Copyright @ 2009 by the Shock Society. Unauthorized reproduction of this article is prohibited. TNF-! INHIBITS TOLL-LIKE RECEPTOR 4 EXPRESSION ON MONOCYTIC CELLS VIA TRISTETRAPROLIN DURING CARDIOPULMONARY BYPASS Chien-Sung Tsai,* Da-Liang Chen, †‡ Sing-Jong Lin, § Jui-Chi Tsai, F Tso-Chou Lin, ¶ Chih-Yuan Lin,* Yung-Hsiang Chen,** Go-Shine Huang, ¶ Hsiao-Ya Tsai, § Feng-Yen Lin, †‡ and Chi-Yuan Li ††‡‡ *Division of Cardiovascular Surgery, Tri-service General Hospital, National Defense Medical Center; † Division of Anesthesiology and Anesthetics and Toxicology Research Center, Taipei Medical University Hospital; ‡ Department of Anesthesiology, School of Medicine, Taipei Medical University; § Institute of Clinical Medicine and Cardiovascular Research Center, National Yang-Ming University; k Graduate Institute of Medical Sciences, ¶ Division of Anesthesiology, Tri-service General Hospital, National Defense Medical Center, Taipei; **Graduate Institute of Integrated Medicine, China Medical University, Taichung; †† Department of Medical Education, Buddhist Tzu-Chi General Hospital, Taipei; and ‡‡ Department of Anesthesia, Pain Service and Critical Care Medicine, China Medical University Hospital, Taichung, Taiwan Received 30 Sept 2008; first review completed 14 Oct 2008; accepted in final form 09 Dec 2008 ABSTRACT—Toll-like receptor 4 (TLR4) plays a major role in regulating the innate immune response, which is related to postoperative complications. Although inflammatory capacity and TNF-! synthesis were altered on monocytes after cardiopulmonary bypass (CPB), whether the CPB and the CPB-induced TNF-! affect TLR4 expression on monocytes have not yet clarified. We speculate that the changing of TNF-! level during CPB may be involved in monocytic TLR4 expression. As previous report, our enzyme-linked immunosorbent assay showed that CPB elevated the plasma level of TNF-!, whereas off-pump cardiac surgery does not. Flow cytometry reported decreased levels of monocytic TLR4 in patients undergoing CPB but not undergoing off-pump cardiac surgery. To elucidate whether the CPB-induced TNF-! is related to TLR4 down-regulation, we used human monocytic THP-1 cells. Actinomycin D chase experiments demonstrated that TNF-! decreased TLR4 expression and TLR4 mRNA stability on THP-1. Confocal microscopy and real-time polymerase chain reaction showed that TNF-! induced intracellular tristetraprolin (TTP) expression. Transfection with TTP siRNA reversed the down-regulation of TLR4 in TNF-!Ystimulated THP-1. Treatment with ERK1/2 inhibitor and SAPK/JNK inhibitor decreased TNF-!Yinduced TTP expression. Immunoprecipitation and Western blot analysis showed that the TNF- !Ymediated activation of TTP might be inhibited by p38 mitogen-activated protein kinase inhibitor and by PD98059. We also demonstrated in clinical samples with confocal microscopy and flow cytometry that CPB led to an elevation of TTP in monocytes. In conclusion, CPB and TNF-! decrease TLR4 expression on monocytes; TTP expression and mitogen- activated protein kinaseYsignaling pathways play critical roles in CPB- and TNF-!Ymediated decreases of TLR4 on monocytes. Our results suggest that using TTP to control cytokine message decay rate may be a promising approach for controlling system inflammation and preventing post-CPB complications. KEYWORDS—Cardiopulmonary bypass, toll-like receptor, tristetraprolin INTRODUCTION Toll-like receptors (TLRs) are type I transmembrane recep- tors expressed on the cell membrane (1). More than 10 types of human TLR have been identified (2). Toll-like receptors are critical for the induction of downstream signals in innate immune responses during pathogen-mediated systemic dis- turbances (3). Toll-like receptor 4 (TLR4) is required for endotoxin to activate mitogen-activated protein kinases (MAPKs) and to stimulate the generation of cytokines (4). Previous studies have demonstrated that TLR4 is abundantly expressed in monocytes and macrophages and mediates TNF- ! and manganese superoxide dismutase expression (5). Toll- like receptor 4 may be a potent stimulator of apoptosis in pathogen-exposed macrophages; in contrast, the regulation and the expression of TLR4 may be involved in changing the actual immune capacity of monocytes and macrophages (3). Previous studies showed that LPS, IFN-+ (6), TNF-! (7), oxi- dized low-density lipoprotein (8), and hypoxia (9) may change the expression and the signaling of TLR4 in endothelial and epithelial cells and leukocytes. In addition to biologic factors, TLR4 expression may be influenced by trauma, hemorrhage (10), and sepsis. The basal expression of proteins associated with inflam- matory responses is likely through the potential stability of mRNAs (11). Unstable mRNAs often contain AU-rich ele- ments (AREs) in their 3¶ untranslated region (UTR). AU-rich elementYregulated mRNA stability is mediated by RNA- binding proteins, such as human antigen R (HuR), AU-binding factor 1 (AUF1), and tristetraprolin (TTP) (12). Human antigen R regulates cyclin B1 (13) and p21 (14) mRNA stability during cell proliferation. AU-binding factor 1 exists in four isoforms 40 SHOCK, Vol. 32, No. 1, pp. 40Y48, 2009 Address reprint requests to Feng-Yen Lin, Department of Anesthesiology, School of Medicine, Taipei Medical University, No. 252, Wu-Hsing St, Taipei 110, Taiwan. E-mail: g870905@tmu.edu.tw; Chi-Yuan Li, Department of Anesthesia, Pain Service and Critical Care Medicine, China Medical University Hospital, No. 2, Yuh-Der Rd, Taichung 404, Taiwan. E-mail: cyli@ndmctsgh.edu.tw. This work was supported by the National Science Council (NSC 94-2314-B-016- 018 and NSC 96-2815-C-039-052-B), the Taipei Medical University (TMU96-AE1- B04), and the China Medical University (CMU95-288, CMU96-188, CMU96-052, and CMU96-251), Taiwan, R.O.C. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the Journal’s Website (www.shockjournal.com). DOI: 10.1097/SHK.0b013e318199608d Copyright Ó 2009 by the Shock Society