Blocking Formyl Peptide Receptor 1 This Information Is Current As of September 25, 2021

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

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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-isoquinolinesulfonamide; 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 determined 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 min at 37˚C, secretion, and chemotaxis by human neutrophils activated with they were treated with propofol (0–50 mmol/L) for 5 min, and then fMLF fMLF. Many of the observations made in the study suggest that the (100 nmol/L) was added for 15 min. The response was terminated by anti-inflammatory effects of propofol are mediated by blocking the placing the cells on ice. The change in fluorescence was analyzed by flow cytometry. interaction between fMLF and its receptor, formyl peptide receptor 1 (FPR1), thus disrupting the receptor-mediated signaling path- Measurement of the elastase released ways. FPR1 is a Gi-coupled G protein–coupled receptor and is This study used elastase release as evidence of the degranulation of the known to be important in neutrophilic inflammatory disorders (21). Downloaded from activated neutrophils. Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide was Notably, our results indicate that propofol may have therapeutic used as the elastase substrate (24). After neutrophils (6 3 105 cells/ml) potential to attenuate neutrophil-mediated inflammatory diseases by weremixedwith100mmol/L substrate and 1 mmol/L CaCl2 at 37˚C, blocking FPR1. they were treated with DMSO (control) or propofol for 5 min, and were then activated by fMLF, MMK-1 (300 nmol/L), NaF (20 mmol/L), or leukotriene B4 (LTB4; 100 nmol/L) in the pretreatment of cytochalasin B Materials and Methods (0.5 mg/ml for fMLF, MMK-1, and LTB4; 2.5 mg/ml for NaF). The change

