Propofol Inhibits Superoxide Production, Elastase Release, and Chemotaxis in Formyl Peptide−Activated Human Neutrophils by Blocking Formyl Peptide Receptor 1 This information is current as of September 25, 2021. Shun-Chin Yang, Pei-Jen Chung, Chiu-Ming Ho, Chan-Yen Kuo, Min-Fa Hung, Yin-Ting Huang, Wen-Yi Chang, Ya-Wen Chang, Kwok-Hon Chan and Tsong-Long Hwang J Immunol published online 13 May 2013 http://www.jimmunol.org/content/early/2013/05/12/jimmun Downloaded from ol.1202215 Supplementary http://www.jimmunol.org/content/suppl/2013/05/13/jimmunol.120221 http://www.jimmunol.org/ Material 5.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 13, 2013, doi:10.4049/jimmunol.1202215 The Journal of Immunology Propofol Inhibits Superoxide Production, Elastase Release, and Chemotaxis in Formyl Peptide–Activated Human Neutrophils by Blocking Formyl Peptide Receptor 1 Shun-Chin Yang,*,† Pei-Jen Chung,‡ Chiu-Ming Ho,*,x Chan-Yen Kuo,‡ Min-Fa Hung,‡ Yin-Ting Huang,‡ Wen-Yi Chang,‡ Ya-Wen Chang,‡ Kwok-Hon Chan,*,x and Tsong-Long Hwang‡,{ Neutrophils play a critical role in acute and chronic inflammatory processes, including myocardial ischemia/reperfusion injury, sepsis, and adult respiratory distress syndrome. Binding of formyl peptide receptor 1 (FPR1) by N-formyl peptides can activate neutrophils and may represent a new therapeutic target in either sterile or septic inflammation. Propofol, a widely used i.v. anesthetic, has been shown to modulate immunoinflammatory responses. However, the mechanism of propofol remains to be Downloaded from established. In this study, we showed that propofol significantly reduced superoxide generation, elastase release, and chemotaxis in human neutrophils activated by fMLF. Propofol did not alter superoxide generation or elastase release in a cell-free system. Neither inhibitors of g-aminobutyric acid receptors nor an inhibitor of protein kinase A reversed the inhibitory effects of propofol. In addition, propofol showed less inhibitory effects in non-FPR1–induced cell responses. The signaling pathways downstream from FPR1, involving calcium, AKT, and ERK1/2, were also competitively inhibited by propofol. These results show that propofol selectively and competitively inhibits the FPR1-induced human neutrophil activation. Consistent with the hypoth- http://www.jimmunol.org/ esis, propofol inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analog of fMLF, to FPR1 in human neutrophils, differentiated THP-1 cells, and FPR1-transfected human embryonic kidney-293 cells. To our knowledge, our results identify, for the first time, a novel anti-inflammatory mechanism of propofol by competitively blocking FPR1 in human neutrophils. Considering the importance of N-formyl peptides in inflammatory processes, our data indicate that propofol may have therapeutic potential to attenuate neutrophil-mediated inflammatory diseases by blocking FPR1. The Journal of Immu- nology, 2013, 190: 000–000. ropofol (2,6-diisopropylphenol) is a widely used i.v. non- animal studies, propofol has decreased cytokine release during by guest on September 25, 2021 opioid anesthetic, and it is mainly administered for the sepsis (2, 3) and reduced neutrophil-mediated inflammation in P sedation of surgical or critically ill patients, usually those acute pulmonary injury (4, 5). In human studies, propofol has with an immunoinflammatory status. As well as its anesthetic effects, attenuated myocardial reperfusion injury and pulmonary dys- there is growing evidence in animal and human studies that propofol function following cardiopulmonary bypass by reducing free radical exerts protective effects during acute inflammatory processes (1). In release and modulating the inflammatory process (6, 7). It is well demonstrated that overwhelming activation of the immune cells may be a major contributor to tissue damage in *Department of Anesthesiology, Taipei Veterans General Hospital, Taipei 112, Tai- inflammatory diseases. It is noteworthy that propofol suppresses wan; †Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang ‡ chemotaxis, phagocytosis, the generation of reactive oxygen species Gung University, Kweishan 333, Taoyuan, Taiwan; Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Kweishan 333, Taoyuan, (ROS), and/or the synthesis of cytokines by