of January 3, 2019.This information is current as

CD36Murine Peritoneal Macrophages: Role of

Release fromβTriterpenoid, Induces IL-1Aggregated Ursolic Acid, a Natural

Ken-ichi Hirano, Shizuya Yamashita and Hajime OhigashiHirofumi Tachibana, Koji Yamada, Daisaku Masuda, Yasutaka Ikeda, Akira Murakami, Yoshinori Fujimura,

http://www.jimmunol.org/content/178/8/4854doi: 10.4049/jimmunol.178.8.4854

2007; 178:4854-4864; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/178/8/4854.full#ref-list-1

, 18 of which you can access for free at: cites 66 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Aggregated Ursolic Acid, a Natural Triterpenoid, InducesIL-1� Release from Murine Peritoneal Macrophages: Role ofCD361

Yasutaka Ikeda,* Akira Murakami,* Yoshinori Fujimura,† Hirofumi Tachibana,†

Koji Yamada,† Daisaku Masuda,‡ Ken-ichi Hirano,‡ Shizuya Yamashita,‡

and Hajime Ohigashi2*

IL-1� has been shown to play a pivotal role in the development of inflammatory disorders. We recently found that a naturaltriterpene, ursolic acid (UA), enhanced MIF release from nonstimulated macrophages. In this study, we examined the effects ofUA on the production of several cytokines in resident murine peritoneal macrophages (pM�). UA increased the protein releaseof IL-1�, IL-6, and MIF, but not of TNF-�, in dose- and time-dependent manners. This triterpene also strikingly induced theactivation of p38 MAPK and ERK1/2 together with that of upstream kinases. The release of UA-induced IL-1� was significantlyinhibited by the inhibitors of p38 MAPK, MEK1/2, ATP-binding cassette transporter, and caspase-1. Furthermore, UA inducedintracellular ROS generation for IL-1� production, which was suppressed by an antioxidant. Pretreatment with an anti-CD36 Absignificantly suppressed IL-1� release, and surface plasmon resonance assay results showed that UA bound to CD36 on macro-phages. In addition, the amount of IL-1� released from UA-treated pM� of CD36-deficient mice was markedly lower than thatfrom those of wild-type mice. Interestingly, UA was found to aggregate in culture medium, and the aggregates were suggested tobe responsible for IL-1� production. In addition, i.p. administration of UA increased the levels of IL-1� secretion and MPOactivity in colonic mucosa of ICR mice. Taken together, our results indicate that aggregated UA is recognized, in part, by CD36on macrophages for generating ROS, thereby activating p38 MAPK, ERK1/2, and caspase-1, as well as releasing IL-1� proteinvia the ATP-binding cassette transporter. The Journal of Immunology, 2007, 178: 4854–4864.

M onocytes/macrophages are key mediators of wound re-pair, tissue remodeling, and inflammation and residentmacrophages) are considered to be the front line of

immunological defense against pathogens. These cells have prom-inent roles such as Ag presentation, phagocytic, microbicidal, tu-moricidal, and secretory functions, as well as innate immunity, byinitiating inflammatory and immune responses (1, 2). Macro-phages produce a variety of inflammatory cytokines, including IL-1�, IL-6, TNF-�, and macrophage migration inhibitory factor(MIF)3 (3), which recruit polymorphonuclear leukocytes fromthe vasculature into the inflammatory site for effective eradica-

tion of offending pathogens (4). Although they are essential forthe host defense system, excessive production at an inflamma-tory site may lead to chronic diseases such as inflammatorybowel disease, which includes ulcerative colitis, Crohn’s dis-ease, and neoplasm (5, 6).

IL-1�, an antiapoptotic and proinflammatory cytokine, is one ofthe most pronounced mediators of inflammatory reactions and isprimarily produced in activated monocytes or macrophages (7).Pro-IL-1�, a precursor of IL-1� (8) that is detected in the cytosolof resident macrophages, is biologically inactive with a molecularmass of �33 kDa. Upon stimulation, it is cleaved via an enzymaticprocession into a 17-kDa mature functional form by the IL-1�-converting enzyme (ICE, also known as caspase-1) (9, 10). Fol-lowing ICE cleavage, active IL-1� is released from macrophagesthrough the ATP binding cassette transporter (ABC)A1-dependentand -independent pathways (11, 12). Enhanced IL-1� productionhas been detected at both mRNA and protein levels in humaninflammatory bowel disease (13), rheumatoid arthritis (14), anddextran sulfate sodium (DSS)-induced colitis murine model (15).In our previous study, IL-1� levels were found to be profoundlyincreased in both colonic mucosa and peritoneal macrophages(pM�) in mice with DSS-induced colitis (16). Thus, pM�-derivedIL-1� may be closely and critically associated with diseasepathology.