Reagents in absorbance was monitored continuously at 405 nm with a spectropho- http://www.jimmunol.org/ tometer. The results are showed as percentages of the elastase released in Propofol (2,6-diisopropylphenol) was purchased from Sigma-Aldrich the control group. (St. Louis, MO). Dihydrorhodamine 123 (DHR 123), fluo-3 acetomethoxyester (fluo-3/AM), and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FNLFNYK) were obtained from Molecular Probes (Eugene, OR). The Analysis of superoxide scavenging cAMP enzyme immunoassay kits were from GE Healthcare (Uppsala, The superoxide-scavenging effect of propofol was determined in a cell-free Sweden). Leu-Glu-Ser-Ile-Phe-Arg-Ser-Leu-Leu-Phe-Arg-Val-Met (MMK-1), xanthine/xanthine oxidase system. The assay buffer contained 50 mmol/L muscimol, baclofen, and CGP52432 were obtained from Tocris Bioscience Tris (pH 7.4), 0.3 mmol/L WST-1, and 0.02 U/ml xanthine oxidase. After (Ellisville, MO). N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 0.1 mmol/L xanthine was added to the assay buffer for 10 min at 30˚C, the (H89), methoxysuccinyl-Ala-Ala-Pro-Val-nitroanilide, and rolipram were change in absorbance reflecting the reduction of WST-1 induced by su- purchased from Calbiochem (La Jolla, CA). Abs directed against phosphor- peroxide was measured at 450 nm. by guest on September 25, 2021 ERK1/2, ERK1/2, AKT (pan), and phosphor-AKT (ser-473) were from Cell Signaling (Beverly, MA). Anti-p38 Ab was obtained from Santa Cruz Bio- Evaluation of lactate dehydrogenase release technology (Santa Cruz, CA). The 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) was obtained from Lactate dehydrogenase (LDH) was used as the symbol of cytotoxicity and Dojindo Laboratories (Kumamoto, Japan). All other pharmacologic agents determined by a commercially available method (Promega, Madison, WI). were purchased from Sigma-Aldrich. The calculation was based on LDH activity in the propofol group (0–100 mmol/L) expressed as a percentage of the total LDH activity. The total Isolation of human neutrophils LDH activity was determined with the lysis of neutrophils with 0.1% Triton X-100 for 30 min at 37˚C. This study was approved by the local institutional review board, and written informed consent was obtained from each healthy volunteer. Neutrophils Chemotaxis assay were isolated from peripheral blood according to the standard method of dextran sedimentation, followed by centrifugation in a Ficoll-Hypaque Cell migration was measured using a 24-well microchemotaxis chamber 3 6 gradient and the hypotonic lysis of the erythrocytes. The purified neu- (pore size 3 mm; Millipore, Darmstadt, Germany). Neutrophils (5 10 trophils contained .98% viable cells, as determined by Trypan blue ex- cells/ml) were pretreated with propofol (0–100 mmol/L) for 5 min at 37˚C clusion, and were suspended in calcium-free HBSS at 4˚C before used. in the top chamber. A total of 30 nmol/L fMLF, 300 nmol/L MMK-1, 100 nmol/L LTB4, or 100 ng/ml IL-8 with propofol was placed in the bottom Differentiation of human monocytic leukemia cells (THP-1) chamber. After incubation for 120 min, the numbers of migrated cells were determined by a Coulter counter (Beckman Coulter, Fullerton, CA) THP-1 were cultured in RPMI 1640 medium supplemented with 2 mmol/L (25, 26). glutamine, 10% FBS, and antibiotics (100 U/ml penicillin, 100 mg/ml strep- tomycin, and 2.5 mg/ml amphotericin B). THP-1 was cultured in the presence Receptor-binding assay of 300 mmol/L dibutyryl cAMP for 48 h to induce cell differentiation (22). Receptor binding was assayed by the FACScan analysis of the binding of Expression of FPR1 in human embryonic kidney cells FNLFNYK, a fluorescent analog of fMLF, as described previously (27, 28). 6 6 (HEK-293) Neutrophils (2 3 10 cells/ml), differentiated THP-1 (2 3 10 cells/ml), or FPR1-transfected HEK-293 (2.5 3 105 cells/ml) were incubated with HEK-293 were maintained in DMEM supplemented with 10% FBS, 2 mmol/L propofol for 5 min at 4˚C and labeled with FNLFNYK. After 30 min, the glutamine, and antibiotics. HEK-293 were stably transfected with the pCMV6- cells were pelleted, resuspended in ice-cold HBSS, and immediately an- AC vector containing the human FPR1 gene (NM_002029; OriGene, alyzed with flow cytometry. Rockville, MD) for 72 h using X-tremeGENE Hp DNA transfection reagent (Roche, Mannheim, Germany), according to the manufacturer’s instruction. Determination of cAMP concentration After transfection, cells were cultured in the medium containing G418 Neutrophils were incubated with propofol (0–50 mmol/L) or DMSO (2 mg/ml). G418-resistant clones were used for further studies. (control) for 5 min and stimulated with fMLF for another 1 min. The reaction Measurement of superoxide generation was then terminated by the addition of 0.5% dodecytrimethylammonium bromide. After centrifugation at 3000 3 g for 5 min at 4˚C, the supernatant The measurement of the superoxide generated by the activated neutrophils was assayed for cAMP with an enzyme immunoassay kit (Amersham was dependent on the reduction of ferricytochrome c. After neutrophils Biosciences, Buckinghamshire, U.K.). The Journal of Immunology 3

