Signal-transducing mechanisms of ketamine-caused inhibition of interleukin-1β gene expression in lipopolysaccharide-stimulated murine macrophage-like Raw 264.7 cells

https://doi.org/10.1016/j.taap.2009.06.013Get rights and content

Abstract

Ketamine may affect the host immunity. Interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) are pivotal cytokines produced by macrophages. This study aimed to evaluate the effects of ketamine on the regulation of inflammatory cytokine gene expression, especially IL-1β, in lipopolysaccharide (LPS)-activated murine macrophage-like Raw 264.7 cells and its possible signal-transducing mechanisms. Administration of Raw 264.7 cells with a therapeutic concentration of ketamine (100 μM), LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. Exposure to 100 μM ketamine decreased the binding affinity of LPS and LPS-binding protein but did not affect LPS-induced RNA and protein synthesis of TLR4. Treatment with LPS significantly increased IL-1β, IL-6, and TNF-α gene expressions in Raw 264.7 cells. Ketamine at a clinically relevant concentration did not affect the synthesis of these inflammatory cytokines, but significantly decreased LPS-caused increases in these cytokines. Immunoblot analyses, an electrophoretic mobility shift assay, and a reporter luciferase activity assay revealed that ketamine significantly decreased LPS-induced translocation and DNA binding activity of nuclear factor-kappa B (NFκB). Administration of LPS sequentially increased the phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK. However, a therapeutic concentration of ketamine alleviated such augmentations. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA reduced cellular TLR4 amounts and ameliorated LPS-induced RAS activation and IL-1β synthesis. Co-treatment with ketamine and TLR4 siRNA synergistically ameliorated LPS-caused enhancement of IL-1β production. Results of this study show that a therapeutic concentration of ketamine can inhibit gene expression of IL-1β possibly through suppressing TLR4-mediated signal-transducing phosphorylations of Ras, Raf, MEK1/2, ERK1/2, and IKK and subsequent translocation and transactivation of NFκB.

Introduction

Ketamine is a widely used intravenous anesthetic agent, which is clinically applied to patients for inducing and maintaining anesthesia during surgical procedures (Himmelseher and Durieux, 2005). Because ketamine has fewer depressant effects on the cardiovascular system resulting in a better patient hemodynamic profile than barbiturates, inhalational anesthetics or propofol, it is often used for anesthesia in severely ill patients (White et al., 1982). Clinical analyses have implied that ketamine possesses immunomodulatory and anti-inflammatory characteristics (Molina et al., 2004). Previous studies showed that ketamine can suppress cocaine-caused immunotoxicity and lipopolysaccharide (LPS)-induced acute lung injury in rats (Rofael et al., 2003, Yang et al., 2005). In cardiac surgical patients, cardiopulmonary bypass-induced inflammation can be attenuated by ketamine administration (Bartoc et al., 2006). A variety of in vitro studies further reported that ketamine can suppress the activities of immune cells. For example, pretreatment with ketamine was shown to decrease leukocyte adherence in rat mesenteric venules (Schmidt et al., 1995). At clinically relevant concentrations, ketamine reduces phagocytotic activity and expressions of the adhesion molecules, CD18 and CD62L, in human neutrophils (Nishina et al., 1998, Weigand et al., 2000). Our previous study further showed that a therapeutic concentration of ketamine induces mitochondrial dysfunction and selectively suppresses the phagocytotic activity and oxidative ability of macrophages (Chang et al., 2005, Wu et al., 2008).

In the innate immune system, macrophages play pivotal roles in mediating a host's response to bacterial infection or tissue injuries (Nathan, 1987, Lee et al., 2009a, Lee et al., 2009b). During inflammation, a body of inflammatory cytokines is produced by macrophages to trigger activation of innate or adaptive-immune cells (Aderem, 2001). Interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) predominantly produced by macrophages participate in regulating innate host defenses and homeostasis (Wewers, 2004). Studies on disease regulation have implied that these inflammatory cytokines represent a common effector in the pathogenesis of inflammatory joint disorders such as rheumatoid arthritis (Firestein, 2004). Production of IL-1β by islet-infiltrating macrophages causes beta-cell dysfunction through nitric oxide production in human insulin-dependent diabetes mellitus (Sjoholm, 1998). A plethora of intrinsic and extrinsic factors can regulate IL-1β gene expression. LPS, a component of gram-negative bacterial cell walls, is a typical stimulator for regulating gene expression of inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in macrophages, and has been implicated as an important contributing factor to the pathogenesis of septic syndrome (Almeida and Gazzinelli, 2001, West and Heagy, 2004, Chen et al., 2005aa,b). Previous studies showed that ketamine suppresses the biosyntheses of TNF-α and IL-6 in LPS-stimulated macrophages (Sun et al., 2004, Yang et al., 2005, Chang et al., 2005, Wu et al., 2008). Meanwhile, there are few studies on the effects of ketamine in regulating IL-1β gene expression.

