LIPOTEICHOIC ACIDYINDUCED TNF-! AND IL-6 GENE EXPRESSIONS AND OXIDATIVE STRESS PRODUCTION IN MACROPHAGES ARE SUPPRESSED BY KETAMINE THROUGH DOWNREGULATING TOLL-LIKE RECEPTOR 2YMEDIATED ACTIVATION OF ERK1/2 AND NF.B Huai-Chia Chang, *† Ke-Hsun Lin, ‡ Yu-Ting Tai, † Juei-Tai Chen, † and Ruei-Ming Chen *† *Graduate Institute of Medical Sciences, Taipei Medical University; † Drug Abuse Research Center, Cell Physiology and Molecular Image Research Center, Department of Anesthesiology, Taipei Medical University-Wan Fang Hospital; and ‡ Department of Urology, Wan-Fang Hospital, Taipei Medical University, Taipei, Taiwan Received 28 Apr 2009; first review completed 26 May 2009; accepted in final form 3 Aug 2009 ABSTRACT—Lipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that ketamine has anti-inflammatory and antioxidant effects on gram-negative LPS-induced macrophage activation. In this study, we further evaluated the effects of ketamine on the regulation of LTA-induced TNF-! and IL-6 gene expressions and oxidative stress production in macrophages and its possible mechanisms. Exposure of macrophages to a therapeutic concentration of ketamine (100 2M) inhibited LTA-induced TNF-! and IL-6 expressions at protein or mRNA levels. In parallel, ketamine at 100 2M reduced LTA-stimulated phosphorylation of extracellular signalYregulated kinase 1/2 (ERK1/2). Sequentially, ketamine reduced the LTA-triggered translocation of nuclear factor-.B (NF.B) from the cytoplasm to nuclei and its transactivation activity. Pretreatment with PD98059, an inhibitor of ERK, decreased LTA-enhanced NF.B activation and TNF-! and IL-6 mRNA syntheses. Cotreatment with ketamine and PD98059 synergistically suppressed the LTA-induced translocation and transactivation of NF.B and biosyntheses of TNF-! and IL-6 mRNA. Application of toll-like receptor 2 (TLR2) small interfering RNA (si)RNA into macrophages decreased the levels of this receptor, and simultaneously ameliorated LTA-augmented NF.B transactivation and consequent production of TNF-! and IL-6 mRNA. Cotreatment with ketamine and TLR2 siRNA synergistically lowered TNF-! and IL-6 mRNA syntheses in LTA-activated macrophages. Ketamine and TLR2 siRNA could reduce the LTA- induced increases in production of nitrite and intracellular reactive oxygen species in macrophages, and their combination had better effects than a single exposure. Thus, this study shows that one possible mechanism involved in ketamine- induced inhibition of LTA-induced TNF-! and IL-6 gene expressions and oxidative stress production is through downregulating TLR2-mediated phosphorylation of ERK1/2 and the subsequent translocation and transactivation of NF.B. KEYWORDS—Ketamine, macrophages, LTA, TLR2, inflammatory cytokines, oxidative stress INTRODUCTION Ketamine, an intravenous anesthetic agent, has more sta- ble hemodynamics than other anesthetic agents, so it is often applied as an inducer of anesthesia in critically ill patients (1). Ketamine can also be a competitive N-methyl-D- aspartate receptor antagonist and modulates central sensiti- zation induced by tissue damage or perioperative analgesics (2). Clinically, induction of anesthesia with ketamine is usually associated with increases in cardiac output, arterial blood pressure, and heart rate (3, 4). In both patients and experimental animals, ketamine has been shown to possess anti-inflammatory effects (5Y7). Previous studies provided in vitro data demonstrating that ketamine can induce dysfunc- tion of lymphocytes, natural killer cells, and neutrophils (8Y10). Recent studies in our laboratory further showed that therapeutic concentrations of ketamine selectively sup- pressed the macrophage functions of phagocytosis, oxida- tive ability, and cytokine production via a mitochondrion- dependent mechanism (11, 12). During recent decades, the incidence of sepsis and septic shock has been increasing (13). Although endotoxin-mediated events are clearly important in gram-negative infection, gram- positive bacteria also have crucial roles (14). The increasing prevalence of sepsis from gram-positive bacterial pathogens necessitates re-evaluation of many of the basic assumptions about the molecular pathogenesis of septic shock. Lipotei- choic acid (LTA), an outer membrane component of gram- positive bacteria, was shown to be one of the critical factors participating in the pathogenesis of sepsis (15). In response to stimuli, LTA can stimulate macrophages to produce massive amounts of inflammatory factors that exhibit systemic effects into the general circulation (16). TNF-! and IL-6, typical and critical inflammatory cytokines predominantly produced by macrophages, have pleiotropic effects on regulating the immune response and acute-phase reactions (17). TNF-! has been reported to contribute to the progression of myocardial infarction, rheumatoid arthritis, and macrophage-mediated tumor cytotoxicity (18, 19). IL-6 can be a regulator of macro- phage development and antitumor activities (20, 21). Effects of ketamine on LTA-induced TNF-! and IL-6 biosyntheses are still unknown. 485 SHOCK, Vol. 33, No. 5, pp. 485Y492, 2010 Address reprint requests to Ruei-Ming Chen, PhD, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Xing St, Taipei 110, Taiwan. E-mail: rmchen@tmu.edu.tw. This study was supported by the Wan-Fang Hospital (grant no. 96WF-EVA-01) and by the National Bureau of Controlled Drug, Department of Health (grant no. DOH98-NNB-1049), Taipei, Taiwan. DOI: 10.1097/SHK.0b013e3181c3cea5 Copyright Ó 2010 by the Shock Society Copyright © 2010 by the Shock Society. Unauthorized reproduction of this article is prohibited.