Measurement of intracellular calcium concentration Neutrophils (3 3 106 cells/ml) or differentiated THP-1 (7 3 105 cells/ml) were labeled with fluo-3/AM (2 mmol/L) for 30 min at 37˚C. The cyto- plasmic calcium levels were measured in a quartz cuvette with a Hitachi F-4500 spectrofluorometer with a thermostat (37˚C), and with continuous stirring. The excitation wavelength was 488 nm, and the emission wavelength was 520 nm. After they were treated with propofol for 5 min, stimulants were added in the presence of 1 mmol/L Ca2+ to increase in- 2+ 2+ tracellular calcium concentration ([Ca ]i). [Ca ]i was calculated from the 2+ fluorescence intensity, as follows: [Ca ]i = Kd 3 [(F 2 Fmin)/ (Fmax 2 F)] ; where F is the observed fluorescence intensity, Fmax and Fmin were obtained by the addition to the neutrophils of 0.05% Triton X-100 and 20 mmol/L EGTA, respectively, and Kd was taken to be 400 nmol/L. Immunoblotting analysis Neutrophils were treated with propofol for 5 min and stimulated with fMLF (10–100 nmol/L) for 30 s. The reaction was terminated by placing the cells on ice. After centrifugation at 4˚C and removal of the supernatant, the pellet was lysed in 150 ml buffer (50 mmol/L HEPES [pH 7.4], 100 mmol/L NaCl,1mmol/LEDTA,2mmol/LNa3VO4, 10 mmol/L p-nitrophenyl phosphate, 5% 2-ME, 1 mmol/L PMSF, 1% dilution of Sigma-Aldrich protease inhibitor cocktails, and 1% Triton X-100). After brief sonica- Downloaded from tion, the samples were centrifuged at 14,000 3 g for 20 min at 4˚C to yield whole-cell lysates. The lysates were used for Western blotting analysis. NaDodSO4-PAGE with 12% polyacrylamide gels was used to separate the proteins. The samples were then blotted onto nitrocellulose membranes. Immunoblotting was performed with the indicated Abs and HRP-conjugated secondary anti-rabbit Abs (Cell Signaling Technology, Beverly, MA). The proteins were detected with the ECL system (Amersham Biosciences). http://www.jimmunol.org/ Statistical analysis All experiments were performed at least three times, and the results are expressed as means 6 SEM. The statistical analyses were based on Student t test or the Mann–Whitney U test, and all calculations were performed with SigmaPlot (Systat Software, San Jose, CA). A p value ,0.05 was FIGURE 1. Propofol inhibits superoxide production and elastase release considered statistically significant. in fMLF-activated human neutrophils, but not in cell-free systems. Neu- trophils were incubated with propofol (PPF; 0–50 mmol/L) for 5 min. (A) Results Superoxide generation and (B) elastase release were induced with fMLF C Propofol inhibits superoxide generation and elastase release in (100 nmol/L). ( ) Neutrophils labeled with DHR 123 were incubated with by guest on September 25, 2021 fMLF-activated neutrophils, but fails in a cell-free system propofol (0–50 mmol/L) for 5 min and stimulated with fMLF (100 nmol/L) for another 15 min. Basal groups (black line) were treated with DMSO To determine whether propofol regulates neutrophil functions, alone. The test groups (other colored lines) were treated with drugs and superoxide generation and elastase release were measured in ac- fMLF. (D) Superoxide scavenging effect of propofol was investigated in tivated cells after the administration of propofol. Propofol (3–50 a cell-free xanthine/xanthine oxidase system. Superoxide dismutase (SOD) mmol/L) had a dose-dependent inhibitory effect on superoxide was used as the positive control. The reduction of WST-1 by propofol was generation in the neutrophils activated by the FPR1 activator, measured by spectrophotometry at 450 nm. (E) The activity of extracellular fMLF (Fig. 1A). The IC of propofol was 10.31 6 3.02 mmol/L. elastase in the presence of propofol was measured at 405 nm. Data are 50 A B Moreover, propofol (3–50 mmol/L) also reduced the elastase representative of six experiments ( ), eight experiments ( ), five experi- C D E release from fMLF-activated neutrophils in a dose-dependent ments ( ), three experiments ( ), or four experiments ( ). All data shown are means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001 versus the control manner, with an IC value of 25.94 6 1.74 mmol/L (Fig. 1B). 