS, or a combination of ketamine and LPS. Thus, one of the major reasons explaining how ketamine can decrease nuclear c-Jun and c-Fos levels is that this intravenous anesthetic agent can suppress the translocation of c-Jun and c-Fos from the cytoplasm to nuclei via downregulation of LPS-stimulated JNK phosphorylation. Extracellular signal-regulated kinases can contribute to activation of AP-1 (Kolch, 2005). Therefore, the other possible reason explaining the ketamine-caused reduction in AP-1 activation is that ketamine may modulate extracellular signal-regulated kinase phosphorylation to downregulate activation of this transcription factor. The AP-1 transcription factor is involved in ketamineinduced suppression of TNF- and IL-6 gene expressions. Jun and Fos are two major components in the heterodimeric structure of AP-1 (Hess et al., 2004). The ketamine-induced suppression of c-Jun and c-Fos translocation from the cytoplasm to nuclei means that this anesthetic agent can decrease AP-1 activation in LPS-stimulated macrophages. The AP-1 consensus DNA motifs are found in the promoter regions of the TNF- and IL-6 genes (Lu et al., 2005). Analysis by EMSA revealed that exposure of macrophages to LPS increased the DNA binding activity of AP-1. The LPS-induced increases in AP-1 DNA binding activity were significantly alleviated following administration of ketamine at a clinically relevant concentration (100 M). In response to stimulation by LPS, there are at least two different mechanisms reported to respectively induce TLR-dependent activation of NFB and AP-1 to regulate the expressions of inflammatory cytokine genes (Jones et al., 2001, Fan et al., 2004). In the intestine of rats, ketamine was shown to inhibit endotoxin-induced TNF- and IL-6 productions via suppression of NFB activity (Sun et al., 2004). Yang et al. (2005) reported that a large dose of ketamine can reduce NFB activation and inhibit expressions of TNF- and IL-6 genes in endotoxininduced rat acute lung injury (Yang et al., 2005). In this study, we further showed that a therapeutic concentration of ketamine can lower LPS-induced TNF- and IL-6 gene expressions through suppressing the translocation and transactivation of AP-1 in LPS-activated macrophages. Meanwhile, this study can not rule out the roles of NFB in ketamine-involved suppression of TNF and IL-6 gene expressions in LPS-activated macrophages. In conclusion, the present study shows that a clinically relevant concentration of ketamine (100 M) can decrease the biosyntheses of TNF- and IL-6 in LPS-activated macrophages. In parallel with decreases in protein levels, administration of ketamine significantly inhibited TNF- and IL-6 mRNA productions. Analysis of RNA interference further revealed that the ketamine-involved suppressions of TNF- and IL-6 syntheses were TLR4-dependent. After exposure to ketamine, the LPS-

induced phosphorylation of JNK and sequent translocation of AP-1 from the cytoplasm to nuclei was significantly ameliorated. Consequently, ketamine can suppress AP-1 DNA binding activity induced by LPS. According to our present data, we suggest that ketamine reduces the biosyntheses of TNF- and IL-6 in LPS-activated macrophages through suppressing TLR4dependent JNK activation and AP-1 translocation and transactivation. The ketamine-induced suppression of inflammatory cytokine syntheses can partially explain its anti-inflammatory and immunosuppressive effects in clinical applications. In the future study, we will evaluate the signal-transducing effects of ketamine on the downstream molecules of TLR4 to further determine the roles of TLR4 in suppression of LPS-induced TNF- and IL-6 gene expressions induced by this intravenous anesthetic agent. Acknowledgments This study was supported by the National Science Council (NSC95-2745-B-038-001-URD) and Shin Kong Wu Ho-Su Memorial Hospital (SKH-TMU-94-04), Taipei, Taiwan. The authors express their gratitude to Ms. Yi-Ling Lin for her technical support and data collection during the experiments. References
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