Phenylpropanoids Inhibit IL-1β and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A 2 This information is current as of September 29, 2021. Andreas Nievergelt, Janine Marazzi, Roland Schoop, Karl-Heinz Altmann and Jürg Gertsch J Immunol 2011; 187:4140-4150; Prepublished online 9 September 2011; doi: 10.4049/jimmunol.1100880 Downloaded from http://www.jimmunol.org/content/187/8/4140

Supplementary http://www.jimmunol.org/content/suppl/2011/09/09/jimmunol.110088 http://www.jimmunol.org/ Material 0.DC1 References This article cites 93 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/187/8/4140.full#ref-list-1

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ginger Phenylpropanoids Inhibit IL-1b and Prostanoid Secretion and Disrupt Arachidonate-Phospholipid Remodeling by Targeting Phospholipases A2

Andreas Nievergelt,* Janine Marazzi,* Roland Schoop,† Karl-Heinz Altmann,‡ and Ju¨rg Gertsch*

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthri- tis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1b expression in monocytes/macrophages. Both the calcium-independent phospholipase A2 (iPLA2)-triggered maturation and the cytosolic phospholipase A2 (cPLA2)-dependent Downloaded from secretion of IL-1b from isolated human monocytes were inhibited. In a fluorescence-coupled PLA2 assay, most major ginger phenylpropanoids directly inhibited i/cPLA2 from U937 macrophages, but not hog pancreas secretory phospholipase A2. The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA2 and methyl arachidonyl fluorophosphonate for cPLA2, with 10-/-shogaol being most effective. Furthermore, a ginger extract (2 mg/ml) and 10-shogaol (2 mM) potently inhibited the release of PGE2 and thromboxane B2 (>50%) and partially also leukotriene B4 in LPS-stimulated macrophages. Intriguingly, the total cellular was increased 2- to http://www.jimmunol.org/ 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA2 and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA2, the resulting attenuation of IL-1b secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically. The Journal of Immunology, 2011, 187: 4140–4150.

he rhizome of ginger (Zingiber officinale) is widely used have been reported to weakly inhibit components of proinflam- in diet, but also in Asian traditional medicine, where it is matory signal transduction pathways in vitro, such as NF-kB, T employed therapeutically to treat mild forms of rheuma- protein kinase C, and MAPKs (8, 9). Moreover, ginger con- by guest on September 29, 2021 toid arthritis, fever, and upper respiratory tract infections (1). In stituents have been shown to inhibit inducible NO synthase, conventional Western medicine, ginger preparations are adminis- cyclooxygenase (COX)-1/-2, and lipoxygenase in vitro (9–12). A tered clinically to treat emesis, nausea, and migraine headache (2, significant inhibition of PGE2 formation by ginger constituents 3). Based on the widespread therapeutic use of ginger in Asia has already been shown in vivo (13). Furthermore, different gin- for the treatment of inflammatory diseases, several studies have gerols and shogaols at low mM concentrations have been shown to been dedicated to the elucidation of potential anti-inflammatory directly bind to and partially activate the cation channel transient mechanisms (4–7). At relatively high mM concentrations, ginger receptor potential vanilloid 1 (14–16), the molecular clue for the constituents such as , shogaols, and diarylheptanoids spiciness of ginger, as well as the serotonin 5-HT1A G protein- coupled receptor (17). Although the latter studies have elucidated feasible molecular mechanisms of action of the major biologically *Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, active ginger constituents, they cannot explain the range of anti- Switzerland; †Bioforce AG, 9325 Roggwil, Switzerland; and ‡Department of Chem- istry and Applied Biosciences, Swiss Federal Institute of Technology, 8093 Zurich, inflammatory effects observed in vivo. In particular, the reported Switzerland antipyretic effects of ginger remain poorly understood. Received for publication March 25, 2011. Accepted for publication August 7, 2011. In this study, we have profiled a standardized food-grade This work was supported by a research grant from Bioforce AG (Roggwil, Switzer- commercial ginger extract in a high content assay using freshly land). The establishment of lipid analyses by gas chromatography/mass spectrometry isolated human peripheral whole blood. Human blood was in- was made possible through a grant from the Novartis Research Foundation. cubated with discrete stimuli targeting specific or nonspecific sig- Address correspondence and reprint requests to Prof. Ju¨rg Gertsch, Institute of Bio- nal transduction pathways, and the resulting cytokine patterns were chemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland. E-mail address: [email protected] quantified. The assay was validated using blood from different The online version of this article contains supplemental material. donors and by profiling the effects of anti-inflammatory drugs with known targets. Using this profiling setup, we were able to identify Abbreviations used in this article: AA, arachidonic acid; BEL, bromoenol lactone; CBA, cytometric bead array; COX, cyclooxygenase; cPLA2, cytosolic phospholipase calcium-independent phospholipase A2 (iPLA2) and cytosolic A ; DBA, 2,49-dibromoacetophenone; iPLA , calcium-independent phospholipase A ; 2 2 2 phospholipase A2 (cPLA2) enzymes as major biological targets MAFP, methyl arachidonyl fluorophosphonate; NMR, nuclear magnetic resonance; of ginger phenylpropanoids. PAK, palmitoyl-6-O-ascorbate potassium salt; PEA, palmitoylethanolamide; PLA2, phospholipase A2; RT, room temperature; sPLA2, secretory PLA2; thio-PC, 1-hexa- PLA2 enzymes play a central role in inflammatory processes decyl-2-arachidonolythio-2-deoxy-sn-glycero-3-phosphorylcholine; TXB2, thrombox- (18, 19) and are classified into three main groups, as follows: ane B2. secretory phospholipase A2 (sPLA2), cPLA2, and iPLA2. These Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 distinctly different enzymes hydrolyze the sn-2 ester bond of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100880 The Journal of Immunology 4141 phospholipids, thus liberating lysophospholipids and free fatty Whole blood, assay conditions, and purification of immune acids, which are precursors for a variety of different mediators cells of physiological and pathological processes (20, 21). PLA2 iso- Human venous whole blood was drawn from healthy volunteers with the enzymes are not only involved in the generation of free arach- BD vacutainer system. Isolation of PBMCs was conducted in accordance idonic acid (AA), which is metabolized by cyclo- and lipoxy- with the guidelines of the World Medical Association’s Declaration of genases to eicosanoids (22), but also in the incorporation of free Helsinki and approved by ETH Zurich. PBMCs were isolated with Opti- + AA into membranes and redistribution into specific compart- Prep (Axis-Shield, Oslo, Norway) density gradient flotation, and CD14 monocytes were enriched by adhesion to culture flasks (FACS analysis was ments (remodeling) (23, 24). Besides regulatory functions in lipid used to check for purity). Whole blood was immediately pipetted in catabolism/metabolism, PLA2 enzymes are critically involved in a 96-well plate at 200 ml/well. Ethanolic extracts, pure compounds, or signal transduction, phospholipid and cell membrane bilayer ethanol as vehicle control were added at different concentrations with a remodeling (25), store-operated cation channels and Ca2+ release- maximal concentration of 0.5% ethanol and incubated for 30 min at 37˚C. 2+ Where indicated, stimuli (LPS [312 ng/ml]) or a combination of PMA activated Ca channels (26), Fas-induced apoptosis (27), cellular [15 ng/ml] and thapsigargin [1 mM], anti-CD3 Ab, or anti-CD28 Ab [each proliferation and differentiation (28, 29), -induced insulin 1 mg/ml]) were added, and blood was incubated for another 18 h at 37˚C. secretion (30), and, most important for this study, they play a For cytokine measurements, the plates were centrifuged, and an ap- central role in acute (18) and chronic inflammation (31). propriate amount (usually 50 ml) of the supernatant serum was removed Both iPLA and cPLA are crucial for IL-1b maturation and for immediate analysis. Isolation of PBMCs and T lymphocytes from 2 2 peripheral whole blood was performed, as previously reported (39). secretion (membrane translocation and fusion of storage vesicles) b Cell cultures. U937 cells (CRL-1593.2) (40) obtained from American Type (32, 33). IL-1 is mainly secreted by monocytes/macrophages and Culture Collection and isolated human monocytes/macrophages were dendritic cells, but also fibroblasts; is involved in a variety of cultured in RPMI 1640 medium containing penicillin (100 U/ml), strep- Downloaded from inflammatory processes, such as costimulus for T cell activation; tomycin (100 mg/ml), 2 mM glutamate (all from Life Technologies Invi- and is one of the key players in rheumatoid arthritis and in- trogen, Basel, Switzerland), amphotericin B (2 mg/ml; Sigma-Aldrich, Steinheim, Germany), and 10% FCS (heat inactivated for 30 min at 56˚C) flammatory bowel disease (34, 35). IL-1b further induces COX-2 at 37˚C, 5% CO2, and humidified atmosphere. In the experiments mea- expression in endothelial cells and is involved in edema forma- suring AA and eicosanoids, the cells were washed and incubated in serum- tion, where it synergizes with PGE2 (36). IL-1b is also a major free medium (see below).