50 group. Propofol (50 mmol/L) did not alter basal superoxide generation and elastase release under resting conditions (Fig. 1A, 1B). In FPR1-activated neutrophils, but that these inhibitory effects do not addition, the superoxide produced by NADPH oxidase in neu- occur in a cell-free system. trophils can be converted to various ROS, which cause extensive tissue damage. The flow cytometric analysis showed that propofol Neither GABAA nor GABAB mediates the inhibitory effects of reduced the intracellular ROS generated by the neutrophils treated propofol with fMLF (Fig. 1C). In contrast, propofol did not induce the It is well known that propofol exerts versatile pharmacologic release of LDH, even at a high concentration (100 mmol/L), functions through the activation of GABA receptors. Therefore, suggesting that inhibition of the neutrophils’ respiratory burst we next investigate whether propofol alters neutrophil functions and degranulation by propofol were not attributable to its cyto- through GABAA or GABAB receptor. To address this question, toxicity (data not shown). The inhibitory effects of propofol were we used a pharmacological approach with GABAA or GABAB re- then tested in cell-free systems to determine whether propofol ceptor agonists and antagonists. Neither muscimol (GABAA re- scavenges superoxide and inhibits elastase activity. We assayed ceptor agonist) nor baclofen (GABAB receptor agonist) altered the the superoxide-scavenging effect of propofol at concentrations of superoxide and elastase secretion by neutrophils under resting up to 100 mmol/L, but superoxide generation was not affected conditions or by fMLF-stimulated neutrophils (Supplemental Fig. in the cell-free xanthine/xanthine oxidase system. Superoxide dis- 1A, 1B). Furthermore, neither SR95531 (GABAA receptor an- mutase was used as the positive control (Fig. 1D). We also found tagonist) nor CGP52432 (GABAB receptor antagonist) affected that propofol (10–100 mmol/L) had no direct inhibitory effect on the propofol-mediated inhibition of these functions (Supplemental the extracellular elastase activity (Fig. 1E). These data indicate Fig. 1C, 1D). These data suggest that the activation of the GABAA that propofol impairs the respiratory burst and the degranulation of and GABAB receptors does not play a role in the generation of 4 PROPOFOL BLOCKS FPR1 OF NEUTROPHILS superoxide or elastase in human neutrophils, which also excludes migration induced by fMLF (30 nmol/L) was significantly reduced the possibility that the GABAA or GABAB receptor mediates the in the presence of propofol (50 and 100 mmol/L) (Fig. 3A). In inhibitory effects of propofol. contrast, propofol failed to inhibit chemotaxis induced by MMK-1 (300 nmol/L), LTB4 (100 nmol/L), and IL-8 (100 ng/ml) (Fig. Propofol less effectively inhibits non-FPR1 agonist-triggered 3B–D). responses We next asked whether propofol impairs the cellular responses Propofol competitively binds FPR1 in human neutrophils of only those neutrophils challenged with fMLF. To answer this To examine whether propofol has a binding affinity for FPR1, the question, neutrophils were stimulated with other non-FPR1 acti- binding of FNLFNYK to the surface of neutrophils was monitored vators, including MMK-1 (FPR2 agonist, 300 nmol/L), LTB4 by flow cytometry. Fig. 4A showed that fMLF (10 mmol/L) com- (BLT1 receptor activator, 100 nmol/L), NaF (direct G protein acti- pletely inhibited the binding of FNLFNYK (4 nmol/L) to neu- vator, 20 mmol/L), and PMA (protein kinase C activator, 5 nmol/L). trophils. Compared with the control group, propofol (5, 50, and Fig. 2 shows that propofol even at high concentration of 100 mmol/L 100 mmol/L) significantly and dose dependently inhibited the exerted only slight inhibitory effects on the MMK-1–, NaF-, LTB4-, binding of FNLFNYK to the fMLF receptor (Fig. 4A). Further- and PMA-induced responses. Taken together, these results suggest more, the specific concentration-binding curve of FNLFNYK (2– that propofol has a selective inhibitory action on FPR1 agonist– 12 nmol/L) was reduced by propofol (50 mmol/L) (Fig. 4B). These activated neutrophils. results indicate that propofol can competitively bind to FPR1. Propofol selectively attenuates fMLF-induced human Propofol exerts competitive inhibitory effects on neutrophil chemotaxis fMLF-activated neutrophil responses Downloaded from The migration of neutrophils into tissues is an essential event in The concentration-response curves of fMLF for superoxide pro- the FPR1-induced inflammatory response. To investigate whether duction and elastase release are shown in Fig. 5, and the EC50 propofol reduces neutrophil migration, we examined the chemotaxis values were 18.94 6 3.35 and 39.94 6 21.78 nmol/L, respectively. of neutrophils in response to different chemoattractants. Neutrophil Significantly, propofol pretreatment produced right shifts in the concentration-response curves of fMLF for superoxide production http://www.jimmunol.org/ and elastase release, with EC50 values of 176.17 6 24.15 and 273.49 6 114.69 nmol/L, respectively. In addition, propofol exerted higher degree of inhibitions in a low concentration of fMLF (10 nmol/L)-treated neutrophils, and the IC50 values for superoxide generation and elastase release were 0.38 6 0.14 and 0.27 6 0.11 mmol/L, respectively (Supplemental Fig. 2). These data support the proposition that propofol is a competitive inhibitor of FPR1.

