This information is current as
of June 17, 2017.
Ginger Phenylpropanoids Inhibit IL-1β and
Prostanoid Secretion and Disrupt
Arachidonate-Phospholipid Remodeling by
Targeting Phospholipases A 2
Andreas Nievergelt, Janine Marazzi, Roland Schoop,
Karl-Heinz Altmann and Jürg Gertsch
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J Immunol 2011; 187:4140-4150; Prepublished online 9
September 2011;
doi: 10.4049/jimmunol.1100880
http://www.jimmunol.org/content/187/8/4140
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
Jürg Gertsch*
T
he rhizome of ginger (Zingiber officinale) is widely used
in diet, but also in Asian traditional medicine, where it is
employed therapeutically to treat mild forms of rheumatoid arthritis, fever, and upper respiratory tract infections (1). In
conventional Western medicine, ginger preparations are administered clinically to treat emesis, nausea, and migraine headache (2,
3). Based on the widespread therapeutic use of ginger in Asia
for the treatment of inflammatory diseases, several studies have
been dedicated to the elucidation of potential anti-inflammatory
mechanisms (4–7). At relatively high mM concentrations, ginger
constituents such as gingerols, shogaols, and diarylheptanoids
*Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern,
Switzerland; †Bioforce AG, 9325 Roggwil, Switzerland; and ‡Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, 8093 Zurich,
Switzerland
Received for publication March 25, 2011. Accepted for publication August 7, 2011.
This work was supported by a research grant from Bioforce AG (Roggwil, Switzerland). The establishment of lipid analyses by gas chromatography/mass spectrometry
was made possible through a grant from the Novartis Research Foundation.
Address correspondence and reprint requests to Prof. Jürg Gertsch, Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland.
E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AA, arachidonic acid; BEL, bromoenol lactone;
CBA, cytometric bead array; COX, cyclooxygenase; cPLA2, cytosolic phospholipase
A2; DBA, 2,49-dibromoacetophenone; iPLA2, calcium-independent phospholipase A2;
MAFP, methyl arachidonyl fluorophosphonate; NMR, nuclear magnetic resonance;
PAK, palmitoyl-6-O-ascorbate potassium salt; PEA, palmitoylethanolamide; PLA2,
phospholipase A2; RT, room temperature; sPLA2, secretory PLA2; thio-PC, 1-hexadecyl-2-arachidonolythio-2-deoxy-sn-glycero-3-phosphorylcholine; TXB2, thromboxane B2.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100880
have been reported to weakly inhibit components of proinflammatory signal transduction pathways in vitro, such as NF-kB,
protein kinase C, and MAPKs (8, 9). Moreover, ginger constituents have been shown to inhibit inducible NO synthase,
cyclooxygenase (COX)-1/-2, and lipoxygenase in vitro (9–12). A
significant inhibition of PGE2 formation by ginger constituents
has already been shown in vivo (13). Furthermore, different gingerols and shogaols at low mM concentrations have been shown to
directly bind to and partially activate the cation channel transient
receptor potential vanilloid 1 (14–16), the molecular clue for the
spiciness of ginger, as well as the serotonin 5-HT1A G proteincoupled receptor (17). Although the latter studies have elucidated
feasible molecular mechanisms of action of the major biologically
active ginger constituents, they cannot explain the range of antiinflammatory effects observed in vivo. In particular, the reported
antipyretic effects of ginger remain poorly understood.
In this study, we have profiled a standardized food-grade
commercial ginger extract in a high content assay using freshly
isolated human peripheral whole blood. Human blood was incubated with discrete stimuli targeting specific or nonspecific signal transduction pathways, and the resulting cytokine patterns were
quantified. The assay was validated using blood from different
donors and by profiling the effects of anti-inflammatory drugs with
known targets. Using this profiling setup, we were able to identify
calcium-independent phospholipase A2 (iPLA2) and cytosolic
phospholipase A2 (cPLA2) enzymes as major biological targets
of ginger phenylpropanoids.
PLA2 enzymes play a central role in inflammatory processes
(18, 19) and are classified into three main groups, as follows:
secretory phospholipase A2 (sPLA2), cPLA2, and iPLA2. These
distinctly different enzymes hydrolyze the sn-2 ester bond of
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The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis 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
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-gingerol/-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 arachidonic acid was increased 2- to
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.
The Journal of Immunology
Materials and Methods
Chemicals and reagents
Ethanol 99%, KOH, NaCl, D-(+)-glucose, EDTA, LPS from Escherichia
coli, ATP, PMA from Euphorbiaceae, BSA fraction V (BSA), and 2,49dibromoacetophenone (DBA) were obtained from Fluka Chemie AG
(Buchs, Switzerland). DMSO, HEPES, MOPS, chloroform-d, monobromobimane, cylcosporin A, aspirin, diclofenac sodium salt, cetirizine,
curcumin, parthenolide, fMLP, and dexamethasone 21-phosphate disodium salt were purchased from Sigma-Aldrich Chemie. All kinase
inhibitors and methyl arachidonyl fluorophosphonate were purchased from
Tocris Biosciences. Thapsigargin was purchased from Alexis Biochem.
Water LiChrosolv, TLC silica aluminum sheets 60F254, silica gel 60 (15–
40 mm), CaCl2, and acetonitrile were from Merck. Chloroform was purchased from Scharlau SA (Barcelona, Spain). The DTT high purity was
obtained from GERBU Biochemicals (Gaiberg, Germany). Authentic
standards of 6-shogaol and 6-, 8-, and 10-gingerol were purchased from
ChromaDex (Santa Ana, CA). Vacutainer LH 170 I.U. and PrecisionGlide were from BD Biosciences (Plymouth, U.K.). KCl was from
Hänseler AG (Herisau, Switzerland), and FCS was from Omnilab. The 1hexadecyl-2-arachidonoylthio-2-deoxy-sn-glycero-3-phosphorylcholine
(thio-PC) was purchased from Cayman Chemicals. The 8- and 10-shogaol
were isolated by liquid chromatography and HPLC, as described previously (17).
Plant material
The extract of Z. officinale (ginger Hot Flavor CO2 extract, type 014.088,
batch number 120507) was provided by FLAVEX (Rehlingen, Germany).