mediator of fever induction (34, 37). In this study, we provide Cytokine measurements. Cytokine quantification in whole blood was done http://www.jimmunol.org/ evidence that the inhibition of i/cPLA by phenylpropanoids is di- with BD cytometric bead array (CBA) human inflammatory cytokine kits 2 b a rectly linked to the global inhibition of IL-1b secretion by ginger (BD kit 551811) for IL-8, IL-1 , IL-6, IL-10, TNF- , IL-12p70, and the BD CBA human allergy mediators kit (BD kit 558022) for IL-3, IL-4, extracts in vitro. Moreover, ginger phenylpropanoids induce dra- IL-5, IL-7, IL-10, and GM-CSF, or with isolated monocytes using the matic changes in arachidonate-phospholipid remodeling, due to BD human IL-1b Flex Set (558279), according to the manufacturer’s their simultaneous inhibition of iPLA2 and prostanoid synthesis. manuals, measured with a FACScan flow cytometer equipped with an This apparently robust mechanism of action could provide a ra- argonlaser,andevaluatedwiththeCBAsoftwareV1.4(allbyBD Biosciences). tional basis for the reported antipyretic and anti-inflammatory ELISA measurements of thromboxane B and leukotriene B . The throm- (i.e., immunomodulatory) effects of ginger preparations. 2 4 boxane B2 (TXB2) ELISA colorimetric kit was from Cayman Chemicals by guest on September 29, 2021 (10004023), limit of detection 35 pg/ml, and leukotriene B4 (LTB4) ELISA Materials and Methods colorometric kit (EHLTB4) was from Thermo Scientific (Pierce Protein Chemicals and reagents Research Products), limit of detection 20 pg/ml. ELISA measurements were carried out as specified by the manufacturer’s instructions. A total of 6 Ethanol 99%, KOH, NaCl, D-(+)-glucose, EDTA, LPS from Escherichia 2 3 10 U937 macrophages was stimulated with LPS (1 mg/ml) to measure 3 6 + coli, ATP, PMA from Euphorbiaceae, BSA fraction V (BSA), and 2,49- TXB2 and 10 10 primary CD14 monocytes/macrophages to measure dibromoacetophenone (DBA) were obtained from Fluka Chemie AG LTB4. Cells were preincubated with DMSO (vehicle) or test compounds m (Buchs, Switzerland). DMSO, HEPES, MOPS, chloroform-d, mono- for 30 min prior to stimulation with LPS (1 g/ml) and for full LTB4 m bromobimane, cylcosporin A, aspirin, diclofenac sodium salt, cetirizine, release costimulation with fMLP (1 M). LTB4 could not be measured , parthenolide, fMLP, and dexamethasone 21-phosphate diso- from stimulated U937 macrophages. dium salt were purchased from Sigma-Aldrich Chemie. All kinase Western blots inhibitors and methyl arachidonyl fluorophosphonate were purchased from Tocris Biosciences. Thapsigargin was purchased from Alexis Biochem. Gels for Western blots were run on denaturing NuPage Novex 4–12% Water LiChrosolv, TLC silica aluminum sheets 60F254, silica gel 60 (15– Bis-Tris precast gels and corresponding MOPS buffer from Invitrogen, m 40 m), CaCl2, and acetonitrile were from Merck. Chloroform was pur- according to the manufacturer’s recommendations, using 10 mlsamples chased from Scharlau SA (Barcelona, Spain). The DTT high purity was dissolved in LDS buffer. Blotting was done on nitrocellulose mem- obtained from GERBU Biochemicals (Gaiberg, Germany). Authentic branes in a NuPage blotting chamber, according to the instruction standards of 6-shogaol and 6-, 8-, and 10-gingerol were purchased from manual. ChromaDex (Santa Ana, CA). Vacutainer LH 170 I.U. and Precision- Staining was done according to the ECL manual from PerkinElmer using Glide were from BD Biosciences (Plymouth, U.K.). KCl was from TBST (pH 7.6 at used temperatures) and nitrocellulose membranes blocked Ha¨nseler AG (Herisau, Switzerland), and FCS was from Omnilab. The 1- with TBST plus 5% defatted milk powder. Ab labeling was done with hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine TBST; 1% defatted milk powder and primary Ab (anti–IL-1b; Abnova (thio-PC) was purchased from Cayman Chemicals. The 8- and 10-shogaol MaxPab and Sigma-Aldrich) were used at 2 ml/ml; and secondary Ab were isolated by liquid chromatography and HPLC, as described pre- (anti-mouse IgG [goat] HRP labeled by GE Healthcare) at 1/8 ml/ml. viously (17). Chemiluminescence detection was done with ECL Plus Western blotting detection reagents (Amersham Biosciences by GE Healthcare). For IL-1b Plant material detection with Western blot analysis, isolated monocytes, in 96-well plates at a density of 2 3 106 cells/ml and 100 ml/well, were stimulated in fresh The extract of Z. officinale (ginger Hot Flavor CO2 extract, type 014.088, batch number 120507) was provided by FLAVEX (Rehlingen, Germany). RPMI 1640 with 100 ng/ml LPS for 4 h. Medium was changed after LPS It was produced by supercritical fluid extraction with carbon dioxide, priming to a gluconate basal salt solution described earlier (33), omitting according to Manninen et al. (38). According to the analysis certificate, glycine. Further processing was in analogy to the CBA (BD Biosciences) m it contains 38.7% 6-, 8-, 10-gingerol, 4.3% 6-, 8-, 10-shogaol, 0.7% zin- protocol, but lyophilisates were directly dissolved in 10 l LDS sample gerone, and 60 ml/g essential oil. Ginger essential oil was a commercial buffer for gel electrophoresis. grade water vapor distillate from Farfalla. Extracts of organic Harpa- Ethidium bromide uptake to measure P2X7-mediated ion fluxes gophytum procumbens were obtained from Bioforce AG (Roggwil, Switzerland). Extract of Salix cortex was obtained from Bionorica SE Ethidium+ uptake was measured in isolated CD14+ human monocytes (Neumarkt, Germany). (according to 41), but without measuring additional cell markers. Primary 4142 GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2 human cells were used for this assay because they gave a better signal-to- at RT under gentle shaking. The enzymatic reaction was stopped after 3 h noise ratio than U937 cells. (.50% hydrolysis) with 16.6 ml ice-cold MeOH. A total of 0.2 ml DTT (100 mM aq. sol., for reduction of dithio-lyso-phosphatidylcholine), 0.2 ml Measurement of IL-1b maturation and secretion Cs4EDTA (100 mM aqueous solution, to chelate free calcium, which otherwise would interfere with the reaction), and 2 mlCs2CO3 (200 mM Cytometric bead arrays. For IL-1b detection with CBA, the isolated . 3 6 m aq. sol., to achieve a pH 8) was added and incubated for 1 h at RT. Then monocytes, in 96-well plates at a density of 2 10 cells/ml and 100 l/ 20 ml monobromobimane (10 mM stock in MeOH) was added and in- well, were stimulated in fresh RPMI 1640 for 4 h under different priming cubated for 1 h at RT. The reaction was stopped and stabilized by acidi- conditions (vehicle, 100 ng/ml LPS and/or 10 mg/ml ginger Hot Flavor m 3 fication with 1 l trichloroacetic acid (2.2 M aqueous solution, to achieve extract). Cells were spun down for 5 min at 200 g, and medium was apH,3) and centrifuged at 500 3 g for 5 min. The supernatant could be replaced (to be processed as described below) with 100 ml fresh one 2 m stored with no detectable decomposition for 3 d at 18˚C until HPLC containing different stimuli (vehicle, 1 mM ATP and/or 10 g/ml ginger measurement. Analytical HPLC was performed on an Elite LaChrome Hot Flavor extract), then incubated for 30 min and centrifuged for 5 min at 3 device (VWR International, Hitachi) equipped with a fluorescence detector 200 g, and supernatant was removed. Medium samples and cells were using a Nucleodur Sphinx column (4 mm particle size, 4.6 3 100-mm shock frozen and lyophilized, to be stored at 280˚C until measurement. m dimension; Macherey-Nagel). A 15-min gradient of 0.25% NH3 in water/ Lyophilisates were reconstituted in 10 l hypotone buffer (10 mM HEPES, acetonitril (LiChrosolv; Merck) from 30:70 to 10:90 was run for analysis, 0.3 mM EGTA, 0.1% Triton X-100, adjusted with KOH to pH 7.4 at RT), l l b b and UV detection at ex 385 nm and em 485 nm was carried out. and IL-1 was quantified with IL-1 BD FlexSet. The enzyme activity was calculated from the area under the curve in absolute values (nmol product total) and (nmol product/min/mg protein). Establishment of PLA assay 2 TLC. TLC was done with a solvent mixture (according to 47). For detection Phospholipase preparation. U-937 cells (106 cells/ml) were kept in culture of phosphate-containing lipids, a molybdenum blue spray solution and medium, according to the recommendations by American Type Culture charring were used. Collection. Twenty-four hours before membrane processing, cells were Downloaded from stimulated with 35 ng/ml PMA, according to published protocols (42, 43). Phospho-MAPKs 3 7 Two times 2.8 10 U937 cells were lysed in a hypotone lysis buffer (340 MAPK phosphorylation in leukocytes was quantified using BD FlexSets, mM , 10 mM HEPES, 1 mM EDTA, 1 mM DTT, pH adjusted with 3 according to the manufacturer’s recommendations, but with 5 times smaller KOH to 7.4 at 4˚C) and centrifuged at 1000 g for 30 min at 4˚C. The volumes. pellets were kept as whole membrane preparations because cPLA2 does Isolated human lymphocytes were adjusted to 4 3 106 cells/ml and colocalize with cell membranes. Supernatants were ultrafiltrated with a 50- aliquoted at 50 ml into 96-well plates. The 50 ml diluted stimuli were kDa molecular mass cut-off, resulting in lysate concentrates of 0.625–1.5 added as 23 stock and incubated at 37˚C for the indicated time. Con- http://www.jimmunol.org/ mg protein/ml for the U937 (quantified with the reducing agent and de- ditions for p38 phosphorylation were 10 ml/ml anti-CD3 Ab and 2 nM tergent-compatible protein assay by Bio-Rad). PMA for 20 min; for ERK1/2 phosphorylation, 10 nM PMA for 30 min; Substrate synthesis and isolation procedure. Palmitoyl-6-O-ascorbic acid and for JNK1/2 phosphorylation, 10 ml/ml anti-CD3 Ab and 10 nM PMA was synthesized (according to 44). Reaction conditions were as follows: for 60 min. Subsequently, samples were cooled on ice and centrifuged at 35˚C was the reaction temperature; 8.8 ml 95% sulphuric acid, 2.29 g 300 3 g for 3 min at 4˚C. The pellets were lysed with 5 ml diluted BD palmitic acid, and 1.76 g ascorbic acid; 100% ultrasonic power output with denaturation buffer containing phosphatase inhibitors (4 mM sodium po- stirring for 15 min. Identity was confirmed by electrospray ionization mass tassium tartrate, 2 mM imidazole, 1.15 mM sodium molybdate, 1 mM spectrometry and two-dimensional nuclear magnetic resonance (NMR). sodium fluoride, and 1 mM sodium orthovanadate) and heated to 80˚C for Palmitoyl-6-O-ascorbic acid potassium salt (PAK) was generated before 5 min. The samples were cooled on ice, diluted with 5 ml assay diluent, use by mixing appropriate solutions of palmitoyl-6-O-ascorbic acid in and shock frozen in liquid nitrogen. After rethawing, the lysates were chloroform with KOH in methanol to result in a 100 mM mixture of 95:5 vigorously pipetted to give a clear solution. Finally, phosphorylation was by guest on September 29, 2021 chloroform to methanol. Crude phosphatidylcholine was isolated from determined with BD FlexSets. commercial grade lecithin (Ha¨nseler AG) by quantitative de-oiling with , enrichment of phosphatidylcholine by quantitative extraction T cell proliferation with boiling ethanol, and finally, precipitation of impurities at 0˚C. Crude CD4+ human lymphocytes were cultured in 96-well plates at 1 3 106 cells/ phosphatidylcholine was separated over a silica gel column with a solvent m m system of dichloromethane/methanol/acetic acid (5:5:1). The extract was ml, and 100 l/well was substituted with additional 50 l RPMI 1640 medium containing 0.625 ml BrdU stock solution and 2 ml anti-CD3 Ab neutralized and dried by filtration over sodium carbonate and anhydrous m magnesium sulfate. The phosphatidylcholine fraction had a TLC purity of per ml or 50 l RPMI 1640 medium containing vehicle control or test .95% and was stabilized with 10 ppm a-tocopherol, dried, and stored at compounds prior to incubation for 5 d. Proliferation was determined with the BD FITC BrdU Flow Kit by FACS, 280˚C. according to the recommendations by the manufacturer (48). m m Buffers. The final phospholipase buffer consisted of 10 l lysate and 10 l A clear correlation between the proliferation rate and residual CD14+ mixed micelle buffer (for iPLA2: 190 mM MOPS, 152 mM KOH, 3 mM monocytes could be observed and is in agreement with earlier reports (49, 50). DTT, 2 mM ATP, 1 mg/ml BSA, and for cPLA2: 190 mM MOPS, 152 mM m KOH, 3 mM DTT, 1.2 mM CaCl2, 1 mg/ml BSA, 20 M bromoenol Characterization of compounds lactone [BEL]) containing mixed micelles (16 mM PAK, 2 mM Tween 80, Nuclear magnetic resonance. Palmitatoyl-6-O-ascorbate was verified by and 2 mM thio-PC, self-forming at ∼T . 35˚C). (Final concentrations are therefore 100 mM Good’s Buffer adjusted with KOH to pH 7.4 at 40˚C, two-dimensional NMR done on a Bruker 400 UltraShield and Bruker 170 mM sucrose, 2 mM DTT, 0.5 mg/ml BSA, 8 mM PAK, 1 mM Tween TOPSPIN 1.3 software. 80, and 1 mM thio-PC, and either 1 mM ATP and 0.5 mM EDTA or 0.1 Mass spectrometry. For in process controls for palmitatoyl-6-O-ascor- mM free calcium and 10 mM BEL.) bate synthesis and phospholipase assay establishment, mass spectrometry (Waters Alliance HT with separation module 2795 coupled to a dual l Mixed micelles. Stock solutions of PAK in chloroform, Tween 80, and absorption detector 2487 and MassLynx V4.0 software) was used. Solvent phospholipid in ethanol were mixed and dried under a nitrogen flow. The m mixed lipid film was hydrated in mixed micelle buffer at 40˚C, briefly was 30% water with 0.1% formic acid and 70% acetonitrile; 20 l sample solution was injected. The mass range measured was m/z 100–500 and/or sonicated to detach film from glass surface if necessary, and gently (to 6 avoid foam formation) shaken until the solution became totally clear. m/z 200–600, electrospray ionization (cone voltage): 20 eV, +40 eV, and Size and z potential of the micelles were analyzed on a zetasizer, giving +80 eV; capillary voltage 3.0 kV; extractor 1 V; and RF lens 0.2 V. Temperature was set at 120˚C and desolvatation temperature at 250˚C. N one single sharp peak in both cases with an average size distribution of 2 was used as carrier gas at a flow rate of 600 l/h for desolvatation and for 32.3 6 9.6 nm by volume (area of 99.3%) and 27.9 6 6.25 nm by number (area of 100%) and an average z potential of 239.4 6 3.25. Estimated by cone flow at 40 l/h. their size, they consist of ∼500 detergent molecules (quod est demon- Quantification of lipids (AA, PGE2, palmitoylethanolamide) by strandum), and their z potential most likely traps c/sPLA2 in the scooting mode (45, 46). Notably, these micelles are self-forming, sonication is not gas chromatography/mass spectrometry mandatory, and they are readily hydrolyzed by all three phospholipase Quantification was performed by a gas chromatography/mass spectrometry subtypes tested. method. A total of 5 3 106 U937 cells was cultured in complete RPMI Fluorescence detection and HPLC. Solutions were mixed at room tem- 1640 medium and differentiated with 2 nM PMA for 48 h and washed with perature (RT), heated to 40˚C (giving a clear solution), and again incubated PBS, and the medium was replaced with FBS-free medium. Cells were The Journal of Immunology 4143