cAMP/Protein kinase A pathway does not mediate the by guest on September 25, 2021 inhibitory effects of propofol In the following experiments, we investigated whether the cAMP/ protein kinase A (PKA) pathway is involved in the inhibitory effects of propofol. Rolipram (phosphodiesterase 4 inhibitor,

FIGURE 2. Inhibitory effects of propofol are less effective in neutrophils activated with non-FPR1 stimulants. Neutrophils were treated with propofol (0–100 mmol/L) for 5 min. Superoxide generation and elastase FIGURE 3. Propofol speciﬁcally inhibits fMLF-induced human neutro- release were triggered with (A) MMK-1 (300 nmol/L) for 10 min or (B) phil chemotaxis. Neutrophils were treated with propofol (0–100 mmol/L) NaF (20 mmol/L) for 30 min. (C) PMA (5 nmol/L) or (D) LTB4 (100 for 5 min in the top compartments of chemotaxis chamber. Migrated neu- nmol/L) for 10 min was used to activate neutrophil superoxide generation trophils in response to (A) fMLF (30 nmol/L), (B) MMK-1 (300 nmol/L), or elastase release in the presence of propofol (0–100 mmol/L). Data are (C)LTB4(100nmol/L),or(D)IL-8(100ng/ml)weredeterminedbya representative of three experiments (A, C), six experiments (B), or seven Coulter counter. Data are representative of ﬁve experiments (A), three ex- experiments (D). All data shown are means 6 SEM. *p , 0.05, **p , periments (B, C), or four experiments (D). All data shown are means 6 0.01 versus the control group. SEM. *p , 0.05, ***p , 0.001 versus the control group. The Journal of Immunology 5

FIGURE 5. Propofol exerts a competitive inhibitory effect on fMLF-

activated neutrophils. Neutrophils were treated with propofol (50 mmol/L) Downloaded from for 5 min. (A) Superoxide generation and (B) elastase release were activated with increasing concentrations of fMLF (1–1,000 or 10,000 nmol/L). Data are representative of four to ﬁve experiments (A) or six to eight experiments (B). Values for superoxide generation are expressed as means 6 SEM. Elastase release is expressed as the mean 6 SEM relative to the

mean maximal change in OD405 (100%). **p , 0.01, ***p , 0.001 versus the corresponding control group. http://www.jimmunol.org/

diminished by propofol (50 mmol/L) (Fig. 7). In contrast, the 2+ peak [Ca ]i induced by MMK-1 (300 nmol/L), IL-8 (100 ng/ml), and LTB4 (100 nmol/L) was unaltered by propofol (50 mmol/L) (Supplemental Fig. 3). These results suggest that propofol speciﬁ- cally attenuates Ca2+ signals in FPR1 agonist–activated neutrophils.

Propofol inhibits AKT and ERK1/2 phosphorylation in by guest on September 25, 2021 fMLF-activated neutrophils It is well known that the PI3K/AKT and MAPK pathways are involved in the downstream signaling of fMLF-stimulated neu- FIGURE 4. Propofol binds the formyl peptide receptor in human neutrophils. Propofol (50 mmol/L) caused a significant reduction in trophils. (A) Neutrophils were incubated with propofol (0–100 mmol/L) or the phosphor-AKT expression in neutrophils in response to dif- fMLF (10 mmol/L) for 5 min and labeled with the FPR1-specific ligand FNLFNYK (4 nmol/L). Basal group (black line) was treated with DMSO ferent concentrations of fMLF (Fig. 8A). In contrast, the ERK1/2 alone in the absence of FNLFNYK. The test groups (other colored lines) activation induced by fMLF at low concentrations (10 and 30 were treated with DMSO, propofol, or fMLF. The mean fluorescence in- mmol/L), but not that induced by a high concentration (100 mmol/L), tensity (MFI) is expressed as the mean 6 SEM relative to the control group was inhibited by propofol (Fig. 8B). However, the administration (100%). (B) Neutrophils pretreated with DMSO (red line) or propofol of propofol induced the expression of phosphor-p38 in resting (50 mmol/L, blue line) were labeled with increasing concentrations of cells, and it failed to suppress fMLF-triggered p38 activation FNLFNYK (2–12 nmol/L). MFI is expressed as the mean 6 SEM relative (Fig. 8C). to the mean maximal MFI (100%). Data are representative of 3 experiments (A) or 7–10 experiments (B). *p , 0.05, **p , 0.01, ***p , 0.001 Propofol binds FPR1 in differentiated THP-1 versus the corresponding control group. and FPR1-transfected HEK-293 The specificity of propofol for FPR1 was examined in dibutyryl 3 mmol/L), but not propofol (10 and 50 mmol/L), increased cAMP-differentiated THP-1 and FPR1-transfected HEK-293. cAMP levels in fMLF-activated human neutrophils (Fig. 6A), fMLF (10 mmol/L) completely inhibited the binding of FNLFNYK suggesting that cAMP is not involved in the inhibitory effects (4 nmol/L) to dibutyryl cAMP-differentiated THP-1. Also, of propofol. Consistent with this result, H89 (3 mmol/L), a PKA propofol dose dependently inhibited the binding of FNLFNYK inhibitor, failed to reverse the inhibitory effects of propofol on (4 nmol/L) to FPR1 in differentiated THP-1 (Fig. 9A). Fur- 2+ superoxide generation and elastase release in activated cells (Fig. thermore, the peak [Ca ]i induced by fMLF (10–100 nmol/L), 6B, 6C). but not MMK-1 (300 nmol/L), was significantly diminished by 2+ propofol (50 mmol/L) (Fig. 9B). These data indicate that pro- Propofol attenuates fMLF-induced Ca mobilization in pofol inhibits fMLF-induced Ca2+ mobilization by blocking human neutrophils FPR1 in differentiated THP-1. Furthermore, the binding of Ca2+ signals play an important role in many neutrophil functions. FNLFNYK (4 nmol/L) to FPR1-transfected HEK-293 was inhibited To determine whether treatment with propofol attenuates Ca2+ by fMLF (10 mmol/L) and propofol (5, 50, and 100 mmol/L) 2+ signals in activated neutrophils, their [Ca ]i was assayed. The (Fig. 10). These results indicate that propofol displays specificity 2+ peak [Ca ]i induced by fMLF (10–1000 nmol/L) was significantly for the FPR1. 6 PROPOFOL BLOCKS FPR1 OF NEUTROPHILS Downloaded from http://www.jimmunol.org/