It was produced by supercritical fluid extraction with carbon dioxide,
according to Manninen et al. (38). According to the analysis certificate,
it contains 38.7% 6-, 8-, 10-gingerol, 4.3% 6-, 8-, 10-shogaol, 0.7% zingerone, and 60 ml/g essential oil. Ginger essential oil was a commercial
grade water vapor distillate from Farfalla. Extracts of organic Harpagophytum procumbens were obtained from Bioforce AG (Roggwil,
Switzerland). Extract of Salix cortex was obtained from Bionorica SE
(Neumarkt, Germany).
Whole blood, assay conditions, and purification of immune
cells
Human venous whole blood was drawn from healthy volunteers with the
BD vacutainer system. Isolation of PBMCs was conducted in accordance
with the guidelines of the World Medical Association’s Declaration of
Helsinki and approved by ETH Zurich. PBMCs were isolated with OptiPrep (Axis-Shield, Oslo, Norway) density gradient flotation, and CD14+
monocytes were enriched by adhesion to culture flasks (FACS analysis was
used to check for purity). Whole blood was immediately pipetted in
a 96-well plate at 200 ml/well. Ethanolic extracts, pure compounds, or
ethanol as vehicle control were added at different concentrations with a
maximal concentration of 0.5% ethanol and incubated for 30 min at 37˚C.
Where indicated, stimuli (LPS [312 ng/ml]) or a combination of PMA
[15 ng/ml] and thapsigargin [1 mM], anti-CD3 Ab, or anti-CD28 Ab [each
1 mg/ml]) were added, and blood was incubated for another 18 h at 37˚C.
For cytokine measurements, the plates were centrifuged, and an appropriate amount (usually 50 ml) of the supernatant serum was removed
for immediate analysis. Isolation of PBMCs and T lymphocytes from
peripheral whole blood was performed, as previously reported (39).
Cell cultures. U937 cells (CRL-1593.2) (40) obtained from American Type
Culture Collection and isolated human monocytes/macrophages were
cultured in RPMI 1640 medium containing penicillin (100 U/ml), streptomycin (100 mg/ml), 2 mM glutamate (all from Life Technologies Invitrogen, Basel, Switzerland), amphotericin B (2 mg/ml; Sigma-Aldrich,
Steinheim, Germany), and 10% FCS (heat inactivated for 30 min at 56˚C)
at 37˚C, 5% CO2, and humidified atmosphere. In the experiments measuring AA and eicosanoids, the cells were washed and incubated in serumfree medium (see below).
Cytokine measurements. Cytokine quantification in whole blood was done
with BD cytometric bead array (CBA) human inflammatory cytokine kits
(BD kit 551811) for IL-8, IL-1b, IL-6, IL-10, TNF-a, IL-12p70, and the
BD CBA human allergy mediators kit (BD kit 558022) for IL-3, IL-4,
IL-5, IL-7, IL-10, and GM-CSF, or with isolated monocytes using the
BD human IL-1b Flex Set (558279), according to the manufacturer’s
manuals, measured with a FACScan flow cytometer equipped with an
argon laser, and evaluated with the CBA software V1.4 (all by BD
Biosciences).
ELISA measurements of thromboxane B2 and leukotriene B4. The thromboxane B2 (TXB2) ELISA colorimetric kit was from Cayman Chemicals
(10004023), limit of detection 35 pg/ml, and leukotriene B4 (LTB4) ELISA
colorometric kit (EHLTB4) was from Thermo Scientific (Pierce Protein
Research Products), limit of detection 20 pg/ml. ELISA measurements
were carried out as specified by the manufacturer’s instructions. A total of
2 3 106 U937 macrophages was stimulated with LPS (1 mg/ml) to measure
TXB2 and 10 3 106 primary CD14+ monocytes/macrophages to measure
LTB4. Cells were preincubated with DMSO (vehicle) or test compounds
for 30 min prior to stimulation with LPS (1 mg/ml) and for full LTB4
release costimulation with fMLP (1 mM). LTB4 could not be measured
from stimulated U937 macrophages.
Western blots
Gels for Western blots were run on denaturing NuPage Novex 4–12%
Bis-Tris precast gels and corresponding MOPS buffer from Invitrogen,
according to the manufacturer’s recommendations, using 10 ml samples
dissolved in LDS buffer. Blotting was done on nitrocellulose membranes in a NuPage blotting chamber, according to the instruction
manual.
Staining was done according to the ECL manual from PerkinElmer using
TBST (pH 7.6 at used temperatures) and nitrocellulose membranes blocked
with TBST plus 5% defatted milk powder. Ab labeling was done with
TBST; 1% defatted milk powder and primary Ab (anti–IL-1b; Abnova
MaxPab and Sigma-Aldrich) were used at 2 ml/ml; and secondary Ab
(anti-mouse IgG [goat] HRP labeled by GE Healthcare) at 1/8 ml/ml.
Chemiluminescence detection was done with ECL Plus Western blotting
detection reagents (Amersham Biosciences by GE Healthcare). For IL-1b
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
RPMI 1640 with 100 ng/ml LPS for 4 h. Medium was changed after LPS
priming to a gluconate basal salt solution described earlier (33), omitting
glycine. Further processing was in analogy to the CBA (BD Biosciences)
protocol, but lyophilisates were directly dissolved in 10 ml LDS sample
buffer for gel electrophoresis.
Ethidium bromide uptake to measure P2X7-mediated ion fluxes
Ethidium+ uptake was measured in isolated CD14+ human monocytes
(according to 41), but without measuring additional cell markers. Primary
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phospholipids, thus liberating lysophospholipids and free fatty
acids, which are precursors for a variety of different mediators
of physiological and pathological processes (20, 21). PLA2 isoenzymes are not only involved in the generation of free arachidonic acid (AA), which is metabolized by cyclo- and lipoxygenases to eicosanoids (22), but also in the incorporation of free
AA into membranes and redistribution into specific compartments (remodeling) (23, 24). Besides regulatory functions in lipid
catabolism/metabolism, PLA2 enzymes are critically involved in
signal transduction, phospholipid and cell membrane bilayer
remodeling (25), store-operated cation channels and Ca2+ releaseactivated Ca2+ channels (26), Fas-induced apoptosis (27), cellular
proliferation and differentiation (28, 29), glucose-induced insulin
secretion (30), and, most important for this study, they play a
central role in acute (18) and chronic inflammation (31).