m m incubated with test compounds (DMSO, 2 M 10-shogaol, 2 g/ml ginger Quantification of TXB2 and LTB4 Hot Flavor extract, 50 and 100 mM aspirin, 0.5 mM methyl arachidonyl fluorophosphonate [MAFP]) and incubated for 30 min. Then LPS (1 ug/ TXB2 and LTB4 were quantified from cell culture supernatants using ml) was added, and the cells were incubated for another 4 h. Cells were commercially available ELISA kits. scraped off and separated from the supernatant by centrifugation for 5 min at 1000 rpm in a Heraeus Biofuge fresco (rotor 3325). Lipids were extracted from the supernatants and the cell fractions as Results follows: 1 ml ethanol was added to internal standards (vide infra) and mixed Setup of whole blood assay and immunopharmacological with 9 ml supernatant. The pH was brought to 3 with and profiling of ginger extract the samples added to C 18 Sep-Pak cartridge (Waters) (preactivated with 3 ml methanol and equilibrated with 3 ml 10% ethanol). Cartridges were A convenient high-content in vitro assay was established to profile washed with 10% ethanol and eluted with 3 ml acetonitrile/ethyl acetate the effects of traditional anti-inflammatory multicomponent agents (1:1). Supernatant samples were evaporated to dryness. (i.e., botanical drugs) with yet unknown molecular mechanisms of Cells were extracted similar to the lipid extraction described by Folch action. It was our aim to employ an assay that was potentially less et al. (51). In short, cell pellets were sonicated for 5 min at 4˚C ice-cold chloroform (1 ml containing the internal standards), methanol (0.5 ml), and susceptible to false positives (e.g., effects only detected in tumor PBS (0.25 ml), and then centrifuged for 5 min at 800 3 g. The organic cell lines, but not primary cells) and more closely associated with phase was dried in a glass vial and dried under N2 and reconstituted in 1 ml physiological parameters like blood plasma components. We used ethanol by vortexing at RT (,5 min), diluted with 9 ml water, and ex- human venous whole blood, which was immediately transferred tracted by solid-phase extraction, as described for the supernatant. to 96-well plates (200-ml aliquots) and incubated with discrete Internal standards were as follows: PGE2-d4, 4 ng/ml (100 ng/sample); AA-d8, 4 ng/ml (100 ng/sample); and palmitoylethanolamide-d5, 1 ng/ml stimuli over 18 h. As the primary readout, we measured differ-

(25 ng/sample). ential Th1/Th2 cytokine expression (Supplemental Fig. 1). To Downloaded from Derivatization of the hydroxyl group of palmitoylethanolamide (PEA) induce patterns of differential cytokine expression, stimuli en- was performed with the silylating agent dimethylisopropylsilyl imidazole. gaging distinct receptors and signaling pathways were applied. As described by Obata et al. (52), the molecular stability is increased and large fragments could be detected. Derivatization of AA was achieved by These stimuli included bacterial LPS acting at CD14/TLR4/ esterification with pentafluorobenzylbromide and N,N-diisopropylethyl- MyD88 (55); PMA that activates protein kinase C (56) and non- amine (53). The hydroxyl groups of PGE2 were readily silylated with specifically amplifies cellular signals; anti-CD28 and anti-CD3 dimethylisopropylsilyl imidazole and the carboxylic acid as for AA with

Abs acting at TCR subtypes (57, 58); and thapsigargin that non- http://www.jimmunol.org/ pentafluorobenzylbromide. The remaining ketone was transformed into an specifically triggers a cytosolic calcium increase in all cells (59). O-methyl oxime by methoxiyamine hydrochloride in pyridine (52). The samples were analyzed by GC/electron ionization mass spectrometry As shown in Supplemental Fig. 1, different stimuli that target using an Agilent 6890N GC equipped with a 30 m HP-5MS column and distinct leukocyte populations triggered significantly distinctive a 5975C MS with triple-axis detector. As carrier, gas helium was used at patterns of cytokine expression. The data show mean values from a constant flow rate of 1.5 ml/min with splitless injection at an inlet independent experiments performed with blood from different temperature of 250˚C. Optimal separation of the three analytes was ach- ieved with the following oven program: initial temperature, 150˚C for female and male donors (n = 18) and clearly demonstrate that the 1 min, followed by an increase to 280˚C at 8˚C/min, with a final time of cytokine network is widely conserved between different individ- 20 min. Specific ions were used for selected ion monitoring (54). uals, thus providing a robust high content readout. As anticipated, by guest on September 29, 2021