Triterpenoids are ubiquitously distributed throughout the plantkingdom, and some are increasingly being used for medicinalpurposes for a variety of clinical diseases in many Asian coun-tries as antitumor, anti-inflammatory, and immunomodulatory

*Division of Food Science and Biotechnology, Graduate School of Agriculture,Kyoto University, Kyoto, Japan; †Department of Bioscience and Biotechnology, Fac-ulty of Agriculture, Kyushu University, Fukuoka, Japan; and ‡Department of Cardio-vascular Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan

Received for publication September 7, 2006. Accepted for publication January30, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by a Grant-in-Aid for Cancer Research from theMinistry of Health, Labor, and Welfare of Japan (to A.M.).2 Address correspondence and reprint requests to Dr. Hajime Ohigashi, Division ofFood Science and Biotechnology, Graduate School of Agriculture, Kyoto University,Kitashirakawa Oiwake-tyo, Sakyo-ku, Kyoto 606-8502, Japan. E-mail address:ohigashi@kais.kyoto-u.ac.jp3 Abbreviations used in this paper: MIF, macrophage migration inhibitory factor;ABC, ATP binding cassette transporter; DCFH-DA, 2,7�-dichlorofluorescein di-acetate; DPI, diphenyleneiodonium; DSS, dextran sulfate sodium; EDTA, ethyl-ene diamine tetraacetic acid; ICE, IL-1�-converting enzyme; iNOS, induciblenitric oxide synthase; MEK, MAPK/ERK kinase; MKK, MAPK kinase; MPO,myeloperoxidase; NAC, N-acetyl-L-cysteine; NOX, NADPH oxidase; oxLDL, ox-idized low-density lipoprotein; PKC, protein kinase C; pM�, peritoneal macro-phage; ROS, reactive oxygen species; SPR, surface plasmon resonance; SR,

scavenger receptor; UA, ursolic acid.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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agents (17–19). However, the molecular mechanisms underlyingthose activities remain to be fully elucidated. Ursolic acid (UA;3�-hydroxy-12-urs-12-en-28-oic acid) is a pentacyclic triterpenecarboxylic acid found in various plants, including Rosmarinus of-ficinalis and Glechoma hederaceae (19, 20), in the form of anaglycones or as glycosides (19–23). It is well known to possessmany important biological functions, such as anticancer, anti-inflammatory, hepatoprotective, antiulcer, hypolipidemic, and anti-atherosclerotic activities, as well as others (17, 20, 24, 25). Fur-thermore, it has been reported that UA attenuated the expression ofinducible NO synthase (iNOS) and cyclooxygenase-2 expressionvia NF-�B repression in LPS- or IFN-�-activated mouse macro-phages (26). In contrast, You et al. (27) recently reported that UAinduced NO and TNF-� production via NF-�B activation in rest-ing RAW264.7 mouse macrophages. Along a similar line, we re-cently reported that UA promoted the release of MIF via ERK2activation in the same cell line (28). Those findings imply that theeffects of UA on NF-�B activities are dependent on the biologicalstatus of the target macrophages. This background led us to thepresent investigation of the potential proinflammatory effects of UA inpM�. Our results indicate for the first time that aggregated UA isrecognized by CD36 for generating ROS, thereby activating p38MAPK, ERK1/2, and caspase-1 and releasing IL-1� protein in pM�.

Materials and MethodsCells and mice

RAW 264.7 macrophages were obtained from American Type Culture Col-lection. Male CD36-deficient (C57BL/6J) mice were provided by Dr.M. W. Freeman (Harvard Medical School, Boston, MA). Specific patho-gen-free 5-wk-old female ICR mice and 9-wk-old male C57BL/6J micewere purchased from Japan SLC. On arrival, the mice were randomizedand transferred to plastic cages containing sawdust bedding (five mice percage), which was changed every third day. They were given MF rodentchow (Oriental Yeast) and fresh tap water ad libitum, which was freshlychanged twice a week, and handled according to the Guidelines of theRegulation of Animals, as provided by the Experimentation Committee ofKyoto University. The mice were maintained in a controlled environmentof 24 � 2°C with a relative humidity of 60 � 5% and a 12-h light/darkcycle (lights on from 06:00 to 18:00). All mice were quarantined for 1 wkbefore starting the experiments.

FIGURE 1. UA releases IL-1� (A), IL-6 (B), and MIF (C) protein inpM� in a concentration- and time-dependent manners. pM� from perito-neal exudates from nontreated female ICR mice were seeded onto a 96-well plate at a density of 1 � 106 cells/dish, then cultured at 37°C for 24 hunder a humidified atmosphere of 5% CO2. After washing, the pM� ob-tained were treated with DMSO (0.1%, v/v) or UA (4 or 20 �M) for 0, 1,3, 6, 12, or 24 h. Supernatants were collected and the amounts of IL-1�,IL-6, and MIF protein were determined by ELISA, as described inMaterials and Methods. (�) DMSO; (Œ) UA (4 �M); (f) UA (20 �M).�, p � 0.01, and ��, p � 0.001 vs DMSO by Student’s t test. Data areshown as the mean � SD of three independent experiments.

Table I. List of primer sequences, product size, cycles, and PCR conditions

Gene/Primer Sequence (5�-3�)Product Size

(bp) CyclesAnnealing ExtensionDenaturation (°C, s)

CXCL16Forward ACT ACA CGA GGT TCC AGC TCC 406 38 94, 30Reverse CTT TGT CCG AGG ACA GTG ATC 60, 30

72, 30SR-A type I

Forward GGG AGA CAG AGG GCT TAC TGG A 389 26 95, 30Reverse TTG TCC AAA GTG AGC TCT CTT G 56, 30

72, 60CD36

Forward GTT TTA TCC TTA CAA TGA CA 420 23 95, 30Reverse GGA AAT GTG GAA GCG AAA TA 50, 30

72, 60CD68

Forward TTG GGA ACT ACA CAC GTG GGC 67 40 95, 15Reverse CGG ATT TGA ATT TGG GCT TG 60, 60