During inflammation, LPS first binds to the LPS-binding protein (LBP) in the bloodstream (Wright et al., 1990). Then, the LPS–LBP complex induces certain gene expressions via toll-like receptor (TLR)-dependent mechanisms (Akira et al., 2006). TLRs, which are type I transmembrane proteins with extracellular domains comprised largely of leucine-rich repeats and intracellular signaling domains, have at least 12 members found in mammalian cells (Akira et al., 2006). TLR4 has been shown to be a major receptor in macrophages responsible for LPS stimulation (Beutler and Rietschel, 2003, Wu et al., 2009). After binding to the LPS–LBP complex, the alteration in TLR4's conformation induces cascade activation of intracellular protein kinases (Schroder et al., 2000). A recent study showed that the Ras protein can mediate the transduction of TLR signaling to diverse biological functions such as cell growth, survival, and differentiation (Kogut et al., 2007). After being phosphorylated by the Ras protein, activated Raf kinase sequentially triggers mitogen-activated protein kinase kinases (MEK) 1/2 and extracellular signal-regulated kinases (ERK) 1/2 (Kolch, 2005, Kholodenko, 2007). Activation of inhibitor kappaB kinase (IKK) by ERK1/2 then stimulates the translocation and transactivation of transcription factor nuclear factor-kappa B (NFκB), which induces the expressions of certain inflammatory genes (Dobrovolskaia et al., 2003, Siwak et al., 2005). NFκB-DNA binding elements are found in promoter regions of these inflammatory cytokine genes (Won et al., 2006). Previous studies showed that ketamine can alleviate LPS-induced NFκB activation (Sun et al., 2004, Yang et al., 2005). However, the role of the ERK1/2 cascade in ketamine-induced regulation of these inflammatory gene expressions is not well understood. Thus in this study, we attempted to evaluate the effects of ketamine on the biosynthesis of inflammatory cytokines, especially IL-1β, in LPS-activated macrophages, and its possible signal-transducing mechanisms in terms of activation of the TLR4-mediated Ras/Raf/MEK/ERK/IKK/NFκB cascade.

Section snippets

Cell culture and drug treatment

A murine macrophage cell line, Raw 264.7, was purchased from the American Type Culture Collection (Rockville, MD, USA). Macrophages 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 μg/ml) in 75-cm2 flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were allowed to grow to confluence prior to ketamine or LPS administration.

Ketamine and LPS were dissolved in

Results

To evaluate the toxicity of ketamine, LPS, and the combination of ketamine and LPS to macrophages, cell viabilities were evaluated (Table 1). Treatment with 1, 10, and 100 μM ketamine for 1, 6, and 24 h did not affect cell viability. In 1- and 6-h-treated macrophages, exposure to 1000 μM ketamine did not cause cell death. However, after administration of 1000 μM ketamine for 24 h, cell viability was significantly reduced by 52%. Exposure to 1, 10, and 100 ng/ml LPS for 1, 6, and 24 h did not

Discussion

This study showed that a therapeutic concentration (100 μM) of ketamine can decrease cellular IL-1β, IL-6, and TNF-α levels in LPS-activated macrophages. Exposure of macrophages to LPS increased cellular IL-1β production in concentration- and time-dependent manners. After ketamine administration, the LPS-caused enhancement of IL-1β synthesis was concentration-dependently decreased. In addition, the expression of IL-6 and TNF-α mRNA was inhibited by ketamine. Ketamine at 20–120 μM corresponds to

Acknowledgments

This study was supported by Wan-Fang Hospital (96WF-EVA-01) and the National Bureau of Controlled Drug, Department of Health (DOH98-NNB-1049), Taipei, Taiwan.

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