Both iPLA2 and cPLA2 are crucial for IL-1b maturation and
secretion (membrane translocation and fusion of storage vesicles)
(32, 33). IL-1b is mainly secreted by monocytes/macrophages and
dendritic cells, but also fibroblasts; is involved in a variety of
inflammatory processes, such as costimulus for T cell activation;
and is one of the key players in rheumatoid arthritis and inflammatory bowel disease (34, 35). IL-1b further induces COX-2
expression in endothelial cells and is involved in edema formation, where it synergizes with PGE2 (36). IL-1b is also a major
mediator of fever induction (34, 37). In this study, we provide
evidence that the inhibition of i/cPLA2 by phenylpropanoids is directly linked to the global inhibition of IL-1b secretion by ginger
extracts in vitro. Moreover, ginger phenylpropanoids induce dramatic changes in arachidonate-phospholipid remodeling, due to
their simultaneous inhibition of iPLA2 and prostanoid synthesis.
This apparently robust mechanism of action could provide a rational basis for the reported antipyretic and anti-inflammatory
(i.e., immunomodulatory) effects of ginger preparations.
4141
4142
human cells were used for this assay because they gave a better signal-tonoise ratio than U937 cells.
Measurement of IL-1b maturation and secretion
Cytometric bead arrays. For IL-1b detection with CBA, the isolated
monocytes, in 96-well plates at a density of 2 3 106 cells/ml and 100 ml/
well, were stimulated in fresh RPMI 1640 for 4 h under different priming
conditions (vehicle, 100 ng/ml LPS and/or 10 mg/ml ginger Hot Flavor
extract). Cells were spun down for 5 min at 200 3 g, and medium was
replaced (to be processed as described below) with 100 ml fresh one
containing different stimuli (vehicle, 1 mM ATP and/or 10 mg/ml ginger
Hot Flavor extract), then incubated for 30 min and centrifuged for 5 min at
200 3 g, and supernatant was removed. Medium samples and cells were
shock frozen and lyophilized, to be stored at 280˚C until measurement.
Lyophilisates were reconstituted in 10 ml hypotone buffer (10 mM HEPES,
0.3 mM EGTA, 0.1% Triton X-100, adjusted with KOH to pH 7.4 at RT),
and IL-1b was quantified with IL-1b BD FlexSet.
Establishment of PLA2 assay
at RT under gentle shaking. The enzymatic reaction was stopped after 3 h
(.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
Cs4EDTA (100 mM aqueous solution, to chelate free calcium, which
otherwise would interfere with the reaction), and 2 ml Cs2CO3 (200 mM
aq. sol., to achieve a pH .8) was added and incubated for 1 h at RT. Then
20 ml monobromobimane (10 mM stock in MeOH) was added and incubated for 1 h at RT. The reaction was stopped and stabilized by acidification with 1 ml trichloroacetic acid (2.2 M aqueous solution, to achieve
a pH ,3) and centrifuged at 500 3 g for 5 min. The supernatant could be
stored with no detectable decomposition for 3 d at 218˚C until HPLC
measurement. Analytical HPLC was performed on an Elite LaChrome
device (VWR International, Hitachi) equipped with a fluorescence detector
using a Nucleodur Sphinx column (4 mm particle size, 4.6 3 100-mm
dimension; Macherey-Nagel). A 15-min gradient of 0.25% NH3 in water/
acetonitril (LiChrosolv; Merck) from 30:70 to 10:90 was run for analysis,
and UV detection at lex 385 nm and lem 485 nm was carried out.
The enzyme activity was calculated from the area under the curve in
absolute values (nmol product total) and (nmol product/min/mg protein).
TLC. TLC was done with a solvent mixture (according to 47). For detection
of phosphate-containing lipids, a molybdenum blue spray solution and
charring were used.
Phospho-MAPKs
MAPK phosphorylation in leukocytes was quantified using BD FlexSets,
according to the manufacturer’s recommendations, but with 5 times smaller
volumes.
Isolated human lymphocytes were adjusted to 4 3 106 cells/ml and
aliquoted at 50 ml into 96-well plates. The 50 ml diluted stimuli were
added as 23 stock and incubated at 37˚C for the indicated time. Conditions for p38 phosphorylation were 10 ml/ml anti-CD3 Ab and 2 nM
PMA for 20 min; for ERK1/2 phosphorylation, 10 nM PMA for 30 min;
and for JNK1/2 phosphorylation, 10 ml/ml anti-CD3 Ab and 10 nM PMA
for 60 min. Subsequently, samples were cooled on ice and centrifuged at
300 3 g for 3 min at 4˚C. The pellets were lysed with 5 ml diluted BD
denaturation buffer containing phosphatase inhibitors (4 mM sodium potassium tartrate, 2 mM imidazole, 1.15 mM sodium molybdate, 1 mM
sodium fluoride, and 1 mM sodium orthovanadate) and heated to 80˚C for
5 min. The samples were cooled on ice, diluted with 5 ml assay diluent,
and shock frozen in liquid nitrogen. After rethawing, the lysates were
vigorously pipetted to give a clear solution. Finally, phosphorylation was
determined with BD FlexSets.
T cell proliferation
CD4+ human lymphocytes were cultured in 96-well plates at 1 3 106 cells/
ml, and 100 ml/well was substituted with additional 50 ml RPMI 1640
medium containing 0.625 ml BrdU stock solution and 2 ml anti-CD3 Ab
per ml or 50 ml RPMI 1640 medium containing vehicle control or test
compounds prior to incubation for 5 d.
Proliferation was determined with the BD FITC BrdU Flow Kit by FACS,
according to the recommendations by the manufacturer (48).
A clear correlation between the proliferation rate and residual CD14+
monocytes could be observed and is in agreement with earlier reports (49, 50).
Characterization of compounds
Nuclear magnetic resonance. Palmitatoyl-6-O-ascorbate was verified by
two-dimensional NMR done on a Bruker 400 UltraShield and Bruker
TOPSPIN 1.3 software.