IL-1�Forward CAG GAC AGG TAT AGA TTC TTT CCT TT 586 22 94, 30Reverse ATG GCA ACT GTT CCT GAA CTC AAC T 56, 30

72, 30Cyclophilin

Forward TTG GGT CGC GTC TCG TTC GA 240 26 95, 30Reverse GCC AGG ACC TGT ATG CTT CA 50, 30

72, 60

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Reagents

DMEM, Opti-MEM, FBS, and TRIzol were purchased from InvitrogenLife Technologies. UA was obtained from Funakoshi. PD98059,SB203580, SP600125, SB202474 (negative control) inhibitors, diphenyle-neiodonium (DPI) chloride, and YVAD-CHO came from Calbiochem.Glibenclamide, human leukocyte myeloperoxidase (MPO), o-dianisidinedihydrochloride, and hexadecyltrimethylammonium were obtained fromSigma-Aldrich. Abs were purchased from the following sources: rat anti-CXCL16 Ab was from TECHNE; goat anti-scavenger receptor (SR)class-A (SR-A), rabbit anti-CD36, rabbit anti-CD68, goat anti-IL-1�, andgoat anti-�-actin Abs from Santa Cruz Biotechnology; rabbit anti-phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti-phospho-p38, rabbit anti-p38, rab-bit anti-active JNK1/2, rabbit anti-JNK1/2, rabbit anti-phospho-MAPK/ERK kinase (MEK)1/2, rabbit anti-MEK1/2, rabbit anti-phospho-MAPKkinase (MKK) 3/6, rabbit anti-MKK3, rabbit anti-phospho-Raf-1, rabbitanti-Raf-1, and anti-rabbit Ab HRP-linked IgG Abs from Cell SignalingTechnology; and anti-goat IgG from DakoCytomation. Oligonucleotideprimers were synthesized by Proligo. Mouse IL-1� ELISA and caspase-1colorimetric assay kits were purchased from R&D Systems. Mouse IL-6and mouse TNF-� ELISA kits were purchased from Endgen. A rat/mouseMIF immunoassay kit came from Sapporo ImmunoDiagnostic Laboratory.All other chemicals were purchased from Wako Pure Chemicals unlessspecified otherwise.

Cell culture

RAW 264.7 macrophages were grown in DMEM supplemented with 10%FBS, L-glutamine (330 �g/ml), penicillin (100 U/ml), and streptomycin(100 �g/ml) at 37°C under a humidified atmosphere of 95% air and 5%CO2. pM� monolayers were prepared as described previously (29), withsome modifications. Briefly, nontreated 6-wk-old female ICR mice werekilled by cervical dislocation, and 10 ml of ice-cold DMEM containing10% FBS, L-glutamine (330 �g/ml), penicillin (100 U/ml), and streptomy-cin (100 �g/ml) was injected i.p. Medium containing peritoneal exudates

cells (PEC) was recollected and kept on ice. The suspended cells thusobtained were centrifuged at 800 � g for 5 min and resuspended withDMEM. Peritoneal exudate cells (5 � 106 cells/ml/well) were then seededonto culture plates and allowed to adhere for 24 h at 37°C under a humid-ified atmosphere of 95% air and 5% CO2. After washing with PBS twice,nonadherent cells were removed, and the remaining monolayers were des-ignated as pM�. Cell viability was �90% in all experiments, unless spec-ified otherwise.

FIGURE 2. UA triggers activation of both the ERK1/2 and p38 MAPK,but not JNK1/2, pathways in pM�. pM� from peritoneal exudates fromnontreated female ICR mice were seeded onto a 12-well plate at a densityof 5 � 106 cells/dish, then cultured at 37°C for 24 h under a humidifiedatmosphere of 5% CO2. After washing, the pM� were treated with UA (4�M) for 0, 2, 5, 10, or 30 min. The intensity of each band was analyzed byWestern blotting, as described in Materials and Methods. The experimentswere repeated three times independently, with representative resultsshown. �-Actin served as the internal standard.

FIGURE 3. A–C, Possible involvement of ERK1/2, p38 MAPK,caspase-1, and ABC transporter in UA-induced IL-1� production pathway.A and B, Effects of kinase inhibitors on UA-induced IL-1� mRNA expres-sion (A) and pro-IL-1� protein (B). pM� from peritoneal exudates fromnontreated female ICR mice were seeded onto a 12-well plate at a densityof 5 � 106 cells/dish, then cultured at 37°C for 24 h under a humidifiedatmosphere of 5% CO2. After washing, the pM� were pretreated withDMSO (0.1%, v/v), 20 �M PD98059 (PD), or 20 �M SB203580 (SB) at37°C for 30 min before exposure to UA (4 �M) for 0, 3, 6, and 12 h. Thenegative control cells were treated only with 0.1% DMSO. Following thespecified times, the cells were lysed, and IL-1� mRNA expression (A) andprotein production (B) were analyzed by RT-PCR and Western blotting,respectively, as described in Materials and Methods. �, p � 0.01; ��, p �0.001 vs DMSO, ���, p � 0.001 vs UA by Student’s t test. Values areshown as the mean � SD of three independent experiments, with repre-sentative results shown. C, Effects of specific and nonspecific inhibitors onUA-induced IL-1� production. pM� from peritoneal exudates from non-treated female ICR mice were seeded onto a 96-well plate at a density of1 � 106 cells/dish, then cultured at 37°C for 24 h under a humidifiedatmosphere of 5% CO2. After washing, the pM� were pretreated withDMSO (0.1%, v/v), 20 �M PD98059 (PD), 20 �M SB203580 (SB), 20�M MAPK-negative control (NEG), 20 �M glibenclamide (GC), or 20�M Ac-YVAD-CHO (YVAD). The negative control cells were treatedonly with 0.1% DMSO (DM). After incubating at 37°C for 30 min, thepM� were treated with UA (4 �M) for 6 h. IL-1� production was exam-ined by ELISA, as described in Materials and Methods. �, p � 0.01 vsDMSO; ��, p � 0.01; ���, p � 0.005 vs UA by Student’s t test. Data areshown as the mean � SD of three independent experiments.