FIGURE 6. cAMP/PKA pathway is not involved in the inhibitory effect of propofol on activated neutrophil functions. (A) Neutrophils were by guest on September 25, 2021 incubated with propofol (0–50 mmol/L) or rolipram (1 mmol/L, positive B control) in the presence of fMLF (100 nmol/L). ( ) Superoxide genera- FIGURE 7. Propofol attenuates intracellular calcium mobilization in C tion and ( ) elastase release from activated neutrophils that had been fMLF-activated neutrophils. (A) Neutrophils labeled with ﬂuo-3/AM (2 preincubated with H89 (3 mmol/L, PKA inhibitor) in the presence of mmol/L) were incubated with propofol (50 mmol/L) and then activated propofol were examined. Data are representative of three experiments with fMLF (10–1000 nmol/L). (B)Peak[Ca2+] after the addition of A B C i ( ), four experiments ( ), or nine experiments ( ). All data shown are fMLF is expressed as the mean 6 SEM. Data are representative of six to 6 , , , means SEM. *p 0.05, **p 0.01, ***p 0.001 versus the control nine independent experiments. *p , 0.05, **p , 0.01 versus the DMSO group. group.

Discussion is known to be involved in the pathogenesis of inflammatory The immunomodulatory effects of propofol have been reported in a diseases. Our data show that propofol significantly inhibits the variety of experimental and functional research over the years (29). superoxide production induced by fMLF in a concentration- However, very little is known about the pharmacologic mecha- dependent manner in intact human neutrophils, which is consis- nisms of propofol, especially in impairing neutrophil functions. To tent with previous reports (33, 34). Propofol has been shown to our knowledge, our study shows, for the first time, that, at thera- display direct scavenging activity for free radical species (35). peutic concentrations, propofol inhibits the respiratory burst, de- However, direct scavenging activity was ruled out because propofol granulation, and chemotaxis of fMLF-activated neutrophils by failed to alter the superoxide generation in the cell-free xanthine/ competitively binding to FPR1 and thus attenuating downstream xanthine oxidase system. These controversial results may be signaling, including in the Ca2+, AKT, and ERK1/2 pathways. explained by the different radical species examined in different There is considerable evidence from clinical and experimental studies. We suggest that propofol inhibits superoxide formation in studies that propofol exerts significant protective effects against activated neutrophils by modulating cellular signaling pathways. inflammatory and cardiovascular diseases, inhibiting the production This hypothesis is supported by the evidence that propofol also of cytokines and the clearance of ROS (3, 30). Propofol has been markedly inhibited elastase release in fMLF-activated cells, but shown to reduce oxidative injury to organ in endotoxemia ani- not in the cell-free system. Neutrophil granules contain many mals (31) and in patients undergoing cardiac surgery (32). Hu- antimicrobial and potentially proinflammatory proteases. Elastase man neutrophils play important roles in the pathogenesis of is a major serine protease secreted by stimulated human neu- various diseases, including acute myocardial infarction, athero- trophils and plays a critical role in inflammatory diseases (36). genesis, ischemic heart disease, and sepsis (15, 18). The oxidative These data also support the proposition that propofol acts as an stress produced by activated human neutrophils