Mass spectrometry. For in process controls for palmitatoyl-6-O-ascorbate synthesis and phospholipase assay establishment, mass spectrometry
(Waters Alliance HT with separation module 2795 coupled to a dual l
absorption detector 2487 and MassLynx V4.0 software) was used. Solvent
was 30% water with 0.1% formic acid and 70% acetonitrile; 20 ml sample
solution was injected. The mass range measured was m/z 100–500 and/or
m/z 200–600, electrospray ionization (cone voltage): 620 eV, +40 eV, and
+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. N2
was used as carrier gas at a flow rate of 600 l/h for desolvatation and for
cone flow at 40 l/h.
Quantification of lipids (AA, PGE2, palmitoylethanolamide) by
gas chromatography/mass spectrometry
Quantification was performed by a gas chromatography/mass spectrometry
method. A total of 5 3 106 U937 cells was cultured in complete RPMI
1640 medium and differentiated with 2 nM PMA for 48 h and washed with
PBS, and the medium was replaced with FBS-free medium. Cells were
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Phospholipase preparation. U-937 cells (106 cells/ml) were kept in culture
medium, according to the recommendations by American Type Culture
Collection. Twenty-four hours before membrane processing, cells were
stimulated with 35 ng/ml PMA, according to published protocols (42, 43).
Two times 2.8 3 107 U937 cells were lysed in a hypotone lysis buffer (340
mM sucrose, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, pH adjusted with
KOH to 7.4 at 4˚C) and centrifuged at 1000 3 g for 30 min at 4˚C. The
pellets were kept as whole membrane preparations because cPLA2 does
colocalize with cell membranes. Supernatants were ultrafiltrated with a 50kDa molecular mass cut-off, resulting in lysate concentrates of 0.625–1.5
mg protein/ml for the U937 (quantified with the reducing agent and detergent-compatible protein assay by Bio-Rad).
Substrate synthesis and isolation procedure. Palmitoyl-6-O-ascorbic acid
was synthesized (according to 44). Reaction conditions were as follows:
35˚C was the reaction temperature; 8.8 ml 95% sulphuric acid, 2.29 g
palmitic acid, and 1.76 g ascorbic acid; 100% ultrasonic power output with
stirring for 15 min. Identity was confirmed by electrospray ionization mass
spectrometry and two-dimensional nuclear magnetic resonance (NMR).
Palmitoyl-6-O-ascorbic acid potassium salt (PAK) was generated before
use by mixing appropriate solutions of palmitoyl-6-O-ascorbic acid in
chloroform with KOH in methanol to result in a 100 mM mixture of 95:5
chloroform to methanol. Crude phosphatidylcholine was isolated from
commercial grade lecithin (Hänseler AG) by quantitative de-oiling with
acetone, enrichment of phosphatidylcholine by quantitative extraction
with boiling ethanol, and finally, precipitation of impurities at 0˚C. Crude
phosphatidylcholine was separated over a silica gel column with a solvent
system of dichloromethane/methanol/acetic acid (5:5:1). The extract was
neutralized and dried by filtration over sodium carbonate and anhydrous
magnesium sulfate. The phosphatidylcholine fraction had a TLC purity of
.95% and was stabilized with 10 ppm a-tocopherol, dried, and stored at
280˚C.
Buffers. The final phospholipase buffer consisted of 10 ml lysate and 10 ml
mixed micelle buffer (for iPLA2: 190 mM MOPS, 152 mM KOH, 3 mM
DTT, 2 mM ATP, 1 mg/ml BSA, and for cPLA2: 190 mM MOPS, 152 mM
KOH, 3 mM DTT, 1.2 mM CaCl2, 1 mg/ml BSA, 20 mM bromoenol
lactone [BEL]) containing mixed micelles (16 mM PAK, 2 mM Tween 80,
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,
170 mM sucrose, 2 mM DTT, 0.5 mg/ml BSA, 8 mM PAK, 1 mM Tween
80, and 1 mM thio-PC, and either 1 mM ATP and 0.5 mM EDTA or 0.1
mM free calcium and 10 mM BEL.)
Mixed micelles. Stock solutions of PAK in chloroform, Tween 80, and
phospholipid in ethanol were mixed and dried under a nitrogen flow. The
mixed lipid film was hydrated in mixed micelle buffer at 40˚C, briefly
sonicated to detach film from glass surface if necessary, and gently (to
avoid foam formation) shaken until the solution became totally clear.
Size and z potential of the micelles were analyzed on a zetasizer, giving
one single sharp peak in both cases with an average size distribution of
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
their size, they consist of ∼500 detergent molecules (quod est demonstrandum), and their z potential most likely traps c/sPLA2 in the scooting
mode (45, 46). Notably, these micelles are self-forming, sonication is not
mandatory, and they are readily hydrolyzed by all three phospholipase
subtypes tested.
Fluorescence detection and HPLC. Solutions were mixed at room temperature (RT), heated to 40˚C (giving a clear solution), and again incubated
GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2
The Journal of Immunology
FIGURE 1. Selected examples of effects 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 inhibitors 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 measured in triplicates. *p , 0.05, **p ,
0.01, ***p , 0.001.
Quantification of TXB2 and LTB4
TXB2 and LTB4 were quantified from cell culture supernatants using
commercially available ELISA kits.
Results
Setup of whole blood assay and immunopharmacological
profiling of ginger extract
A convenient high-content in vitro assay was established to profile
the effects of traditional anti-inflammatory multicomponent agents
(i.e., botanical drugs) with yet unknown molecular mechanisms of
action. It was our aim to employ an assay that was potentially less
susceptible to false positives (e.g., effects only detected in tumor
cell lines, but not primary cells) and more closely associated with
physiological parameters like blood plasma components. We used
human venous whole blood, which was immediately transferred
to 96-well plates (200-ml aliquots) and incubated with discrete
stimuli over 18 h. As the primary readout, we measured differential Th1/Th2 cytokine expression (Supplemental Fig. 1). To
induce patterns of differential cytokine expression, stimuli engaging distinct receptors and signaling pathways were applied.
These stimuli included bacterial LPS acting at CD14/TLR4/
MyD88 (55); PMA that activates protein kinase C (56) and nonspecifically amplifies cellular signals; anti-CD28 and anti-CD3
Abs acting at TCR subtypes (57, 58); and thapsigargin that nonspecifically triggers a cytosolic calcium increase in all cells (59).