FIGURE 1. Selected examples of ef- fects on differentially stimulated whole blood by different inhibitors (10 mM) and the experimental samples ginger Hot Flavor extract, two H. procumbens extracts, and a Salix sp. extract (each 50 mg/ml). Human whole blood from different donors was treated with in- hibitors and incubated for 18 h with LPS (312 ng/ml) (upper two graphs) and PMA/aCD3 (1 mg/ml) (lower two graphs), respectively. Data are mean values (+SD) of blood samples from at least three different donors, each mea- sured in triplicates. *p , 0.05, **p , 0.01, ***p , 0.001. 4144 GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2

LPS stimulation induced mainly Th1 monokines with strong TNF- sporine potently inhibited the expression of CD3/CD28-induced a, IL-1b, IL-6, IL-8, and IL-10 expressions (.10,000 pg/ml), but factors, as exemplified by GM-CSF and TNF-a (Fig. 1), but also weak IL-12 expression (Supplemental Fig. 1). Under these con- other calcium-dependent cytokines (IL-3, IL-4, IL-5, IL-7) (data ditions, the T cell cytokines IL-3, IL-4, IL-5, IL-7, and GM-CSF not shown). The COX-2 inhibitor diclofenac and the H1-antag- were not induced. In contrast, PMA/anti-CD3 Ab stimulation led onist cetirizine were largely ineffective, whereas the broad- to a robust Th2 cell response with similar IL-8, IL-12, and TNF-a spectrum enzyme inhibitor DBA significantly inhibited most cy- expressions; less pronounced IL-1b, IL-6, and IL-10 expressions; tokines (Fig. 1). As blood from different donors was employed, we but significantly stronger IL-3, IL-4, IL-5, IL-7, and GM-CSF concluded that the inhibitory effects observed were robust and expressions. meaningful. To validate our assay, we applied specific inhibitors of key in- Ginger extract globally inhibits IL-1b expression in human flammatory processes or signal transduction events. Clinically and whole blood and in primary monocytes experimentally used inhibitors were dexamethasone, cyclosporin, diclofenac, cetirizine, parthenolide, the MAPK kinase inhibitors Intriguingly, the ginger extract (50 mg/ml) potently inhibited IL-1b SB203580 (p38 MAPK inhibitor), SB202190 (p38 MAPK in- expression ($35%) in all experiments in which whole blood was hibitor), SP600125 (JNK inhibitor), U0126 (MEK inhibitor), and stimulated, irrespective of the stimulus applied (Fig. 2). In con- PD98059 (selective MEK1 inhibitor) (Fig. 1). As expected, dexa- trast, the other cytokines were not or only partially inhibited (Figs. methasone potently inhibited the expression of proinflammatory 1, 2), also reflecting the lack of general cytotoxicity of ginger cytokines independent of the stimulus used. Intriguingly, in this extracts under these assay conditions (data not shown). Based on assay, only dexamethasone strongly inhibited LPS-stimulated this finding, we concluded that ginger constituents could selec- Downloaded from TNF-a expression. The MEK1/2 inhibitor U0126 and the p38 tively and globally interfere with the IL-1b expression machinery inhibitor SB203580 weakly inhibited TNF-a. Distinct MEK and by concrete, yet unknown mechanisms (Supplemental Fig. 2). p38 inhibitors like U0126 and PD98059, as well as the broad- Because the stimuli employed induced distinctly different signal spectrum enzyme inhibitor 2,49-dibromoacetophenone (4-bromo- phenacyl bromide or DBA), significantly inhibited LPS-stimulated

IL-1b expression, but with a high interassay variability. Based on http://www.jimmunol.org/ our assumption that certain ginger constituents may be able to modulate proinflammatory cytokine expression (vide supra), the commercial food-grade ginger Hot Flavor extract (50 mg/ml) was analyzed in this assay. Additional anti-inflammatory medicinal plant extracts, like Harpagopyhtum procumbens and Salix spp., were tested in the same setup. The ginger extract potently in- hibited IL-1b expression with little interassay variability, whereas the H. procumbens and Salix extracts (both 50 mg/ml) and cur- (25 mg/ml) were largely ineffective (Fig. 1). Moreover, the by guest on September 29, 2021 ginger extract differentially modulated the expression of several cytokines, depending on the stimulus used (vide infra). All in- hibitors of kinases (SB203580, SB202190, SP600125, U0126) and NF-kB (parthenolid and curcumin), as well as DBA and cetirizine, potently inhibited the PMA/anti-CD3 Ab-stimulated GM-CSF (Fig. 1), which may be explained by the fact that this factor is least strongly induced (,1500 pg/ml). As anticipated, cyclo-

FIGURE 3. A, Relative expression of IL-1b in culture medium of hu- man monocytes. Cells were incubated for 4 h with vehicle control, LPS (312 ng/ml), ginger Hot Flavor extract (10 mg/ml), or both, and then stimulated for 30 min (e.g., with vehicle control, ATP [2 mM], ginger Hot Flavor extract [10 mg/ml], or both). Data show mean values + SD (n = 4); *p , 0.05. B, Representative graphs of the time-resolved (x-axis) ethidium bromide fluorescence (y-axis) of vehicle control (1) and ginger Hot Flavor extract (10 mg/ml) in LPS-primed and ATP-stimulated monocytes (ATP added at 0 s). The time versus fluorescence mean value plots were iden- tical, and an inhibition of either ATP at its receptor or fast ion fluxes FIGURE 2. Global inhibition of IL-1b expression by ginger Hot Flavor (potassium out, calcium in) can be excluded. C, Western blot for IL-1b extract in differentially stimulated whole blood. A total of 50 mg/ml extract after 60-min stimulation in gluconate basal salt solution in the cytosol (a) was incubated together with different stimuli (z-axis) for 18 h, and cyto- and in supernatant medium (b) of isolated monocytes. Upper row, 31-kDa kine expression was determined by CBA. Arrow indicates that IL-1b pro–IL-1b; lower row, 17-kDa mature IL-1b. Lane 1, Vehicle control; is robustly inhibited by 30–50%, independent of the stimulus applied, lanes 2–4, priming with ginger extract, LPS, and ginger extract prior to whereas TNF-a, IL-6, and IL-10 are only partially inhibited. IL-8 ex- LPS; lane 5, LPS priming plus ginger extract stimulation; lane 6, only ATP pression was not modulated by ginger. Data are the mean values from stimulation; lanes 7–9, LPS priming with ATP stimulation; alone, with blood of at least three different donors, each measured in triplicates. The ginger extract prior to LPS, and ginger extract prior to ATP; and lane 10, SD was ,20%. pro- and mature IL-1b standards. The Journal of Immunology 4145

caspase-1 that cleaves pro–IL-1b into the mature IL-1b. In par- 2+ allel, this causes an increase in [Ca ]i, which triggers the release of the IL-1b storage vesicles. As shown in Fig. 3A, the ginger extract significantly and specifically inhibited the ATP/LPS- stimulated IL-1b secretion from U937 cells, thus indicating po- tential effects on P2X7, PLA2 enzymes, and/or caspase-1. The level of ethidium bromide uptake after ATP stimulation can be FIGURE 4. Chemical structures of ginger phenylpropanoids. The alkyl regarded as equivalent to general cation influx through large P2X7 side chain length varies by two carbon atoms between 6-(n = 1), 8-(n = 2), receptor adjacent pores (41). The assay was performed in isolated and 10-(n = 3) gingerol and shogaol, respectively. human monocytes, as they showed a better signal-to-noise ratio than U937 cells. In this assay, P2X7 activity was not inhibited by transduction events, we excluded the possibility that upstream the ginger extract (Fig. 3B). events, such as, for example, inhibition of MAPKs or transcription Western blot and cytometric bead analyses showed that the factors, could be responsible for this effect. ginger extract inhibits maturation and release of IL-1b from ac- tivated monocytes by ∼60% when the ginger extract was added b The inhibition of IL-1 expression by ginger extract is before ATP (Fig. 3C). Under nonstimulated conditions, the iso- mediated by phenylpropanoids that inhibit i/cPLA2, but not lated human monocytes (2 3 105) secrete ∼4 pg mature IL-1b sPLA2 within 4 h in culture and ∼60 pg/ml when incubated (primed) with

Both the purinoreceptor P2X7 and PLA2 enzymes have been LPS. The constitutive secretion was statistically unchanged by 10 Downloaded from shown to be crucial for efficient IL-1b expression (maturation and mg/ml ginger extract (Fig. 3A). A total of 1 mM ATP did not secretion) from monocytes/macrophages (60). To explore the modulate cytokine secretion in nonprimed cells. However, stim- effects of ginger on IL-1b, experiments in which LPS stimulation ulating LPS-primed cells with ATP caused an increase in cytokine was coactivated by ATP were performed in U937 cells, a human secretion (180 pg/ml) (Fig. 3A). This stimulated secretion was monocyte/macrophage cell line, to differentiate between IL-1b significantly reduced by .50% when ginger extract (10 mg/ml)

maturation and secretion. U937 cells that are stimulated by LPS was added prior to the LPS priming and by ∼40% when added http://www.jimmunol.org/ empty the IL-1b stores (by yet unknown mechanisms), leading after LPS and prior to ATP. These levels were significantly dif- only to partial IL-1b section (Fig. 3A) (61). When the cells are ferent between LPS plus ATP, but insignificant when compared costimulated by ATP (which is elevated under inflammatory with LPS alone, ruling out an effect on caspase-1. A differential conditions and present in whole blood), the purinoceptor P2X7, effect on intra- and extracellular pro- and mature IL-1b species a ligand-gated ion channel, is activated, leading to activation of was clearly visible in Western blots (Fig. 3C) even though the by guest on September 29, 2021

FIGURE 5. A, Fluorescence-coupled PLA2 assay. The substrate thio-PC is incorporated into mixed micelles and incubated with a PLA2 containing cell lysate. The reaction is quenched with methanol, and the product, a free thiol, is coupled to mono- bromobimane and quantified by HPLC with fluo- rescence detection. B, Size and homogeneity of mixed micelles were measured by zetasizer. C,