can directly or anti-inflammatory agent. Furthermore, the recruitment of neu- indirectly cause damage by destroying the surrounding tissue and trophils to sites of inflammation is one of the major biological The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 8. Propofol attenuates the phosphorylation of AKT and ERK1/2 but not p38 in activated neutrophils. Neutrophils were treated FIGURE 9. Propofol blocks FPR1 and inhibits fMLF-induced calcium A with propofol (50 mmol/L) for 5 min, and then activated with fMLF mobilization in dibutyryl cAMP-differentiated THP-1. ( ) The differen- (10–100 nmol/L) for 30 s. Phosphorylation of AKT, ERK1/2, and p38 tiated THP-1 were incubated with propofol (0–100 mmol/L) or fMLF (10 mmol/L) for 5 min and labeled with the FPR1-specific ligand FNLFNYK was assessed with NaDodSO4-PAGE and immunoblotting. Represen- tative Western blots are shown for phosphorylated AKT and total AKT (4 nmol/L). Basal group (black line) was treated with DMSO alone in the protein [p-AKT and AKT, respectively (A)]; phosphorylated ERK1/2 absence of FNLFNYK, and the test groups (other colored lines) were and total ERK1/2 protein [p-ERK1/2 and ERK1/2, respectively (B)]; treated with DMSO, propofol, and fMLF in the presence of FNLFNYK. 6 and phosphorylated p38 and total p38 protein [p-p38 and p38, respec- The mean fluorescence intensity (MFI) is expressed as the mean SEM tively (C)]. Densitometric analysis of all samples was normalized to the relative to the control group (100%). Data are representative of three B corresponding total protein. All data are summarized as means 6 SEM experiments. ( ) The differentiated THP-1 labeled with fluo-3/AM (2 relative to the mean maximal ratio. Data are representative of four in- mmol/L) was incubated with propofol (50 mmol/L) and then activated with dependent experiments. *p , 0.05, ***p , 0.001 versus the corre- fMLF (10, 30, and 100 nmol/L) or MMK-1 (300 nmol/L). Traces shown , sponding control group. are from three independent experiments. ***p 0.001 versus the control group. functions for FPR1 (37). Our results show that propofol reduces neutrophil chemotaxis by fMLF in a concentration-dependent are still unknown. Data from the current study show that the manner, which is consistent with previous reports (38, 39). GABAA and GABAB agonists, muscimol and baclofen, respectively, GABA is an inhibitory neurotransmitter in the CNS and can do not induce superoxide production or elastase release in either modulate autoimmune inflammation (40). Through the activation resting or activated human neutrophils, suggesting that the GABA of the GABA receptors, propofol exerts various pharmacological receptors do not have a significant effect on human neutrophils. A effects, including the inhibition of chemotaxis, phagocytosis, role for the GABAA and GABAB receptor in the inhibitory action ROS generation, and/or cytokine synthesis in monocytes and mac- of propofol was excluded because either GABAA or GABAB rophages (8, 9). Another research has demonstrated that human antagonists failed to reverse the inhibitory effects of propofol. neutrophils express GABAB, but few GABAA receptors (41). The molecular and functional responses for human neutrophil However, the roles of the GABAA and GABAB receptors in the recognition of formyl peptides are their binding to FPRs. Human respiratory burst and degranulation of activated human neutrophils neutrophils express two members of this family, FPR1 and FPR2 8 PROPOFOL BLOCKS FPR1 OF NEUTROPHILS