monocytes and mac- x Taiwan; School of Medicine, National Yang-Ming University, Taipei 112, Taiwan; rophages, which are mediated by activation of the g-aminobutyric and {Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan 333, Taoyuan, Taiwan acid (GABA)A receptor (8, 9); reduces the mitochondria potential (10); and inhibits the AKT/IKKb/NF-kB pathways (11). Propofol Received for publication August 8, 2012. Accepted for publication April 16, 2013. impairs the chemotaxis and respiratory burst of neutrophils in re- This work was supported by Chang Gung University Grants EMRPD1A0881 and CMRPD1B0481; Taipei Veterans General Hospital Grant V100A-023; and National sponse to fMLF (12, 13). However, other research revealed that Science Council Grant NSC 100-2628-B-182-001-MY3, Taiwan. propofol fails to alter respiratory burst in PMA-activated neu- Address correspondence and reprint requests to Dr. Tsong-Long Hwang, Graduate trophils (14). Indeed, the cellular mechanisms responsible for the Institute of Natural Products, College of Medicine, Chang Gung University, 259 pharmacological effects of propofol in human neutrophils are Wen-Hwa 1st Road, Kweishan 333, Taoyuan, Taiwan. E-mail address: [email protected]. edu.tw controversial and remain to be established. The online version of this article contains supplemental material. Neutrophils are a major cell population in the human innate 2+ immune system, which is the first line of defense against bacterial Abbreviations used in this article: [Ca ]i, intracellular calcium concentration; DHR 123, dihydrorhodamine 123; fluo-3/AM, fluo-3 acetomethoxyester; FNLFNYK, invasion (15). However, neutrophils are regarded as destructive N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein; FPR1, formyl peptide receptor 1; cells, releasing toxic ROS and proteolysis enzymes that destroy GABA, g-aminobutyric acid; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquino- linesulfonamide; HEK, human embryonic kidney; LDH, lactate dehydrogenase; LTB4, the surrounding tissue (16, 17). For example, accumulated neu- leukotriene B4; MMK-1, Leu-Glu-Ser-Ile-Phe-Arg-Ser-Leu-Leu-Phe-Arg-Val-Met; trophils induce endothelial dysfunction and microcirculatory col- PKA, protein kinase A; ROS, reactive oxygen species; WST-1, 2-(4-iodophenyl)-3-(4- lapse in acute coronary syndrome and in a myocardial ischemia/ nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt. reperfusion injury model (18). Similarly, in lung injury models, in- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 appropriately activated neutrophils have resulted in acute respiratory www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202215 2 PROPOFOL BLOCKS FPR1 OF NEUTROPHILS distress syndrome or transfusion-related acute lung injury (19, 20). (6 3 105 cells/ml) were mixed with 0.5 mg/ml ferricytochrome c and 1 Interestingly, it has been shown in animal and human studies that mmol/L CaCl2 at 37˚C, they were treated with DMSO (as control) or propofol may diminish the oxidative or inflammatory injury in- propofol for 5 min, and were activated with fMLF, MMK-1 (300 nmol/L), or sodium fluoride (NaF; 20 mmol/L) in the pretreatment of cytochalasin B duced by neutrophils (2, 4, 7). These observations are interpreted (1 mg/ml for fMLF and MMK-1; 2.5 mg/ml for NaF) or PMA (5 nmol/L). to mean that propofol has an antioxidant capacity. Clearly, addi- The change in absorbance was monitored continuously at 550 nm with tional research is required to define the mechanisms of the action a spectrophotometer (U-3010; Hitachi, Tokyo, Japan). Calculation was of propofol on activated neutrophils. dependent on the statement in the previous study (23). In this study, we examined the pharmacological roles of propofol Measurement of ROS production in the respiratory burst, degranulation, and chemotaxis of human neutrophils, and explored its potential anti-inflammatory mecha- The intracellular ROS production by the activated neutrophils was deter- mined from the conversion of nonfluorescent DHR 123 to fluorescent nisms. Our results demonstrate that, at clinical concentrations, 6 rhodamine 123, detected with flow cytometry. After neutrophils (2.5 3 10 propofol inhibits superoxide production, ROS generation, elastase cells/ml) were incubated with DHR 123 (2 mmol/L) for 15