As shown in Supplemental Fig. 1, different stimuli that target
distinct leukocyte populations triggered significantly distinctive
patterns of cytokine expression. The data show mean values from
independent experiments performed with blood from different
female and male donors (n = 18) and clearly demonstrate that the
cytokine network is widely conserved between different individuals, thus providing a robust high content readout. As anticipated,
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incubated with test compounds (DMSO, 2 mM 10-shogaol, 2 mg/ml ginger
Hot Flavor extract, 50 and 100 mM aspirin, 0.5 mM methyl arachidonyl
fluorophosphonate [MAFP]) and incubated for 30 min. Then LPS (1 ug/
ml) was added, and the cells were incubated for another 4 h. Cells were
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
follows: 1 ml ethanol was added to internal standards (vide infra) and mixed
with 9 ml supernatant. The pH was brought to 3 with hydrochloric acid and
the samples added to C 18 Sep-Pak cartridge (Waters) (preactivated with
3 ml methanol and equilibrated with 3 ml 10% ethanol). Cartridges were
washed with 10% ethanol and eluted with 3 ml acetonitrile/ethyl acetate
(1:1). Supernatant samples were evaporated to dryness.
Cells were extracted similar to the lipid extraction described by Folch
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
PBS (0.25 ml), and then centrifuged for 5 min at 800 3 g. The organic
phase was dried in a glass vial and dried under N2 and reconstituted in 1 ml
ethanol by vortexing at RT (,5 min), diluted with 9 ml water, and extracted by solid-phase extraction, as described for the supernatant.
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
(25 ng/sample).
Derivatization of the hydroxyl group of palmitoylethanolamide (PEA)
was performed with the silylating agent dimethylisopropylsilyl imidazole.
As described by Obata et al. (52), the molecular stability is increased and
large fragments could be detected. Derivatization of AA was achieved by
esterification with pentafluorobenzylbromide and N,N-diisopropylethylamine (53). The hydroxyl groups of PGE2 were readily silylated with
dimethylisopropylsilyl imidazole and the carboxylic acid as for AA with
pentafluorobenzylbromide. The remaining ketone was transformed into an
O-methyl oxime by methoxiyamine hydrochloride in pyridine (52).
The samples were analyzed by GC/electron ionization mass spectrometry
using an Agilent 6890N GC equipped with a 30 m HP-5MS column and
a 5975C MS with triple-axis detector. As carrier, gas helium was used at
a constant flow rate of 1.5 ml/min with splitless injection at an inlet
temperature of 250˚C. Optimal separation of the three analytes was achieved with the following oven program: initial temperature, 150˚C for
1 min, followed by an increase to 280˚C at 8˚C/min, with a final time of
20 min. Specific ions were used for selected ion monitoring (54).
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FIGURE 2. Global inhibition of IL-1b expression by ginger Hot Flavor
extract in differentially stimulated whole blood. A total of 50 mg/ml extract
was incubated together with different stimuli (z-axis) for 18 h, and cytokine expression was determined by CBA. Arrow indicates that IL-1b
is robustly inhibited by 30–50%, independent of the stimulus applied,
whereas TNF-a, IL-6, and IL-10 are only partially inhibited. IL-8 expression was not modulated by ginger. Data are the mean values from
blood of at least three different donors, each measured in triplicates. The
SD was ,20%.
sporine potently inhibited the expression of CD3/CD28-induced
factors, as exemplified by GM-CSF and TNF-a (Fig. 1), but also
other calcium-dependent cytokines (IL-3, IL-4, IL-5, IL-7) (data
not shown). The COX-2 inhibitor diclofenac and the H1-antagonist cetirizine were largely ineffective, whereas the broadspectrum enzyme inhibitor DBA significantly inhibited most cytokines (Fig. 1). As blood from different donors was employed, we
concluded that the inhibitory effects observed were robust and
meaningful.
Ginger extract globally inhibits IL-1b expression in human
whole blood and in primary monocytes
Intriguingly, the ginger extract (50 mg/ml) potently inhibited IL-1b
expression ($35%) in all experiments in which whole blood was
stimulated, irrespective of the stimulus applied (Fig. 2). In contrast, the other cytokines were not or only partially inhibited (Figs.
1, 2), also reflecting the lack of general cytotoxicity of ginger
extracts under these assay conditions (data not shown). Based on
this finding, we concluded that ginger constituents could selectively and globally interfere with the IL-1b expression machinery
by concrete, yet unknown mechanisms (Supplemental Fig. 2).
Because the stimuli employed induced distinctly different signal
FIGURE 3. A, Relative expression of IL-1b in culture medium of human 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 identical, and an inhibition of either ATP at its receptor or fast ion fluxes
(potassium out, calcium in) can be excluded. C, Western blot for IL-1b
after 60-min stimulation in gluconate basal salt solution in the cytosol (a)
and in supernatant medium (b) of isolated monocytes. Upper row, 31-kDa
pro–IL-1b; lower row, 17-kDa mature IL-1b. Lane 1, Vehicle control;
lanes 2–4, priming with ginger extract, LPS, and ginger extract prior to
LPS; lane 5, LPS priming plus ginger extract stimulation; lane 6, only ATP
stimulation; lanes 7–9, LPS priming with ATP stimulation; alone, with
ginger extract prior to LPS, and ginger extract prior to ATP; and lane 10,
pro- and mature IL-1b standards.
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LPS stimulation induced mainly Th1 monokines with strong TNFa, IL-1b, IL-6, IL-8, and IL-10 expressions (.10,000 pg/ml), but
weak IL-12 expression (Supplemental Fig. 1). Under these conditions, the T cell cytokines IL-3, IL-4, IL-5, IL-7, and GM-CSF
were not induced. In contrast, PMA/anti-CD3 Ab stimulation led
to a robust Th2 cell response with similar IL-8, IL-12, and TNF-a
expressions; less pronounced IL-1b, IL-6, and IL-10 expressions;
but significantly stronger IL-3, IL-4, IL-5, IL-7, and GM-CSF
expressions.