as the ERK1/2 and p38 MAPK cascades, is mediated by the interactions between fMLF and FPR1 for multiple intracellular activities (49). In fact, propofol inhibits fMLF-induced neutrophil 2+ ROS production and chemotaxis by suppressing the [Ca ]i and phosphorylation of ERK (34, 39). The present study shows that 2+ propofol reduces the fMLF-induced peak [Ca ]i and phosphorylation of AKT and ERK1/2. The inhibitory potency of propofol is inversely related to the fMLF concentration, providing evidence that propofol inhibits fMLF-caused cell signals in a competitive manner. In contrast, we unexpectedly showed that propofol itself can induce the phosphorylation of p38 MAPK in human neutrophils. Nowadays, there are strong evidences supporting that propofol has anti-inﬂammatory effects in human and animal studies (1–7, 29, 30). However, studies showed that propofol in- creases neutrophil respiratory burst in the bronchoalveolar lavage ﬂuid from patients undergoing tympanoplasty surgery (50), and it fails to affect neutrophil oxidative response in patients undergoing cataract surgery (51). Together, these studies demonstrate that

FIGURE 10. Propofol binds the formyl peptide receptor in FPR1- the anti-inflammatory effects of propofol may differ by diseases. Downloaded from transfected HEK-293. FPR1-transfected HEK-293 were incubated with Obviously, further research is required to clarify the effects and propofol (0–100 mmol/L) or fMLF (10 mmol/L) for 5 min and labeled action mechanisms of propofol in various in vivo models of with FNLFNYK (4 nmol/L). Basal group (black line) was treated with inflammation. DMSO alone in the absence of FNLFNYK, and the test groups (other The growing evidences have supported that FPR1 plays critical colored lines) were treated with DMSO, propofol, and fMLF in the pres- N ence of FNLFNYK. The mean fluorescence intensity (MFI) is expressed as roles in sterile and septic inflammation. FPR1 is activated by - the mean 6 SEM relative to the control group (100%). Data are repre- formyl peptides, which are derived from either bacterial peptides http://www.jimmunol.org/ sentative of three experiments. *p , 0.05, ***p , 0.001 versus the control or mitochondrial proteins (52, 53). The endogenous damage- group. associated molecular patterns from bone and liver mitochondria can activate neutrophils through FPR1 and induce severe inflam- (21, 42). In our study, the superoxide generation, elastase re- matory response syndrome (54–56). Therefore, concerns have lease, and chemotaxis induced by MMK-1, a FPR2 agonist, are been raised about the potential of functional FPR1 as a therapeutic less sensitive to inhibition by propofol than those induced by target for the development of new drugs to treat inflammatory fMLF, a FPR1 agonist. In addition, the results obtained using diseases (21, 57). In conclusion, to our knowledge, our results different receptor activators, NaF, LTB4, PMA, and IL-8, clearly clearly demonstrate for the first time that propofol inhibits human confirm that propofol selectively inhibits FPR1-mediated effects. It neutrophil activations by selective and competitive blockade of by guest on September 25, 2021 is noteworthy that propofol produced a parallel rightward shift in FPR1. Given the importance of FPR1 in inflammatory diseases, the fMLF concentration-response curves, whereas the maximum these results also suggest that propofol may have potential benefits response remained unchanged. Besides, propofol exerted higher in protecting against FPR1-involved inflammatory diseases. degree of inhibitions in a low concentration of fMLF-treated neutrophils. Based on these observations, we postulate that propofol Disclosures has a selective and competitive blocking effect on FPR1. Our data The authors have no financial conflicts of interest. confirm that propofol blocks the binding of FNLFNYK to FPR1 in human neutrophils in a concentration-dependent manner. Also, the specificity of propofol for FPR1 is obtained in the differentiated References THP-1 and the FPR1-transfected HEK-293. To the best of our 1. Marik, P. E. 2005. Propofol: an immunomodulating agent. Pharmacotherapy 25: 28S–33S. knowledge, this is the first study to show that propofol has a com- 2. Taniguchi, T., K. Yamamoto, N. Ohmoto, K. Ohta, and T. Kobayashi. 2000. petitive binding affinity for FPR1. Effects of propofol on hemodynamic and inflammatory responses to endotoxemia in rats. Crit. Care Med. 28: 1101–1106. The intracellular signaling mechanisms responsible for neu- 3. Taniguchi, T., H. Kanakura, and K. Yamamoto. 2002. Effects of posttreatment trophil activation are very complex and remain elusive. 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