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Tissue harvest

At the end of each experiment, mice were killed by cervical dislocation,and the large intestines without the cecum were removed. After washing inice-cold PBS, the specimens were placed on filter papers and opened withsurgical scissors to remove their contents. The colonic mucosa wasscraped off using a razor while the specimen was on ice, then frozen inliquid nitrogen until use, according to a method previously reported byPerdue et al. (30), with some modifications.

Protein determination

Total protein concentrations in the pM� and tissue supernatants were de-termined using a DC protein assay (Bio-Rad) according to the protocol ofthe manufacturer (dilution factor � 50), with gammaglobulin used as thestandard.

ELISA

pM� were seeded onto a 96-well plate at a density of 1 � 106 cells/200 �lin 200 �l of DMEM, which included 10% FBS, L-glutamine (330 �g/ml),penicillin (100 U/ml), and streptomycin (100 �g/ml). The cells were cul-tured at 37°C under a humidified atmosphere of 95% air and 5% CO2. After24 h, the cells were washed twice with PBS, and a serum-free medium wasadded. The cells were then treated with UA (4 or 20 �M), which wasdissolved in DMSO (0.1% v/v, as a final concentration). Control cells weretreated only with 0.1% (v/v) DMSO and showed no significant effect on theassay systems (data not shown). After incubation for 0, 1, 3, 6, 12, and24 h, the supernatants, 5 �l for MIF and 50 �l for TNF-�, IL-1�, and IL-6,were used for ELISA and examined according to the protocol of the ap-propriate kit. Alternatively, pM� were pretreated with each specific inhib-itor (see Fig. 4B) or Ab (see Fig. 5B) 30 min before UA treatment.

Western blotting

To determine the expression of phospho-ERK1/2 (Thr202/Tyr204), ERK,phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNK1/2

(Thr183/Tyr185), JNK1/2, phospho-MEK1/2 (Ser217/221), MEK1/2, phos-pho-Raf (Ser259), Raf, phospho-MKK3/6 (Ser189/207), MKK3/6, proIL-1�,SR-A, CD36, CD68, and CXCL16 in pM�, the total cell lysate was sub-jected to Western blotting. �-Actin served as the internal control. Cells(5.0 � 106) were treated with lysis buffer (protease inhibitor mixture, phos-phatase inhibitor mixture (TaKaRa Bio), 10 mM Tris (pH 7.4), 1% SDS,and 1 mM sodium vanadate (V)), and the lysates were boiled for 5 min.Denatured proteins (40 �g) were separated using SDS-PAGE on a 10%polyacrylamide gel and then transferred onto Immobilon-P membranes(Millipore). After blocking overnight at 4°C in Block Ace (DainipponPharmaceutical), the membranes were first incubated with each Ab at di-lutions of 1/1000. The second incubation was performed with HRP-con-jugated secondary anti-rabbit IgG or anti-goat IgG Ab (1/1000 dilutioneach). The blots were developed using an ECL Advance Western blottingdetection reagent (Amersham Biosciences). The intensity of each band wasanalyzed using NIH Image.

RT-PCR

Steady-state mRNA levels of CXCL16, SR-A type I, CD36, CD 68, andIL-1� were detected by RT-PCR. pM� were cultured at a density of 5 �106 cells/2 ml for 24 h. Total cellular RNA was extracted from the cellsusing TRIzol reagent, then precipitated with isopropanol, washed with 70%(v/v) ethanol, and treated with DNase I (Invitrogen Life Technologies) toremove genomic DNA. A cyclophilin transcript served as the internal con-trol. The primer sequences, PCR product sizes, and PCR conditions arelisted in Table I. cDNA was synthesized using 1 �g of total RNA and anRNA PCR kit (Takara). Amplified cDNA was electrophoresed on 2% aga-rose gels and stained with SYBR Gold (Molecular Probes). Image analysiswas performed using NIH Image. No PCR saturation was confirmed bytitration of each cDNA amount (data not shown).