To validate our assay, we applied specific inhibitors of key inflammatory processes or signal transduction events. Clinically and
experimentally used inhibitors were dexamethasone, cyclosporin,
diclofenac, cetirizine, parthenolide, the MAPK kinase inhibitors
SB203580 (p38 MAPK inhibitor), SB202190 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), U0126 (MEK inhibitor), and
PD98059 (selective MEK1 inhibitor) (Fig. 1). As expected, dexamethasone potently inhibited the expression of proinflammatory
cytokines independent of the stimulus used. Intriguingly, in this
assay, only dexamethasone strongly inhibited LPS-stimulated
TNF-a expression. The MEK1/2 inhibitor U0126 and the p38
inhibitor SB203580 weakly inhibited TNF-a. Distinct MEK and
p38 inhibitors like U0126 and PD98059, as well as the broadspectrum enzyme inhibitor 2,49-dibromoacetophenone (4-bromophenacyl bromide or DBA), significantly inhibited LPS-stimulated
IL-1b expression, but with a high interassay variability. Based on
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 inhibited IL-1b expression with little interassay variability, whereas
the H. procumbens and Salix extracts (both 50 mg/ml) and curcumin (25 mg/ml) were largely ineffective (Fig. 1). Moreover, the
ginger extract differentially modulated the expression of several
cytokines, depending on the stimulus used (vide infra). All inhibitors 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-
GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2
The Journal of Immunology
FIGURE 4. Chemical structures of ginger phenylpropanoids. The alkyl
side chain length varies by two carbon atoms between 6-(n = 1), 8-(n = 2),
and 10-(n = 3) gingerol and shogaol, respectively.
transduction events, we excluded the possibility that upstream
events, such as, for example, inhibition of MAPKs or transcription
factors, could be responsible for this effect.
The inhibition of IL-1b expression by ginger extract is
mediated by phenylpropanoids that inhibit i/cPLA2, but not
sPLA2
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 monobromobimane and quantified by HPLC with fluorescence detection. B, Size and homogeneity of
mixed micelles were measured by zetasizer. C,
Absolute activities of iPLA2 (left) and cPLA2
(right) in absence and presence of either 10 mg/ml
ginger Hot Flavor extract or 10 mM pure compounds 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).
caspase-1 that cleaves pro–IL-1b into the mature IL-1b. In parallel, this causes an increase in [Ca2+]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/LPSstimulated IL-1b secretion from U937 cells, thus indicating potential effects on P2X7, PLA2 enzymes, and/or caspase-1. The
level of ethidium bromide uptake after ATP stimulation can be
regarded as equivalent to general cation influx through large P2X7
receptor adjacent pores (41). The assay was performed in isolated
human monocytes, as they showed a better signal-to-noise ratio
than U937 cells. In this assay, P2X7 activity was not inhibited by
the ginger extract (Fig. 3B).
Western blot and cytometric bead analyses showed that the
ginger extract inhibits maturation and release of IL-1b from activated monocytes by ∼60% when the ginger extract was added
before ATP (Fig. 3C). Under nonstimulated conditions, the isolated human monocytes (2 3 105) secrete ∼4 pg mature IL-1b
within 4 h in culture and ∼60 pg/ml when incubated (primed) with
LPS. The constitutive secretion was statistically unchanged by 10
mg/ml ginger extract (Fig. 3A). A total of 1 mM ATP did not
modulate cytokine secretion in nonprimed cells. However, stimulating LPS-primed cells with ATP caused an increase in cytokine
secretion (180 pg/ml) (Fig. 3A). This stimulated secretion was
significantly reduced by .50% when ginger extract (10 mg/ml)
was added prior to the LPS priming and by ∼40% when added
after LPS and prior to ATP. These levels were significantly different between LPS plus ATP, but insignificant when compared
with LPS alone, ruling out an effect on caspase-1. A differential
effect on intra- and extracellular pro- and mature IL-1b species
was clearly visible in Western blots (Fig. 3C) even though the
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Both the purinoreceptor P2X7 and PLA2 enzymes have been
shown to be crucial for efficient IL-1b expression (maturation and
secretion) from monocytes/macrophages (60). To explore the
effects of ginger on IL-1b, experiments in which LPS stimulation
was coactivated by ATP were performed in U937 cells, a human
monocyte/macrophage cell line, to differentiate between IL-1b
maturation and secretion. U937 cells that are stimulated by LPS
empty the IL-1b stores (by yet unknown mechanisms), leading
only to partial IL-1b section (Fig. 3A) (61). When the cells are
costimulated by ATP (which is elevated under inflammatory
conditions and present in whole blood), the purinoceptor P2X7,
a ligand-gated ion channel, is activated, leading to activation of
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FIGURE 6. A, Proliferation of anti-CD3 Abstimulated human CD4+ T lymphocytes, as determined by BrdU incorporation. The ginger Hot
Flavor extract (10 mg/ml) and 10-shogaol (5–10
mM) show weak, but significantly reduced proliferation, 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 phosphorylation in isolated human CD4+ lymphocytes stimulated 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.
To assess the specificity of this effect, we also measured sPLA2.
Porcine pancreatic sPLA2 activity was tested using isolated phosphatidylcholine, and an established TLC detection method (65,
66) was not inhibited by ginger constituents up to 20 mM, but
strongly inhibited by the nonspecific enzyme inhibitor DBA (data
not shown).
Weak effects of ginger extract on T cell proliferation and
MAPKs
iPLA2 inhibition has been shown to contribute to the proliferation of lymphocytes, Jurkat T cells (28), and monocytes (29). As
shown in Fig. 6, the proliferation of anti-CD3 Ab-stimulated
human lymphocytes was weakly, but significantly inhibited by the
ginger CO2 extract (10 mg/ml) and 10-shogaol (.5 mM), but not
by a totum extract containing a higher essential oil content. To
address the effect of ginger constituents on MAPKs previously
reported with cancer cells (54), we also measured the effects of
gingerols and shogaols on ERK, JNK, and p38 in primary human
T cells. PMA/anti-CD3 Ab-stimulated lymphocytes were used to
determine the modulation of MAPKs by measuring kinase phosphorylation states using a commercial CBA assay (from BD
Biosciences). As shown in Fig. 6, experiments using primary
human T cells did not show significant modulations. Phosphorylation of p38 was even increased by ginger constituents. Only
10-shogaol significantly inhibited JNK phosphorylation by ∼35%,
but the whole extract showed a trend toward activation of this
kinase, and thus, no conclusive picture.