Absolute activities of iPLA2 (left)andcPLA2 (right) in absence and presence of either 10 mg/ml ginger Hot Flavor extract or 10 mM pure com- pounds or controls are shown as mean values + SD (n = 3). If not indicated otherwise, mean values are significantly different from controls (p , 0.05). D, Concentration-dependent inhibition of 10-shogaol

(mM) and ginger extract (mg/ml) on iPLA2 and cPLA2, showing positive controls BEL and MAFP, respectively. Data show mean values 6 SD (n = 3). 4146 GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2

inhibition of maturation and secretion was less pronounced than in To assess the specificity of this effect, we also measured sPLA2. the assay conditions used for quantitative CBA (Fig. 3A). In iso- Porcine pancreatic sPLA2 activity was tested using isolated phos- lated human monocytes, the iPLA2-dependent IL-1b maturation phatidylcholine, and an established TLC detection method (65, and the cPLA2-dependent IL-1b secretion were reduced to ∼60% 66) was not inhibited by ginger constituents up to 20 mM, but (at 10 mg/ml ginger extract), whereas transcription/translation strongly inhibited by the nonspecific enzyme inhibitor DBA (data and constitutive maturation/secretion were unaffected (Fig. 3C). not shown). Therefore, we concluded that the major ginger constituents (Fig. Weak effects of ginger extract on T cell proliferation and 4) could be potential inhibitors of PLA2 enzymes (Supplemental Fig. 2). MAPKs To measure i/cPLA2 enzyme activities, a suitable assay was iPLA2 inhibition has been shown to contribute to the prolifera- established using mixed micelles and a fluorescence-coupled as- tion of lymphocytes, Jurkat T cells (28), and monocytes (29). As say (Fig. 5A,5B) (for experimental details, see Materials and shown in Fig. 6, the proliferation of anti-CD3 Ab-stimulated Methods). The major lipophilic ginger constituents (Fig. 4) were human lymphocytes was weakly, but significantly inhibited by the purchased or isolated, as previously reported (17), and tested at 10 ginger CO2 extract (10 mg/ml) and 10-shogaol (.5 mM), but not mM. As shown in Fig. 5C, the ginger extract and its main ginger by a totum extract containing a higher essential oil content. To constituents inhibited both iPLA2 and cPLA2 enzyme activities. address the effect of ginger constituents on MAPKs previously Overall, the phenylpropanoids more strongly inhibited iPLA2. The reported with cancer cells (54), we also measured the effects of inhibition of ∼50% was in the same range as the inhibition by the gingerols and shogaols on ERK, JNK, and p38 in primary human irreversible PLA2 inhibitors MAFP and BEL. The relatively low T cells. PMA/anti-CD3 Ab-stimulated lymphocytes were used to Downloaded from efficacy of the latter two may be due to the preincubation scheme determine the modulation of MAPKs by measuring kinase phos- used (62, 63) or more likely residual calcium- and phospholipase- phorylation states using a commercial CBA assay (from BD independent hydrolytic activities. Nevertheless, the assay employed Biosciences). As shown in Fig. 6, experiments using primary was robust enough to detect i/cPLA2 inhibitors and to compare human T cells did not show significant modulations. Phosphory- potencies of individual compounds. The inhibitor concentration lation of p38 was even increased by ginger constituents. Only

was as low as 0.1 molar percent of the total detergent molecules 10-shogaol significantly inhibited JNK phosphorylation by ∼35%, http://www.jimmunol.org/ a possible inhibition by surface dilution kinetics could be ex- but the whole extract showed a trend toward activation of this cluded (64). The ginger extract and 10-shogaol concentration- kinase, and thus, no conclusive picture. dependently inhibited both iPLA2 and cPLA2 with similar poten- cies as BEL and MAFP (Fig. 5D). However, the inhibition Inhibition of prostanoid secretion and modulation of of iPLA was clearly stronger at lower concentrations than the arachidonate-phospholipid remodeling by ginger 2 phenylpropanoids inhibition of cPLA2, in particular with the ginger extract (EC50 values 0.7 mg/ml versus 3 mg/ml). For iPLA2, the eight homologs Based on the finding that ginger phenylpropanoids (Fig. 4) inhibit were less active than the controls at equimolar concentrations the PLA2 enzymes in the mixed micelles assay (Fig. 5), we next (10 mM), but 6-shogaol and 10-gingerol were somewhat more assessed the effects of ginger extract and 10-shogaol on free AA by guest on September 29, 2021 potent, with an overall inhibition of ∼65% (Fig. 5C). Although levels and PGE2 release directly in differentiated U937 macro- 6-,8-gingerols did not inhibit cPLA2 and 10-shogaol was the most phages (differentiated with 5 nM PMA for 48 h). A quantitative potent inhibitor, structure-activity relationships were not apparent. gas chromatography/mass spectrometry analysis was employed to

FIGURE 6. A, Proliferation of anti-CD3 Ab- stimulated human CD4+ T lymphocytes, as de- termined by BrdU incorporation. The ginger Hot Flavor extract (10 mg/ml) and 10-shogaol (5–10 mM) show weak, but significantly reduced pro- liferation, whereas essential ginger oil, a ginger totum extract, and the other phenylpropanoids had no effect. Shown are the mean values + SEM of triplicates performed with lymphocytes of at least three different donors. B–D, MAPK phosphoryla- tion in isolated human CD4+ lymphocytes stimu- lated with PMA/anti-CD3 Ab together with the ginger Hot Flavor extract (10 mg/ml) (2) and four of its main constituents (10 mM). Phosphorylation of kinases was measured using selective Abs and CBA (BD Biosciences). Shown are the mean values 6 SEM of triplicates performed with lymphocytes from at least three different donors. *p , 0.05. The Journal of Immunology 4147

determine both AA and PGE2 levels in cell supernatants (cell (2 mM) inhibited the release of PGE2 and the prostanoid metab- medium) and in the cellular fractions (see Materials and Meth- olite TXB2 by .50% (Figs. 7A,7B, 8). In nonstimulated U937 ods). Whereas free AA could be detected in both supernatant (5.4 macrophages, 10-shogaol inhibited constitutive PGE2 expression nmol/1 3 107 cells) and in the thoroughly washed cellular fraction by .50% (Fig. 7A). Noteworthily, even concentrations of ginger 7 (0.4 nmol/1 3 10 cells) of undifferentiated U937 cells, PGE2 was extract as low as 2 mg/ml showed a significant inhibition of PGE2 only found in the supernatant of U937 macrophages. U937 mac- and TXB2 release, whereas LTB4 was only weakly inhibited. As 7 rophages constitutively released PGE2 (0.1 nmol/1 3 10 cells) expected, BEL, which more specifically inhibits iPLA2, had no even without LPS stimulation (Fig. 7A). Upon stimulation by LPS, effect on PGE2, TXB2,orLTB4 production, whereas MAFP also the PGE2 levels stably increased ∼3-fold. As shown in Fig. 7B, the inhibited LTB4 (Figs. 7, 8). Moreover, PEA, which is also con- positive control acetyl salicylic acid (aspirin) at concentrations at stitutively released by U937 macrophages (0.1 nmol/1 3 107 which COX-1/2 is fully inhibited in vitro (.10 mM) inhibited cells), was not modulated by ginger extract or 10-shogaol (Fig. 7F, PGE2 release from LPS-stimulated U937 cells. The PLA2 and 7G). LPS stimulation did not increase PEA secretion. Somewhat nonspecific hydrolase inhibitor MAFP (0.5 mM) and 10-shogaol unexpectedly, ginger extract and 10-shogaol significantly increased Downloaded from http://www.jimmunol.org/

FIGURE 7. Effects of ginger extract and 10-sho- by guest on September 29, 2021 gaol on PEG2, AA, and PEA levels in LPS-stimulated U937 macrophages (PMA differentiated). A and B,

PGE2 levels in supernatant. C and D, Free AA levels in supernatant. E, Free AA levels in cells. F, PEA levels in supernatant. G, PEA levels in cells. Super- natant levels of AA, PGE2, and PEA and the in- tracellular level of AA and PEA were measured by GC/MC. Ginger Hot Flavor extract (ginger, 2 mg/ml), 10-shogaol (2 mM), MAFP (0.5 mM), and acetyl sal- icylic acid (aspirin, 50 and 100 mM) were used as controls. Indicated are the relative mean values + SD of three independent experiments compared with un- treated controls (one sample t test). *p , 0.05, **p , 0.01, ***p , 0.001. 4148 GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2

the inhibition of iPLA2 leads to a dramatic and unexpected in- crease of free AA. This was corroborated in an experiment in which BEL and aspirin were coincubated, and in this combination also led to significant increase of free AA levels (Fig. 7). Free fatty acids are first bound to CoA and then incorporated into lipids by either de novo synthesis of triglycerides (Kennedy pathway) or remodeling of phospholipids (Lands cycle) (23, 24) (Supplemental Fig. 3). In the case of AA, the latter involves incorporation of arachidonate into phosphatidylcholine by a deacetylation/reacety- lation reaction, followed by a phospholipid class switch via CoA- independent transacylation (73, 74). This is the main pathway for AA incorporation in most cell types (75). In both of these pro- cesses, phospholipases, and especially iPLA2, play major regulatory roles by providing free fatty acid acceptor molecules (e.g., lyso- FIGURE 8. Effects of ginger extract and 10-shogaol and PLA2 inhib- phosphatidylcholine) (25). In contrast, immune cells generate free itors MAFP and BEL on production of TXB2 and LTB4 in LPS-stimulated AA mainly from phospholipids by cPLA IVA, the only PLA 6 6 2 2 macrophages. A total of 2 3 10 U937 macrophages (TXB2) and 10 3 10 + with a preference for AA (46, 76–79), and only under certain con- primary human CD14 monocytes/macrophages (LTB4) was incubated ditions by sPLA IIA, V, and X and iPLA VIA (74). Because we with test compounds for 30 min prior to stimulation by LPS (1 mg/ml). To 2 2 observed a significant increase of free cellular AA by ginger phenyl- Downloaded from measure LTB4, the cells were costimulated with fMLP (1 mM). After 3 h, propanoids in U937 macrophages (Fig. 7E), the iPLA2 inhibition TXB2 and LTB4 were quantified from cell-free culture supernatants by ELISA. Data show mean values of three independent experiments + SD, is likely to be predominant in cells, and sPLA2 enzymes are *p , 0.05, ***p , 0.001. not affected. The sPLA2 enzymes may be the cause of the free AA observed in our assays. The reason that MAFP at concentra- tions around the IC50 of reported i/cPLA2 inhibition (80, 81) only