Determination of intracellular ROS generation

pM� were cultured at a density of 1 � 106 cells/200 �l for 24 h onone-chamber slides (GLASS/PS, 9 � 9; IWAKI). After washing, the cells

FIGURE 4. UA may activate NOX for generating intracellular ROS generation which, in turn, phosphorylates MAPKs in pM�. A, UA inducesDCFH-detectable peroxide generation in pM�. pM� from peritoneal exudates from nontreated female ICR mice were seeded onto one-chamber slides ata density of 1 � 106 cells/chamber, then cultured at 37°C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM� were treated withDMSO (0.1%, v/v) or UA (4 or 20 �M) for 5 min. ROS generation was determined using a fluorescent probe, DCFH-DA, as described in Materials andMethods. The negative control cells were treated only with DMSO. �, p � 0.01 vs DMSO by Student’s t test. The experiments were repeated three timesindependently. B and C, Effects of an antioxidant, NOX inhibitor, and Ca2� chelator on UA-induced IL-1� production (B), and activation of Raf, MEK1/2,ERK1/2, MKK3/6, and p38 MAPK (C). pM� from peritoneal exudates from nontreated female ICR mice were seeded onto 96- and 12-well plates at adensity of 1 � 106 cells/dish (B) or 5 � 106 cells/dish (C), then cultured at 37°C for 24 h under a humidified atmosphere of 5% CO2. After washing, thepM� were pretreated with DMSO (0.1%, v/v), 1 mM NAC, 10 �M DPI, or 5 mM EDTA for 30 min, followed by exposure to UA (4 �M) for 6 h (B)or 5 min (C). IL-1� production (B) and MAPK activation (C) were examined by ELISA and Western blotting, respectively, as described in Materials andMethods. �, p � 0.01 vs DMSO; ��, p � 0.05; ���, p � 0.01 vs UA by Student’s t test. Data are shown as the mean � SD of three independent experiments,with one representative result shown (C).

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were suspended in 200 �l of PBS and treated with 10 �M of 2,7�-dichlo-rofluorescein diacetate (DCFH-DA; Molecular Probes) for 5 min. There-after, the medium was discarded and the pM� were washed twice withPBS, followed by exposure to DMSO or UA (final 4 or 20 �M) for 5 min.Negative control cells were treated only with DMSO and without DCFH-DA. UA-induced intracellular ROS generation was detected using a fluo-rescence microscope (Olympus).

Binding analysis using surface plasmon resonance (SPR)biosensor

Analysis of the interaction between UA and CD36 on the cell surface ofmacrophages was performed using the SPR biosensor SPR670 (Moritex).Mouse macrophage RAW264.7 cells (5 � 105 cells/ml) and pM� (1.5 �106 cells/ml) were immobilized on sensor chips, which had been pretreatedwith nonspecific IgG and anti-CD36 Ab (2.5 �g/ml each) in serum-freeDMEM for 30 min. Following pretreatment, the medium was discardedand the chips were equilibrated in SPR running buffer, and 0.1% DMSO(v/v) in PBS (pH 7.4, flow rate � 30 �l/min). UA was diluted at 0 or 20�M in SPR running buffer in 60-�l injection volumes and run on the chipat a flow rate of 30 �l/min. Binding was measured at 25°C for 2 min,followed by dissociation. The value of the angle was deducted from thebinding signal of 0 �M UA and used as the binding strength.

HPLC analysis

UA (4 �M) was dissolved in DMSO (0.05–1%, v/v) and added to 50 ml ofserum-free DMEM in a 50-ml centrifuge tube. After centrifugation at2,500 � g for 5 min, UA was extracted separately from the supernatant and

pellet with chloroform, with a recovery rate of �85% in each experiment.Each extract was concentrated in vacuo and dissolved in 200 �l of chlo-roform. The amount of UA was quantified by HPLC analysis on a YMC-Pack ODS-AQ column (150 � 4.6 mm inside diameter; YMC), which waseluted with 10% methanol in water at a flow rate of 1.0 ml/min. A standardcurve was made using 0–8 �g of UA, with the peak area based on ab-sorption of 210 nm. The amounts of UA derivatives (4 �M, 0.1% DMSOv/v) were analyzed under the same experimental conditions. Data are ex-pressed by the distribution rates, which were calculated from the amountsof each fraction.

Animal treatment

Corn oil alone (n � 8) or UA suspended in 200 �l of corn oil was admin-istrated by i.p. injection to specific pathogen-free 5-wk-old female ICRmice daily at a dose of 50 (n � 6), 100 (n � 6), or 200 (n � 9) mg/kg bodyweight for 8 days. Twenty-four hours after the final administration, pM�monolayers were separately prepared as described above and seeded ontoa 96-well plate at a density of 1 � 106 cells/200 �l, followed by incubationfor 24 h at 37°C under a humidified atmosphere of 95% air and 5% CO2.After the cells were washed twice with PBS, serum-free medium (200 �l)was added. The cells were then incubated for another 24 h, and the super-natant (50 �l) was used for measuring IL-1� with ELISA, as describedabove. After preparation of pM�, the mucosa layer removed from thecolon was homogenized (10 mg/300 �l) in ice-cold PBS using a homog-enizer (UP 50H; Hielscher). Homogenates were frozen in liquid nitrogen andthawed using a sonicator (EYELA). These procedures were repeated threetimes, then the homogenate was centrifuged at 20,000 � g for 30 min at 4°C