Inhibition of prostanoid secretion and modulation of
arachidonate-phospholipid remodeling by ginger
phenylpropanoids
Based on the finding that ginger phenylpropanoids (Fig. 4) inhibit
the PLA2 enzymes in the mixed micelles assay (Fig. 5), we next
assessed the effects of ginger extract and 10-shogaol on free AA
levels and PGE2 release directly in differentiated U937 macrophages (differentiated with 5 nM PMA for 48 h). A quantitative
gas chromatography/mass spectrometry analysis was employed to
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inhibition of maturation and secretion was less pronounced than in
the assay conditions used for quantitative CBA (Fig. 3A). In isolated human monocytes, the iPLA2-dependent IL-1b maturation
and the cPLA2-dependent IL-1b secretion were reduced to ∼60%
(at 10 mg/ml ginger extract), whereas transcription/translation
and constitutive maturation/secretion were unaffected (Fig. 3C).
Therefore, we concluded that the major ginger constituents (Fig.
4) could be potential inhibitors of PLA2 enzymes (Supplemental
Fig. 2).
To measure i/cPLA2 enzyme activities, a suitable assay was
established using mixed micelles and a fluorescence-coupled assay (Fig. 5A, 5B) (for experimental details, see Materials and
Methods). The major lipophilic ginger constituents (Fig. 4) were
purchased or isolated, as previously reported (17), and tested at 10
mM. As shown in Fig. 5C, the ginger extract and its main ginger
constituents inhibited both iPLA2 and cPLA2 enzyme activities.
Overall, the phenylpropanoids more strongly inhibited iPLA2. The
inhibition of ∼50% was in the same range as the inhibition by the
irreversible PLA2 inhibitors MAFP and BEL. The relatively low
efficacy of the latter two may be due to the preincubation scheme
used (62, 63) or more likely residual calcium- and phospholipaseindependent hydrolytic activities. Nevertheless, the assay employed
was robust enough to detect i/cPLA2 inhibitors and to compare
potencies of individual compounds. The inhibitor concentration
was as low as 0.1 molar percent of the total detergent molecules
a possible inhibition by surface dilution kinetics could be excluded (64). The ginger extract and 10-shogaol concentrationdependently inhibited both iPLA2 and cPLA2 with similar potencies as BEL and MAFP (Fig. 5D). However, the inhibition
of iPLA2 was clearly stronger at lower concentrations than the
inhibition of cPLA2, in particular with the ginger extract (EC50
values 0.7 mg/ml versus 3 mg/ml). For iPLA2, the eight homologs
were less active than the controls at equimolar concentrations
(10 mM), but 6-shogaol and 10-gingerol were somewhat more
potent, with an overall inhibition of ∼65% (Fig. 5C). Although
6-,8-gingerols did not inhibit cPLA2 and 10-shogaol was the most
potent inhibitor, structure-activity relationships were not apparent.
GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2
The Journal of Immunology
determine both AA and PGE2 levels in cell supernatants (cell
medium) and in the cellular fractions (see Materials and Methods). Whereas free AA could be detected in both supernatant (5.4
nmol/1 3 107 cells) and in the thoroughly washed cellular fraction
(0.4 nmol/1 3 107 cells) of undifferentiated U937 cells, PGE2 was
only found in the supernatant of U937 macrophages. U937 macrophages constitutively released PGE2 (0.1 nmol/1 3 107 cells)
even without LPS stimulation (Fig. 7A). Upon stimulation by LPS,
the PGE2 levels stably increased ∼3-fold. As shown in Fig. 7B, the
positive control acetyl salicylic acid (aspirin) at concentrations at
which COX-1/2 is fully inhibited in vitro (.10 mM) inhibited
PGE2 release from LPS-stimulated U937 cells. The PLA2 and
nonspecific hydrolase inhibitor MAFP (0.5 mM) and 10-shogaol
(2 mM) inhibited the release of PGE2 and the prostanoid metabolite TXB2 by .50% (Figs. 7A, 7B, 8). In nonstimulated U937
macrophages, 10-shogaol inhibited constitutive PGE2 expression
by .50% (Fig. 7A). Noteworthily, even concentrations of ginger
extract as low as 2 mg/ml showed a significant inhibition of PGE2
and TXB2 release, whereas LTB4 was only weakly inhibited. As
expected, BEL, which more specifically inhibits iPLA2, had no
effect on PGE2, TXB2, or LTB4 production, whereas MAFP also
inhibited LTB4 (Figs. 7, 8). Moreover, PEA, which is also constitutively released by U937 macrophages (0.1 nmol/1 3 107
cells), was not modulated by ginger extract or 10-shogaol (Fig. 7F,
7G). LPS stimulation did not increase PEA secretion. Somewhat
unexpectedly, ginger extract and 10-shogaol significantly increased
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FIGURE 7. Effects of ginger extract and 10-shogaol 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. Supernatant levels of AA, PGE2, and PEA and the intracellular 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 salicylic acid (aspirin, 50 and 100 mM) were used as
controls. Indicated are the relative mean values + SD
of three independent experiments compared with untreated controls (one sample t test). *p , 0.05, **p ,
0.01, ***p , 0.001.
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the free AA levels in U937 macrophages in the cellular fraction
and in the supernatant under both untreated and LPS-stimulated
conditions (Fig. 7C–E). A PLA2 inhibitor would be expected
to rather decrease free AA levels. MAFP reduced free AA in
the supernatant, but aspirin, as expected, did not influence free AA
levels. Because the ginger extract and 10-shogaol inhibit both
iPLA2 and COX, we speculated that this double effect may lead
to an increase of free AA from the pool that constitutively feeds
COX catalyzing prostanoid synthesis. We therefore combined the
iPLA2 inhibitor BEL and aspirin. As shown in Fig. 7C and 7D,
this combination led to an increase of free AA similar to what was
observed with ginger phenylpropanoids. We concluded that the
disruption of arachidonate-phospholipid remodeling, that is, the
blockage of phospholipid reacylation by iPLA2 inhibition, and the
concurrent inhibition of COX is responsible for the unexpected
effect on free AA by ginger phenylpropanoids (Supplemental
Fig. 3).