the free AA levels in U937 macrophages in the cellular fraction weakly inhibits free AA in the supernatant is not clear, but may http://www.jimmunol.org/ and in the supernatant under both untreated and LPS-stimulated be due to the combined i/cPLA2 isoform inhibition and a result- conditions (Fig. 7C–E). A PLA2 inhibitor would be expected ing indirect reduction of extracellular sPLA2 activity. Moreover, to rather decrease free AA levels. MAFP reduced free AA in a fraction of the AA that is released by PLA2 activation will be the supernatant, but aspirin, as expected, did not influence free AA rapidly reincorporated into phospholipid, whereas the remainder levels. Because the ginger extract and 10-shogaol inhibit both will be lost by conversion to eicosanoids or other products, or iPLA2 and COX, we speculated that this double effect may lead by b-oxidation (82, 83). Unesterified AA in plasma rapidly to an increase of free AA from the pool that constitutively feeds replaces the amount lost, and this replacement is proportional COX catalyzing prostanoid synthesis. We therefore combined the to PLA2 activation (84, 85). In our experiments, BEL alone in- iPLA2 inhibitor BEL and aspirin. As shown in Fig. 7C and 7D, hibited free AA, but increased free AA when COX was inhibited by guest on September 29, 2021 this combination led to an increase of free AA similar to what was at the same time. This dramatic change (Fig. 7C,7D) shows observed with ginger phenylpropanoids. We concluded that the the role of iPLA2 for AA phospholipid reacylation. Therefore, disruption of arachidonate-phospholipid remodeling, that is, the in the presence of COX inhibitors, blockage of iPLA2 deprives blockage of phospholipid reacylation by iPLA2 inhibition, and the the cell of phospholipid acceptor molecules and subsequently concurrent inhibition of COX is responsible for the unexpected augments the intracellular AA concentration (75). effect on free AA by ginger phenylpropanoids (Supplemental Given that free AA in cells induces apoptosis (29), the pro- Fig. 3). nounced increase of free cellular AA may also explain some of the differential antiproliferative effects of gingerols and shogaols Discussion on cancer cells (86, 87). In contrast, PEA levels were not affected A validated high-content whole blood assay was established to by ginger phenylpropanoids. Its biosynthesis mainly relies on profile robust cytokine modulating effects of reported immuno- N-acyltransferases, N-acylphosphatidylethanolamine phospholi- modulatory agents, including botanical drugs. This has led to the pase D, and fatty acid amide hydrolases, and only to a minor elucidation of a new anti-inflammatory molecular mechanism of extent on lyso-phospholipase D and sPLA2, but not on other PLA2 action of gingerols and shogaols, which are the major phenyl- classes (88, 89). Consequently, our data indicate selectivity toward propanoids in dietary ginger (67, 68). Our study indicates that the i/cPLA2 of the ginger extract and its main constituents and ex- potent inhibition of i/cPLA2 by phenylpropanoids is related to the clude unspecific perturbation of lipid homeostasis. The action of specific inhibition of IL-1b secretion from monocytes/macrophages ginger phenylpropanoids is in line with the already known anti- by ginger (Supplemental Fig. 2). Moreover, our data confirm the oxidant and radical scavenging effects of ginger (90, 91) and may inhibition of COX (10, 11, 67–69) by ginger phenylpropanoids. further increase its effect on iPLA2 in cells. Reactive oxygen This is shown by the inhibition of LPS-stimulated PGE2 and TXB2 species produced by cyclo-/lipoxygenases are known to either secretion (Figs. 7, 8) from U937 macrophages, indicating non- activate iPLA2 in a positive feedback loop (42) or act as mediator selective inhibition of COX enzymes. LPS-stimulated LTB4 from between cPLA2 and sPLA2 (92–95). primary human monocytes/macrophages (U937 macrophages did The ginger rhizome and its extracts have been shown to be not secrete LTB4) was only weakly inhibited by the ginger extract. safe, as exemplified by its widespread dietary use in Asia (1). In The double inhibition of prostanoid secretion and IL-1b by ginger contrast, many tested synthetic i/cPLA2 inhibitors exert unwanted phenylpropanoids is interesting because IL-1b is known to re- side effects due to unspecific toxicity/reactivity (e.g., BEL or MAFP), inforce PGE2 synthesis (70), which plays multiple roles in in- poor selectivity (e.g., MAFP and arachidonyl trifluoromethyl ke- flammatory processes (71, 72). tone), or lack of oral availability [e.g., EXPLIS (96)]. Ginger Our study further shows that the significant cellular inhibition of extracts or isolated compounds show similar in vitro potencies prostanoid production by ginger phenylpropanoids together with against iPLA2 enzymes as standard PLA2 inhibitors, but are The Journal of Immunology 4149 nontoxic. Ginger as a botanical drug has a great acceptance in 20. Honda, Z., M. Nakamura, I. Miki, M. Minami, T. Watanabe, Y. Seyama, H. Okado, H. Toh, K. Ito, T. Miyamoto, et al. 1991. Cloning by functional ex- the population, and might therefore be used as a physically and pression of platelet-activating factor receptor from guinea-pig lung. Nature 349: mentally well-tolerated augmentation to conventional anti-inflam- 342–346. matory medication in cases where first-line therapy is not suffi- 21. Caughey, G. E., M. J. James, and L. G. Cleland. 2005. Prostaglandins and leu- kotrienes. In Encyclopedia of Human Nutrition, 2nd Ed. B. Caballero, L. Allen, cient. In particular, treatment of inflammatory bowel syndrome and A. Prentice, eds. Elsevier, Amsterdam, p. 42–45. and celiac disease in which IL-1b and PGE2 play a major role 22. Smith, W. L., and R. C. Murphy. 2004. Prostaglandins and Leukotrienes. In (97) or autoimmune inner ear disease (98) could be novel thera- Encyclopedia of Biological Chemistry. W. J. Lennarz and M. D. Lane, eds. Elsevier, Amsterdam, p. 452–456. peutic applications of ginger. Overall, the inhibition of PLA2 en- 23. Lands, W. E. 1958. Metabolism of glycerolipides; a comparison of lecithin and zymes provides a rational basis for several reported properties triglyceride synthesis. J. Biol. Chem. 231: 883–888. of ginger [anti-inflammatory, antipyretic, analgesic, or cardio- 24. Lands, W. E. 1960. Metabolism of glycerolipids. 2. The enzymatic acylation of lysolecithin. J. Biol. Chem. 235: 2233–2237. vascular effects (86, 87)]. 25. Balsinde, J., I. D. Bianco, E. J. Ackermann, K. Conde-Frieboes, and E. A. Dennis. 1995. Inhibition of calcium-independent phospholipase A2 pre- vents arachidonic acid incorporation and phospholipid remodeling in P388D1 Acknowledgments macrophages. Proc. Natl. Acad. Sci. USA 92: 8527–8531. We acknowledge the numerous blood donors and medical assistants, in 26. Smani, T., S. I. Zakharov, E. Leno, P. Csutora, E. S. Trepakova, and particular Maria Feher. The ginger extracts were provided by FLAVEX. V. M. Bolotina. 2003. Ca2+-independent phospholipase A2 is a novel de- terminant of store-operated Ca2+ entry. J. Biol. Chem. 278: 11909–11915. 27. Atsumi, G., M. Murakami, K. Kojima, A. Hadano, M. Tajima, and I. Kudo. 2000. Disclosures Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway: proteolytic fragment of type IVA cytosolic phospholipase The authors have no financial conflicts of interest. A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release. J. Downloaded from Biol. Chem. 275: 18248–18258. References 28. Roshak, A. K., E. A. Capper, C. Stevenson, C. Eichman, and L. A. Marshall. 1. Duke, J. A., and E. S. Ayensu. 1985. Medicinal Plants of China. Reference 2000. Human calcium-independent phospholipase A2 mediates lymphocyte Publications, Algonac, MI. proliferation. J. Biol. Chem. 275: 35692–35698. ´ 2. Boone, S. A., and K. M. Shields. 2005. Treating pregnancy-related nausea and 29. Balboa, M. A., R. Perez, and J. Balsinde. 2008. Calcium-independent phos- vomiting with ginger. Ann. Pharmacother. 39: 1710–1713. pholipase A2 mediates proliferation of human promonocytic U937 cells. FEBS J. 3. Bryer, E. 2005. A literature review of the effectiveness of ginger in alleviating 275: 1915–1924. 30. Poitout, V. 2008. Phospholipid hydrolysis and insulin secretion: a step toward http://www.jimmunol.org/ mild-to-moderate nausea and vomiting of pregnancy. J. Midwifery Womens solving the Rubik’s cube. Am. J. Physiol. Endocrinol. Metab. 294: E214–E216. Health 50: e1–e3. 31. Malaviya, R., J. Ansell, L. Hall, M. Fahmy, R. L. Argentieri, G. C. Olini, Jr., 4. Altman, R. D., and K. C. Marcussen. 2001. Effects of a ginger extract on knee D. W. Pereira, R. Sur, and D. Cavender. 2006. Targeting cytosolic phospholipase pain in patients with osteoarthritis. Arthritis Rheum. 44: 2531–2538. A2 by arachidonyl trifluoromethyl ketone prevents chronic inflammation in 5. Bliddal, H., A. Rosetzsky, P. Schlichting, M. S. Weidner, L. A. Andersen, mice. Eur. J. Pharmacol. 539: 195–204. H. H. Ibfelt, K. Christensen, O. N. Jensen, and J. Barslev. 2000. A randomized, 32. Kahlenberg, J. M., K. C. Lundberg, S. B. Kertesy, Y. Qu, and G. R. Dubyak. placebo-controlled, cross-over study of ginger extracts and ibuprofen in osteo- 2005. Potentiation of caspase-1 activation by the P2X7 receptor is dependent on arthritis. Osteoarthritis Cartilage 8: 9–12. TLR signals and requires NF-kappaB-driven protein synthesis. J. Immunol. 175: 6. Penna, S. C., M. V. Medeiros, F. S. Aimbire, H. C. Faria-Neto, J. A. Sertie´, and 7611–7622. R. A. Lopes-Martins. 2003. Anti-inflammatory effect of the hydralcoholic ex- 33. Qu, Y., L. Franchi, G. Nunez, and G. R. Dubyak. 2007. Nonclassical IL-1 beta tract of Zingiber officinale rhizomes on rat paw and skin edema. Phytomedicine secretion stimulated by P2X7 receptors is dependent on inflammasome activa-