FIGURE 5. Possible involvement of CD36 in UA-induced IL-1� production. A, SR mRNA and protein expression in UA-stimulated pM�. pM� fromperitoneal exudates from nontreated female ICR mice were seeded onto a 12-well plate at a density of 5 � 106 cells/dish, then cultured at 37°C for 24 hunder a humidified atmosphere of 5% CO2. After washing, the pM� were treated with UA (4 �M) for 0 or 6 h. SR mRNA and proteins were examinedby RT-PCR and Western blotting, respectively, as described in Materials and Methods. The arrow indicates the band for CXCL16 protein. The experimentswere repeated three times independently, with one representative result shown for each. Cyclophilin and �-actin served as the internal standards. B, Effectsof anti-SR Abs on IL-1� production in pM�. pM� from peritoneal exudates from nontreated female ICR mice were seeded onto a 96-well plate at a densityof 1 � 106 cells/dish, then cultured at 37°C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM� were pretreated with the vehicle(nonspecific IgG), anti-CXCL16, anti-SR-A, anti-CD36, or anti-CD68 Abs (2.5 �g/ml each), followed by exposure to UA (4 �M) for 6 h. IL-1� productionwas examined by ELISA, as described in Materials and Methods. �, p � 0.0005 vs DMSO; ��, p � 0.005 vs UA by Student’s t test. Each value is shownas the mean � SD of three replicated experiments. C, pM� from CD36-deficient mice had lower levels of IL-1�, IL-6, and MIF production as comparedwith those from wild-type mice. pM� from peritoneal exudates from nontreated male C57BL/6J (wild-type) and CD36-deficient mice were seeded ontoa 96-well plate at a density of 1 � 106 cells/dish, then cultured at 37°C for 24 h under a humidified atmosphere of 5% CO2. After washing, the pM� weretreated with DMSO (0.1%, v/v), UA (4 or 20 �M), or LPS (100 ng/ml) for 12 h. IL-1�, IL-6, and MIF production was examined by ELISA, as describedin Materials and Methods. �, wild type; f, CD36-deficient mice. �, p � 0.05 vs wild-type mice by Student’s t test. Each value is shown as the mean �SD of three replicated experiments. D, UA binds to cell surface CD36 on mouse macrophages. RAW264.7 cells and pM� were pretreated with nonspecificIgG or anti-CD36 Ab, then fixed on the sensor chip. The cell surface interactions of UA with immobilized RAW264.7 cells and pM� were measured usinga SPR biosensor, as described in Materials and Methods. UA was injected at a concentration of 0 or 20 �M for the indicated intervals.

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to obtain a supernatant. Each supernatant (50 �l) was subjected to ELISA, andthe amounts of IL-1� were measured according to the protocol of the kit.

MPO assay

MPO activity was measured as an index of inflammatory cell infiltration inthe colonic mucosa using a previously reported method (31), with somemodifications. The mucosal layer (�10 mg) was homogenized in 500 �l of50 mM potassium phosphate buffer containing 0.5% hexadecyltrimethyl-ammonium bromide (pH 6.0) using a homogenizer. Homogenates werefrozen in liquid nitrogen and thawed using a sonicator (EYELA). Thefreeze-thaw cycle was repeated three times, then each homogenate wascentrifuged at 20,000 � g for 30 min at 4°C to obtain a supernatant, whichwas used to measure MPO activity. MPO in the sample was activated by0.0005% H2O2 in potassium phosphate buffer solution containing 0.5 mMo-dianisidine dihydrochloride (pH 6.0). The change in absorbance at 460nm was measured using a spectrophotometer (Smart Spec; Bio-Rad) andconverted to MPO activity using the standard curve for human leukocyteMPO. MPO activity was normalized further to the total protein content ofthe supernatant, as measured with a DC protein assay. Activity is expressedas units of MPO activity per milligram of protein.

Statistical analysis

Each experiment was performed at least three times, and the data are shownas the mean � SD where applicable. Statistically significant differencesbetween groups in each assay were determined using Student’s t test(two-sided).

ResultsUA causes release of IL-1�, IL-6, and MIF protein from pM�

To investigate the effects of UA on proinflammatory cytokine re-lease into medium, pM� were treated with UA (0, 4, and 20 �M)for 0–24 h and examined with ELISA. As shown in Fig. 1, thelevels of IL-1�, IL-6, and MIF protein in pM� treated with UA (4and 20 �M) were increased in concentration- and time-dependentmanners in the medium (14- to �44-fold for IL-1�; 2.7- to �9.8-fold for IL-6; 7.4- to �28-fold for MIF from 6 to 24 h), as com-pared with the vehicle-treated cells. In contrast, TNF-� was notdetectable (�50 pg/mg) under any of the experimental condi-tions (data not shown). Although viability of the cells treatedwith UA (20 �M) for 24 h was decreased by 60%, that of theothers was maintained at �90% in each experiment (data notshown).

UA activates Raf/MEK/ERK and MKK3/p38 MAPK pathways

Subsequently, we explored the molecular mechanisms underlyingIL-1� production by UA. To determine whether UA activatesMAPK pathways, which are known to regulate the induction andproduction of proinflammatory cytokines, pM� were treated withUA (4 �M) for 0–30 min, and both the inactive and activatedforms of Raf-1, MEK1/2, ERK1/2, MKK3/6, p38 MAPK, andJNK1/2 were analyzed by Western blotting using Abs specific foreach target protein. UA strikingly induced both Raf-1 and MEK1/2activation within 2 min and that of ERK1/2 within 5 min, as com-pared with the nontreated cells (Fig. 2). Similarly, both MKK3/6and p38 MAPK were activated within 2 min, whereas phosphor-ylation of JNK1/2 was not observed. The expression levels of theinactive forms of each protein kinase remained constant.