Discussion
A validated high-content whole blood assay was established to
profile robust cytokine modulating effects of reported immunomodulatory agents, including botanical drugs. This has led to the
elucidation of a new anti-inflammatory molecular mechanism of
action of gingerols and shogaols, which are the major phenylpropanoids in dietary ginger (67, 68). Our study indicates that the
potent inhibition of i/cPLA2 by phenylpropanoids is related to the
specific inhibition of IL-1b secretion from monocytes/macrophages
by ginger (Supplemental Fig. 2). Moreover, our data confirm the
inhibition of COX (10, 11, 67–69) by ginger phenylpropanoids.
This is shown by the inhibition of LPS-stimulated PGE2 and TXB2
secretion (Figs. 7, 8) from U937 macrophages, indicating nonselective inhibition of COX enzymes. LPS-stimulated LTB4 from
primary human monocytes/macrophages (U937 macrophages did
not secrete LTB4) was only weakly inhibited by the ginger extract.
The double inhibition of prostanoid secretion and IL-1b by ginger
phenylpropanoids is interesting because IL-1b is known to reinforce PGE2 synthesis (70), which plays multiple roles in inflammatory processes (71, 72).
Our study further shows that the significant cellular inhibition of
prostanoid production by ginger phenylpropanoids together with
the inhibition of iPLA2 leads to a dramatic and unexpected increase 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/reacetylation reaction, followed by a phospholipid class switch via CoAindependent transacylation (73, 74). This is the main pathway for
AA incorporation in most cell types (75). In both of these processes, phospholipases, and especially iPLA2, play major regulatory
roles by providing free fatty acid acceptor molecules (e.g., lysophosphatidylcholine) (25). In contrast, immune cells generate free
AA mainly from phospholipids by cPLA2 IVA, the only PLA2
with a preference for AA (46, 76–79), and only under certain conditions by sPLA2 IIA, V, and X and iPLA2 VIA (74). Because we
observed a significant increase of free cellular AA by ginger phenylpropanoids in U937 macrophages (Fig. 7E), the iPLA2 inhibition
is likely to be predominant in cells, and sPLA2 enzymes are
not affected. The sPLA2 enzymes may be the cause of the free
AA observed in our assays. The reason that MAFP at concentrations around the IC50 of reported i/cPLA2 inhibition (80, 81) only
weakly inhibits free AA in the supernatant is not clear, but may
be due to the combined i/cPLA2 isoform inhibition and a resulting indirect reduction of extracellular sPLA2 activity. Moreover,
a fraction of the AA that is released by PLA2 activation will be
rapidly reincorporated into phospholipid, whereas the remainder
will be lost by conversion to eicosanoids or other products, or
by b-oxidation (82, 83). Unesterified AA in plasma rapidly
replaces the amount lost, and this replacement is proportional
to PLA2 activation (84, 85). In our experiments, BEL alone inhibited free AA, but increased free AA when COX was inhibited
at the same time. This dramatic change (Fig. 7C, 7D) shows
the role of iPLA2 for AA phospholipid reacylation. Therefore,
in the presence of COX inhibitors, blockage of iPLA2 deprives
the cell of phospholipid acceptor molecules and subsequently
augments the intracellular AA concentration (75).
Given that free AA in cells induces apoptosis (29), the pronounced increase of free cellular AA may also explain some of
the differential antiproliferative effects of gingerols and shogaols
on cancer cells (86, 87). In contrast, PEA levels were not affected
by ginger phenylpropanoids. Its biosynthesis mainly relies on
N-acyltransferases, N-acylphosphatidylethanolamine phospholipase D, and fatty acid amide hydrolases, and only to a minor
extent on lyso-phospholipase D and sPLA2, but not on other PLA2
classes (88, 89). Consequently, our data indicate selectivity toward
i/cPLA2 of the ginger extract and its main constituents and exclude unspecific perturbation of lipid homeostasis. The action of
ginger phenylpropanoids is in line with the already known antioxidant and radical scavenging effects of ginger (90, 91) and may
further increase its effect on iPLA2 in cells. Reactive oxygen
species produced by cyclo-/lipoxygenases are known to either
activate iPLA2 in a positive feedback loop (42) or act as mediator
between cPLA2 and sPLA2 (92–95).
The ginger rhizome and its extracts have been shown to be
safe, as exemplified by its widespread dietary use in Asia (1). In
contrast, many tested synthetic i/cPLA2 inhibitors exert unwanted
side effects due to unspecific toxicity/reactivity (e.g., BEL or MAFP),
poor selectivity (e.g., MAFP and arachidonyl trifluoromethyl ketone), or lack of oral availability [e.g., EXPLIS (96)]. Ginger
extracts or isolated compounds show similar in vitro potencies
against iPLA2 enzymes as standard PLA2 inhibitors, but are
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FIGURE 8. Effects of ginger extract and 10-shogaol and PLA2 inhibitors MAFP and BEL on production of TXB2 and LTB4 in LPS-stimulated
macrophages. A total of 2 3 106 U937 macrophages (TXB2) and 10 3 106
primary human CD14+ monocytes/macrophages (LTB4) was incubated
with test compounds for 30 min prior to stimulation by LPS (1 mg/ml). To
measure LTB4, the cells were costimulated with fMLP (1 mM). After 3 h,
TXB2 and LTB4 were quantified from cell-free culture supernatants by
ELISA. Data show mean values of three independent experiments + SD,
*p , 0.05, ***p , 0.001.
GINGER PHENYLPROPANOIDS INHIBIT PHOSPHOLIPASES A2
The Journal of Immunology
nontoxic. Ginger as a botanical drug has a great acceptance in
the population, and might therefore be used as a physically and
mentally well-tolerated augmentation to conventional anti-inflammatory medication in cases where first-line therapy is not sufficient. In particular, treatment of inflammatory bowel syndrome
and celiac disease in which IL-1b and PGE2 play a major role
(97) or autoimmune inner ear disease (98) could be novel therapeutic applications of ginger. Overall, the inhibition of PLA2 enzymes provides a rational basis for several reported properties
of ginger [anti-inflammatory, antipyretic, analgesic, or cardiovascular effects (86, 87)].
Acknowledgments
We acknowledge the numerous blood donors and medical assistants, in
particular Maria Feher. The ginger extracts were provided by FLAVEX.
Disclosures
The authors have no financial conflicts of interest.
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