10: 381–385. by guest on September 29, 2021 tion and correlated with exosome release in murine macrophages. J. Immunol. 7. Wigler, I., I. Grotto, D. Caspi, and M. Yaron. 2003. The effects of Zintona EC 179: 1913–1925. (a ginger extract) on symptomatic gonarthritis. Osteoarthritis Cartilage 11: 783– 34. Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87: 789. 2095–2147. 8. Kim, J. K., Y. Kim, K. M. Na, Y. J. Surh, and T. Y. Kim. 2007. [6]-Gingerol 35. Fernandes, J. C., J. Martel-Pelletier, and J. P. Pelletier. 2002. The role of cyto- prevents UVB-induced ROS production and COX-2 expression in vitro and kines in osteoarthritis pathophysiology. Biorheology 39: 237–246. in vivo. Free Radic. Res. 41: 603–614. 36. Molina-Holgado, E., S. Ortiz, F. Molina-Holgado, and C. Guaza. 2000. Induction 9. Lee, T. Y., K. C. Lee, S. Y. Chen, and H. H. Chang. 2009. 6-Gingerol inhibits of COX-2 and PGE(2) biosynthesis by IL-1beta is mediated by PKC and mitogen- ROS and iNOS through the suppression of PKC-alpha and NF-kappaB pathways activated protein kinases in murine astrocytes. Br. J. Pharmacol. 131: 152–159. in lipopolysaccharide-stimulated mouse macrophages. Biochem. Biophys. Res. 37. Dinarello, C. A. 1998. Interleukin-1, interleukin-1 receptors and interleukin-1 Commun. 382: 134–139. receptor antagonist. Int. Rev. Immunol. 16: 457–499. 10. Nurtjahja-Tjendraputra, E., A. J. Ammit, B. D. Roufogalis, V. H. Tran, and 38. Manninen, P., E. Ha¨iva¨la¨, S. Sarimo, and H. Kallio. 1997. Distribution of C. C. Duke. 2003. Effective anti-platelet and COX-1 enzyme inhibitors from microbes in supercritical CO2 extraction of sea buckthorn (Hippophae¨ rham- pungent constituents of ginger. Thromb. Res. 111: 259–265. noides) oils. Zeitschrift fu¨r Lebensmitteluntersuchung und -Forschung A 204: 11. van Breemen, R. B., Y. Tao, and W. Li. 2011. Cyclooxygenase-2 inhibitors in 202–205. DOI: 10.1007/s002170050063. ginger (Zingiber officinale). Fitoterapia 82: 38–43. 39. Gertsch, J., R. Schoop, U. Kuenzle, and A. Suter. 2004. Echinacea alkylamides 12. Heubl, G., G. Abel, W. Blaschek, and H. Hager. 1997. Hagers Handbuch der modulate TNF-alpha gene expression via receptor CB2 and multiple Pharmazeutischen Praxis. Springer, Berlin. signal transduction pathways. FEBS Lett. 577: 563–569. 13. Aimbire, F., S. C. Penna, M. Rodrigues, K. C. Rodrigues, R. A. Lopes-Martins, 40. Sundstro¨m, C., and K. Nilsson. 1976. Establishment and characterization of and J. A. Sertie´. 2007. Effect of hydroalcoholic extract of Zingiber officinalis a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17: 565–577. rhizomes on LPS-induced rat airway hyperreactivity and lung inflammation. 41. Jursik, C., R. Sluyter, J. G. Georgiou, S. J. Fuller, J. S. Wiley, and B. J. Gu. 2007. Prostaglandins Leukot. Essent. Fatty Acids 77: 129–138. A quantitative method for routine measurement of cell surface P2X7 receptor 14. Denniff, P., I. Macleod, and D. A. Whiting. 1981. Syntheses of the (6)-[n]- function in leucocyte subsets by two-colour time-resolved flow cytometry. J. gingerols (pungent principles of ginger) and related compounds through regio- Immunol. Methods 325: 67–77. selective aldol condensations: relative assays. J.C.S. Perkin I: 82–87. 42. Balboa, M. A., and J. Balsinde. 2002. Involvement of calcium-independent 15. Iwasaki, Y., A. Morita, T. Iwasawa, K. Kobata, Y. Sekiwa, Y. Morimitsu, phospholipase A2 in -induced accumulation of free fatty K. Kubota, and T. Watanabe. 2006. A nonpungent component of steamed ginger— acids in human U937 cells. J. Biol. Chem. 277: 40384–40389. [10]-shogaol—increases adrenaline secretion via the activation of TRPV1. 43. Pe´rez, R., R. Melero, M. A. Balboa, and J. Balsinde. 2004. Role of group VIA Nutr. Neurosci. 9: 169–178. calcium-independent phospholipase A2 in arachidonic acid release, phospholipid 16. Morita, A., Y. Iwasaki, K. Kobata, H. Yokogoshi, and T. Watanabe. 2007. Newly fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen synthesized oleylgingerol and oleylshogaol activate TRPV1 ion channels. Biosci. peroxide. J. Biol. Chem. 279: 40385–40391. Biotechnol. Biochem. 71: 2304–2307. 44. Wen, B., W. Eli, Q. Xue, X. Dong, and W. Liu. 2007. Ultrasound accelerated 17. Nievergelt, A., P. Huonker, R. Schoop, K. H. Altmann, and J. Gertsch. 2010. esterification of palmitic acid with vitamin C. Ultrason. Sonochem. 14: 213–218. Identification of serotonin 5-HT1A receptor partial agonists in ginger. Bioorg. 45. Jain, M. K., B. Z. Yu, M. H. Gelb, and O. G. Berg. 1992. Assay of phospholi- Med. Chem. 18: 3345–3351. pases A(2) and their inhibitors by kinetic analysis in the scooting mode. Medi- 18. Gilroy, D. W., J. Newson, P. Sawmynaden, D. A. Willoughby, and J. D. Croxtall. ators Inflamm. 1: 85–100. 2004. A novel role for phospholipase A2 isoforms in the checkpoint control of 46. Bayburt, T., and M. H. Gelb. 1997. Interfacial catalysis by human 85 kDa cy- acute inflammation. FASEB J. 18: 489–498. tosolic phospholipase A2 on anionic vesicles in the scooting mode. Biochemistry 19. Amandi-Burgermeister, E., U. Tibes, B. M. Kaiser, W. G. Friebe, and 36: 3216–3231. W. V. Scheuer. 1997. Suppression of cytokine synthesis, integrin expression and 47. Weerheim, A. M., A. M. Kolb, A. Sturk, and R. Nieuwland. 2002. Phospholipid chronic inflammation by inhibitors of cytosolic phospholipase A2. Eur. J. composition of cell-derived microparticles determined by one-dimensional high- Pharmacol. 326: 237–250. performance thin-layer chromatography. Anal. Biochem. 302: 191–198. 4150 GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2