Involvement of ERK1/2, p38 MAPK, caspase-1, and ABCtransporter in UA-induced IL-1� production

The production of a number of proinflammatory cytokines is reg-ulated by transcription, translation, and posttranslation mecha-nisms. Caspase-1 is the rate-limiting enzyme responsible for theconversion of pro-IL-1� to its active form. The effects of UA onthe levels of IL-1� mRNA, proIL-1� protein, and caspase-1 acti-vation were examined using RT-PCR, Western blotting, andELISA, respectively. As shown in Fig. 3, A and B, IL-1� mRNAand proIL-1� protein were detected in a constitutive manner at low

levels in nontreated pM�. Those treated with 4 �M UA for 3 and6 h were markedly up-regulated, whereas, intriguingly, the levelsdiminished after 12 h. Furthermore, to determine whether theERK1/2 and p38 pathways are associated with those up-regula-tions, a pharmacological approach was used using each kinase-specific inhibitor. Pretreatment with 20 �M PD98059 (a MEK1/2inhibitor) and SB203580 (a p38 MAPK inhibitor) for 30 min abol-ished the UA-induced increase in expression of IL-1� mRNA andpro-IL-1� protein (Fig. 3, A and B). In addition, PD98059 (20�M), SB203580 (20 �M), glibenclamide (ABC transporter inhib-itor, 20 �M), and Ac-YVAD-CHO (caspase-1 inhibitor, 20 �M),but not SB202474 (MAPK negative control, 20 �M), markedlyreduced the level of UA-induced IL-1� secretion (Fig. 3C).

UA-generated ROS mediate IL-1� production

Oxidative stress has been demonstrated to induce the activation ofMAPKs in many cell types; therefore, we investigated ROS gen-eration in pM� treated with UA (4 and 20 �M) for 5 min usingDCFH-DA, a fluorescent probe. The rate of ROS-positive cellswas increased in a concentration-dependent manner (31 and 43%at 4 and 20 �M, respectively) as compared with the vehicle-treated cells (Fig. 4A). Subsequently, we pretreated pM� withthe vehicle, N-acetyl-L-cysteine (NAC (an antioxidant), 1 mM),DPI (NADPH oxidase (NOX) inhibitor, 10 �M), or EDTA

FIGURE 6. Aggregated UA may be responsible for IL-1� production.Upper graph, Amounts of aggregated UA in culture medium. UA (finalconcentration 4 �M) dissolved in DMSO (0.05–1%, v/v) was added to 50ml of serum-free DMEM in a 50-ml centrifuge tube. After centrifugationat 2500 � g for 5 min, UA was extracted with chloroform from the su-pernatant and pellet. Each extract was concentrated in vacuo and dissolvedin 200 �l of chloroform. The amount of UA was quantified by HPLCanalysis using a YMC-Pack ODS-AQ column, which was eluted with 10%methanol in water at a flow rate of 1.0 ml/min and absorption wasmonitored at 210 nm. A standard curve using 0 – 8 �g of UA was pre-pared from the peak areas. The data are expressed as distribution rates,which were determined from the amount of UA in each fraction.�, dissolved UA; �, aggregated UA; p, dissolved plus aggregated UA.a, p � 0.01 vs corresponding rate of UA (0.05%, v/v) by Student’s t test.Each value is shown as the mean � SD of three replicated experiments.Lower graph, pM� from peritoneal exudates from nontreated female ICRmice were seeded onto a 96-well plate at a density of 1 � 106 cells/dish,then cultured at 37°C for 24 h under a humidified atmosphere of 5% CO2.After washing, the pM� were treated with UA (final concentration 4 �M)dissolved in DMSO (0.05–1%, v/v) for 6 h. The supernatant was collected,and the amount of IL-1� protein was determined by ELISA, as describedin Materials and Methods. �, DMSO; f, UA. a, p � 0.01 vs correspond-ing DMSO; b, p � 0.01 vs UA (0.05%, v/v) by Student’s t test. Data areshown as the mean � SD of three independent experiments.

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(Ca2� chelator, 5 mM) for 30 min, followed by exposure to UA(4 �M) for 6 h. NAC, DPI, and EDTA markedly reduced UA-induced IL-1� secretions by 52, 69, and 80%, respectively, to-gether with a dramatic suppression of ERK1/2 and p38 MAPKpathways (Fig. 4, B and C).

Involvement of CD36 in UA-induced IL-1� production

SRs, which mediate the endocytic uptake of modified forms oflow-density lipoprotein, apoptotic cells, glycated proteins, andbacteria, are expressed on monocytes/macrophages, platelets,and certain microvascular endothelium (32, 33). Recently, wefound that DSS induces IL-1� production though a SR-mediatedmechanism (K. H. Kwon, A. Murakami, and H. Ohigashi, submit-ted for publication). In this study, we examined the status of themRNA and protein expressions of CXCL16, SR-A, CD36, andCD68 in pM� with or without UA treatment. As shown in Fig. 5A,the expression of each was detected in a constitutive manner, andthe levels were not changed by treatment with UA for 6 h. Next,we examined the effects of neutralizing Abs for SRs on UA-in-duced IL-1� production. When pM� were pretreated with an anti-CD36 Ab (2.5 �g/ml) for 30 min, IL-1� production was signifi-cantly reduced by 56% as compared with the PBS-treated control,whereas nonspecific IgG and other SR Abs were inactive (Fig. 5B).In addition, when pM� from CD36-deficient mice were treatedwith UA (4 or 20 �M) for 12 h, and the levels of IL-1�, IL-6, andMIF released were markedly lower than from those of wild-type

mice (Fig. 5C). In contrast, CD36 deficiency did not have an effecton LPS-induced cytokine production. Furthermore, SPR analysisshowed that UA was bound to the cell surface of RAW264.7 cellsand pM� (Fig. 5D, nonspecific IgG), which was suppressed bytreatment with the Ab for CD36, suggesting that UA interacts withCD36 located on the cell surface of macrophages.

