ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expression.pdf

7/28/2019 Ketamine inhibits tumor necrosis factor- and interleukin-6 gene expression.pdf

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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,

a Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwanb 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, Taiwand Department of Anesthesiology, Taipei Medical University Hospital, Taipei Medi cal 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

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 signal-

transducing 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 1000M of ketamine alone or in

combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF- and IL-6 protein levelsin concentration-

and 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

inhibitTNF- andIL-6gene 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

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 pres-

sure, and heart rate (Reich and Silvay, 1989; Chen et al., 2005b).

In addition, ketamine has also been shown to possess anti-

Available online at www.sciencedirect.com

Toxicology and Applied Pharmacology 228 (2008) 105 113www.elsevier.com/locate/ytaap

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: [email protected] (R.-M. Chen).

0041-008X/$ - see front matter 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.taap.2007.11.027
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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 therapeuticconcentration 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, macro-

phages 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 inflamma-

tory cytokines predominantly produced by macrophages, are

reported to have pleiotropic effects on regulating the immuneresponse, 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 erythema-

tosus, 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 dif-

ferentiation, and endometriosis (Sachs et al., 1989; Bonta and

Ben-Efraim, 1993; Sidell et al., 2002). Levels of TNF- and IL-

6 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 pro-

teins with extracellular domains comprised largely of leucine-

rich 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, lipopolysacchar-

ide (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 acti-

vation (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 macro-

phages and its possible signal-transducing mechanisms from

the viewpoints of cell viability, protein and RNA expressions,

TLR4 knockdown, JNK phosphorylation, and AP-1 transloca-

tion 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 mMNa2HPO4, 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-(4,5-

dimethylthiazol-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. Theblueformazanproducts in macrophages were dissolvedin dimethylsulfoxide 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. Messen-

ger 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-ATGAGCACA-

GAAAGCATGATCCGC-3 and 3-CTCAGGCCCGTCCAGATGAAACC-5

for TNF-, 5-ATGAAGTTCCTCTCTGCAAGAGACT-3 and 3-CACTAG-

GTTTGCCGAGTAGATCTC-5 for IL-6, and 5-GTGGGCCGCTCTAGG-

CACCAA-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 Biotechnol-

ogy (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 macro-

phages. 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 describedmethod (Tai et al., 2007). After drug treatment, cell lysates were prepared in ice-

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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

ofWu 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 c-Jun 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 assa y (EMSA). An EMSAwas 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 chemilu-

minescent 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 ofb0.05.

Results

To evaluate the toxic effects of ketamine and LPS on macro-

phages, 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 LPS-

induced TNF- synthesis. Treatment with 10, 100, and 1000M

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 concentra-

tion 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 adminis-

tered for 6 and 24 h, a clinically relevant concentration ofketamine 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

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 1000M

(B) for 1, 6, and 24 h. Cell viability was determined using a colorimetric method.

Each value represents the meanSEM for n =6. The asterisk () indicates that

the value significantly differs from the respective control, pb0.05. O.D., optical

density.

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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 1000M KTM for 24 h (A and C), or to 100 ng/ml LPS, 100M 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 meanSEM for n =6. The symbols, and #, indicate that a value significantly (pb0.05) 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 100ng/ml LPS, 100M

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 mRNAwas detected as an internal standard (bottom panels). These DNAbands were quantifiedand analyzed (B andD). Each valuerepresents the meanSEM forn =4. The symbols, and #, indicate that a value significantly (pb0.05) 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 siRNAwas 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 forn =6. The symbols, , #, and indicate that a

value significantly (pb0.05) 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 100ng/mlLPS, 100M KTM, or a combination of KTMand LPSfor 1 h. Phosphorylated JNK(P-JNK) wasimmunodetectedusinga rabbit polyclonal antibody with

a synthetic phospho-peptidecorresponding to residues Thr183/Tyr185 of human JNK(A, top panel). JNKwas 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 valuerepresents the meanSEM for n =4. The symbols, and #, indicate that a value significantly (pb0.05) 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 suppres-

sion of TNF- and IL-6 syntheses in LPS-activated macro-

phages, the RNA levels of these two cytokines were quantified

(Fig. 3). In untreated macrophages, low levels of TNF- and IL-6 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 IL-

6 mRNA (Figs. 3A, C, top panels, lane 3). Meanwhile, ketamine

at a clinically relevant concentration (100 M) inhibited LPS-

induced 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). Administra-

tion of LPS caused significant 63- and 20-fold increases in

cellular TNF- and IL-6 mRNA levels, respectively. A thera-peutic concentration (100 M) of ketamine significantly in-

hibited 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 suppres-

sion of TNF- and IL-6 syntheses in LPS-activated macro-

phages (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 administra-

tion 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 keta-mine-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 2.2-

fold, 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, theLPS-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). Admin-

istration of LPS caused significant 2.8- and 2.2-fold enhance-

ments 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 down-

stream events of ketamine-caused suppression of the phosphor-ylation 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 wereexposedto 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 meanSEM forn = 4. The symbols, and #, indicate that a

value significantly (pb

0.05) differed from the control or LPS-treated groups,respectively.



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binding activity of nuclear extracts to AP-1 consensus oligo-

nucleotides 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 consensusoligonucleotides 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 decreasesin 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 concentra-

tions (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 bio-

syntheses 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 bio-

syntheses contributed to its anti-inflammatory effects in LPS-

activated 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 IL-

6 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 biosynth-

eses 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 IL-

6 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 transcrip-

tion 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 (Luet al., 2005). Therefore, ketamine inhibits thebiosyntheses 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 LPS-caused enhancements of TNF- and IL-6 syntheses are TLR4-

dependent. 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 inhibitTNF-

and IL-6 gene expressions and macrophage activation. Expo-

sure 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, proteinkinase 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 LPS-

activated macrophages. Thus, the ketamine-induced reduction

of JNK phosphorylation may be due to suppression of TLR4-

involved 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 rathippocampus, pretreatment with ketamine was shown to

decrease JNK phosphorylation induced by cerebral ischemia-

reperfusion (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



8/9

increased the nuclear levels of c-Jun and c-Fos. After admin-

istration of a therapeutic concentration of ketamine, the LPS-

caused 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 activa-

tion 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 tran-scription factor.

The AP-1 transcription factor is involved in ketamine-

induced 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 ofketamine 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 endotoxin-

inducedrat acute lung injury (Yang et al., 2005). In this study, we

further showed that a therapeutic concentration of ketamine canlower 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 ofNFB 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 pro-

ductions. 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 amelio-

rated. 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 TLR4-

dependent JNK activation and AP-1 translocation and transac-tivation. 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.

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