48. Eidukevicius, R., D. Characiejus, R. Janavicius, N. Kazlauskaite, V. Pasukoniene, 73. Balsinde, J. 2002. Roles of various phospholipases A2 in providing lysophos- M. Mauricas, and W. Den Otter. 2005. A method to estimate cell cycle time and pholipid acceptors for fatty acid phospholipid incorporation and remodelling. growth fraction using bromodeoxyuridine-flow cytometry data from a single Biochem. J. 364: 695–702. sample. BMC Cancer 5: 122. 74. Pe´rez-Chaco´n, G., A. M. Astudillo, D. Balgoma, M. A. Balboa, and J. Balsinde. 49. Thiele, D. L., M. Kurosaka, and P. E. Lipsky. 1983. Phenotype of the accessory 2009. Control of free arachidonic acid levels by phospholipases A2 and lyso- cell necessary for mitogen-stimulated T and B cell responses in human periph- phospholipid acyltransferases. Biochim. Biophys. Acta 1791: 1103–1113. eral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucine 75. Chilton, F. H., A. N. Fonteh, M. E. Surette, M. Triggiani, and J. D. Winkler. methyl ester. J. Immunol. 131: 2282–2290. 1996. Control of arachidonate levels within inflammatory cells. Biochim. Bio- 50. Dixon, J. F., and K. Uyemura. 1987. Highly purified human T-lymphocytes do phys. Acta 1299: 1–15. not respond to mitogens—including calcium ionophore and phorbol ester. 76. Diez, E., P. Louis-Flamberg, R. H. Hall, and R. J. Mayer. 1992. Substrate spe- Immunol. Invest. 16: 189–200. cificities and properties of human phospholipases A2 in a mixed vesicle model. 51. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the J. Biol. Chem. 267: 18342–18348. isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: 77. Burdge, G. C., A. Creaney, A. D. Postle, and D. C. Wilton. 1995. Mammalian 497–509. secreted and cytosolic phospholipase A2 show different specificities for phos- 52. Obata, T., Y. Sakurai, Y. Kase, Y. Tanifuji, and T. Horiguchi. 2003. Simulta- pholipid molecular species. Int. J. Biochem. Cell Biol. 27: 1027–1032. neous determination of endocannabinoids (arachidonylethanolamide and 2-arach- 78. Burke, J. R., M. R. Witmer, and J. A. Tredup. 1999. The size and curvature of idonylglycerol) and isoprostane (8-epiprostaglandin F2alpha) by gas chromato- anionic covesicle substrate affects the catalytic action of cytosolic phospholipase graphy-mass spectrometry-selected ion monitoring for medical samples. J. A2. Arch. Biochem. Biophys. 365: 239–247. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 792: 131–140. 79. Ghosh, M., D. E. Tucker, S. A. Burchett, and C. C. Leslie. 2006. Properties of the 53. Balazy, M. 1991. Metabolism of 5,6- by the human group IV phospholipase A2 family. Prog. Lipid Res. 45: 487–510. platelet: formation of novel thromboxane analogs. J. Biol. Chem. 266: 23561– 80. Lio, Y. C., L. J. Reynolds, J. Balsinde, and E. A. Dennis. 1996. Irreversible 23567. inhibition of Ca(2+)-independent phospholipase A2 by methyl arachidonyl flu- 54. Wu¨bert, J., E. Reder, A. Kaser, P. C. Weber, and R. L. Lorenz. 1997. Simulta- neous solid phase extraction, derivatization, and gas chromatographic mass orophosphonate. Biochim. Biophys. Acta 1302: 55–60. spectrometric quantification of thromboxane and prostacyclin metabolites, 81. Surette, M. E., N. Dallaire, N. Jean, S. Picard, and P. Borgeat. 1998. Mechanisms of prostaglandins, and isoprostanes in urine. Anal. Chem. 69: 2143–2146. the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in Downloaded from 55. Aderem, A., and R. J. Ulevitch. 2000. Toll-like receptors in the induction of the chemotactic peptide-stimulated human neutrophils. FA SE B J. 12: 1521–1531. innate immune response. Nature 406: 782–787. 82. Rapoport, S. I. 2001. In vivo fatty acid incorporation into brain phosholipids in 56. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, and Y. Nishizuka. relation to plasma availability, signal transduction and membrane remodeling. J. 1982. Direct activation of calcium-activated, phospholipid-dependent protein Mol. Neurosci. 16: 243–261, discussion 279–284. kinase by tumor-promoting . J. Biol. Chem. 257: 7847–7851. 83. Rapoport, S. I. 2003. In vivo approaches to quantifying and imaging brain 57. Los, M., W. Dro¨ge, and K. Schulze-Osthoff. 1994. Inhibition of activation of arachidonic and docosahexaenoic acid metabolism. J. Pediatr. 143: S26–S34. transcription factor AP-1 by CD28 signalling in human T-cells. Biochem. J. 302: 84. Holman, R. T. 1986. Control of polyunsaturated acids in tissue lipids. J. Am. Coll. Nutr. 5: 183–211. 119–123. http://www.jimmunol.org/ 58. Nu¨sslein, H. G., K. H. Frosch, W. Woith, P. Lane, J. R. Kalden, and B. Manger. 85. Basselin, M., L. Chang, R. Seemann, J. M. Bell, and S. I. Rapoport. 2005. 1996. Increase of intracellular calcium is the essential signal for the expression Chronic lithium administration to rats selectively modifies 5-HT2A/2C receptor- of CD40 ligand. Eur. J. Immunol. 26: 846–850. mediated brain signaling via arachidonic acid. Neuropsychopharmacology 30: 59. Inesi, G., R. Wade, and T. Rogers. 1998. The sarcoplasmic reticulum Ca2+ 461–472. pump: inhibition by thapsigargin and enhancement by adenovirus-mediated gene 86. Aggarwal, B. B., A. B. Kunnumakkara, K. B. Harikumar, S. T. Tharakan, transfer. Ann. N. Y. Acad. Sci. 853: 195–206. B. Sung, and P. Anand. 2008. Potential of spice-derived phytochemicals for 60. Andrei, C., P. Margiocco, A. Poggi, L. V. Lotti, M. R. Torrisi, and A. Rubartelli. cancer prevention. Planta Med. 74: 1560–1569. 2004. Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: 87. Aggarwal, B. B., and S. Shishodia. 2006. Molecular targets of dietary agents for implications for inflammatory processes. Proc. Natl. Acad. Sci. USA 101: 9745– prevention and therapy of cancer. Biochem. Pharmacol. 71: 1397–1421. 9750. 88. Schmid, H. H. 2000. Pathways and mechanisms of N-acylethanolamine bio- 61. Perregaux, D. G., R. E. Laliberte, and C. A. Gabel. 1996. Human monocyte synthesis: can be generated selectively? Chem. Phys. Lipids 108: 71–87.

interleukin-1beta posttranslational processing: evidence of a volume-regulated 89. LoVerme, J., M. Guzma´n, S. Gaetani, and D. Piomelli. 2006. Cold exposure by guest on September 29, 2021 response. J. Biol. Chem. 271: 29830–29838. stimulates synthesis of the bioactive lipid in rat adipose 62. Yang, H. C., M. Mosior, C. A. Johnson, Y. Chen, and E. A. Dennis. 1999. Group- tissue. J. Biol. Chem. 281: 22815–22818. specific assays that distinguish between the four major types of mammalian 90. Dugasani, S., M. R. Pichika, V. D. Nadarajah, M. K. Balijepalli, S. Tandra, and phospholipase A2. Anal. Biochem. 269: 278–288. J. N. Korlakunta. 2010. Comparative antioxidant and anti-inflammatory effects 63. Lucas, K. K., and E. A. Dennis. 2005. Distinguishing phospholipase A2 types in of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol. J. Ethnopharmacol. biological samples by employing group-specific assays in the presence of 127: 515–520. inhibitors. Prostaglandins Other Lipid Mediat. 77: 235–248. 91. Patro, B. S., S. Rele, G. J. Chintalwar, S. Chattopadhyay, S. Adhikari, and 64. Carman, G. M., R. A. Deems, and E. A. Dennis. 1995. Lipid signaling enzymes T. Mukherjee. 2002. Protective activities of some phenolic 1,3-diketones against and surface dilution kinetics. J. Biol. Chem. 270: 18711–18714. lipid peroxidation: possible involvement of the 1,3-diketone moiety. Chem- 65. Skipski, V. P., R. F. Peterson, and M. Barclay. 1964. Quantitative analysis of BioChem 3: 364–370. Biochem. J. phospholipids by thin-layer chromatography. 90: 374–378. 92. Balboa, M. A., R. Pe´rez, and J. Balsinde. 2003. Amplification mechanisms of 66. Nakagawa, Y., and K. Waku. 1989. Phospholipids. In Lipids and Related inflammation: paracrine stimulation of arachidonic acid mobilization by secreted Compounds. Springer Protocols, Berlin, p. 149–178. phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydro- 67. Jolad, S. D., R. C. Lantz, G. J. Chen, R. B. Bates, and B. N. Timmermann. 2005. peroxyeicosatetraenoic acid. J. Immunol. 171: 989–994. Commercially processed dry ginger (Zingiber officinale): composition and 93. Kuwata, H., S. Yamamoto, Y. Miyazaki, S. Shimbara, Y. Nakatani, H. Suzuki, effects on LPS-stimulated PGE2 production. Phytochemistry 66: 1614–1635. 68. Jolad, S. D., R. C. Lantz, A. M. Solyom, G. J. Chen, R. B. Bates, and N. Ueda, S. Yamamoto, M. Murakami, and I. Kudo. 2000. Studies on a mecha- B. N. Timmermann. 2004. Fresh organically grown ginger (Zingiber officinale): nism by which cytosolic phospholipase A2 regulates the expression and function composition and effects on LPS-induced PGE2 production. Phytochemistry 65: of type IIA secretory phospholipase A2. J. Immunol. 165: 4024–4031. 1937–1954. 94. Murakami, M., Y. Nakatani, H. Kuwata, and I. Kudo. 2000. Cellular components 69. Lantz, R. C., G. J. Chen, M. Sarihan, A. M. So´lyom, S. D. Jolad, and that functionally interact with signaling phospholipase A(2)s. Biochim. Biophys. B. N. Timmermann. 2007. The effect of extracts from ginger rhizome on in- Acta 1488: 159–166. flammatory mediator production. Phytomedicine 14: 123–128. 95. Burke, J. E., and E. A. Dennis. 2009. Phospholipase A2 structure/function, 70. Dinarello, C. A. 1985. An update on human interleukin-1: from molecular bi- mechanism, and signaling. J. Lipid Res. 50: S237–S242. ology to clinical relevance. J. Clin. Immunol. 5: 287–297. 96. Chakraborti, S. 2003. Phospholipase A(2) isoforms: a perspective. Cell. Signal. 71. Ha, H., J. H. Lee, H. N. Kim, H. M. Kim, H. B. Kwak, S. Lee, H. H. Kim, and 15: 637–665. Z. H. Lee. 2006. alpha-Lipoic acid inhibits inflammatory bone resorption by 97. Harris, K. M., A. Fasano, and D. L. Mann. 2008. Cutting edge: IL-1 controls the suppressing prostaglandin E2 synthesis. J. Immunol. 176: 111–117. IL-23 response induced by gliadin, the etiologic agent in celiac disease. J. 72. White, K. E., Q. Ding, B. B. Moore, M. Peters-Golden, L. B. Ware, Immunol. 181: 4457–4460. M. A. Matthay, and M. A. Olman. 2008. Prostaglandin E2 mediates IL-1beta- 98. Pathak, S., E. Goldofsky, E. X. Vivas, V. R. Bonagura, and A. Vambutas. 2011. related fibroblast mitogenic effects in acute lung injury through differential IL-1b is overexpressed and aberrantly regulated in corticosteroid nonresponders utilization of prostanoid receptors. J. Immunol. 180: 637–646. with autoimmune inner ear disease. J. Immunol. 186: 1870–1879.