Aggregated UA may induce IL-1� production

The above results led us to hypothesize that UA aggregates inculture medium and is then recognized by CD36, leading to therelease of IL-1� protein, because SRs largely recognize macro-molecules. To test our hypothesis, we prepared serum-free DMEMin which the amounts of aggregated UA were changed by increas-ing the concentration of DMSO (0.05–1%). After centrifugation,UA (4 �M) distribution throughout the supernatant and pellet wasquantified by HPLC analysis (Fig. 6, upper panel). The amounts ofaggregated UA were significantly decreased (47.0317.8%) asDMSO concentration increased (0.0531.0%). Interestingly, therewas a marked decrease in UA-induced IL-1� production when thepM� were exposed to lower levels of aggregated UA (0.5 and1.0% DMSO; Fig. 6, lower panel), suggesting that aggregated UAis in an active form for IL-1� production.

Effects of UA derivatives on UA-induced IL-1� release

It is important to understand whether IL-1� inducibility is specificfor UA in light of the structural diversity of natural triterpenoids.

FIGURE 7. UA, but not its derivatives, induce IL-1� release. A, Chemical structures of UA and UA derivatives. B, upper panel, Amounts of aggregatedUA and its derivatives in culture medium. UA and its derivatives (final concentration 4 �M) were dissolved in DMSO (0.1%, v/v) and added to 50 ml ofserum-free DMEM in a 50-ml centrifuge tube. After centrifugation at 2500 � g for 5 min, UA and its derivatives were extracted with chloroform fromthe supernatant and pellet. Each extract was concentrated in vacuo and dissolved in 200 �l of chloroform. The amounts of UA and its derivatives werequantified by HPLC analysis using a YMC-Pack ODS-AQ column, which was eluted with 10% methanol in water at a flow rate of 1.0 ml/min, andabsorption was monitored at 210 nm. Standard curves using 0–8 �g of UA, and its derivatives were prepared from the peak areas. The data are expressedas the distribution rates, which were determined by the amount of UA or derivative in each fraction. �, dissolved triterpenes; f, aggregated triterpenes;p, dissolved plus aggregated triterpenes. Each value is shown as the mean � SD of three replicated experiments. Lower panel, pM� from peritonealexudates from nontreated female ICR mice were seeded onto a 96-well plate at a density of 1 � 106 cells/dish, then cultured at 37°C for 24 h under ahumidified atmosphere of 5% CO2. After washing, the pM� were treated with UA, and its derivatives (final concentration 4 �M) dissolved in DMSO (0.1%,v/v) for 6 h. The supernatant was collected, and the amount of IL-1� protein was determined by ELISA, as described in Materials and Methods. a, p �0.05; b, p � 0.005 vs 0 �M of UA (DMSO, 0.1%, v/v); c, p � 0.05 vs 4 �M UA; d, p � 0.001 vs 20 �M UA, by Student’s t test. Data are shown asthe mean � SD of three independent experiments.

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To investigate this issue, we selected six natural and synthetictriterpenoids (Fig. 7A) and investigated their properties for aggre-gation and IL-1� production in medium using HPLC analysis andELISA, respectively. As shown in the upper panel of Fig. 7B, fiveUA derivatives and glycyrrhetinic acid exhibited distribution pat-terns in the supernatant and pellets similar to UA. However, sur-prisingly, they showed no notable potential for IL-1� production(Fig. 7B, lower panel).

IL-1� production and MPO activity in UA-administrated mice

Finally, we investigated whether UA is able to stimulate pM�(Fig. 8A) and colonic mucosa (Fig. 8, B and C) when injected i.p.into female ICR mice. As shown in Fig. 8A, a 24-h incubation ofpM� from mice administrated UA (50, 100, or 200 mg/kg) once aday for 8 days led to a marked dose-dependent increase in IL-1�production (1.6-, 2.3-, and 3.3-fold, respectively) as comparedwith that of pM� from vehicle-treated mice. In parallel, the levelsof IL-1� protein and MPO activity in colonic mucosa from miceadministrated UA at 200 mg/kg were significantly increased by3.0- and 2.0-fold, respectively, as compared with that of mucosafrom vehicle-treated mice (Fig. 8, B and C).

DiscussionIn the present experiments, we found that UA markedly amplifiedABC transporter-mediated IL-1� secretion from murine pM� attranscriptional, translational, and posttranslational levels, presum-ably through binding to CD36, ROS generation, the resultant ac-tivation of both the p38 MAPK and ERK1/2 pathways, andcaspase-1 activation (Fig. 9). The action mechanisms underlyingUA-induced IL-6 and MIF protein release may be similar to thatfor IL-1� release (Fig. 1) because IL-6 was previously shown to bestrongly induced by ROS generation and the p38 MAPK, ERK1/2,

FIGURE 8. Production of IL-1� protein and MPO activity in pM� (A)and colonic mucosa (B and C) from non-, vehicle-, and UA-treated ICRmice. A, UA administration (i.p.) to ICR mice induced IL-1� production inpM�. Corn oil alone or UA suspended in 200 �l of corn oil was admin-istrated by i.p. injection to specific pathogen-free 5-wk-old female ICRmice daily at a dose of 50, 100, or 200 mg/kg body weight for 8 days.Twenty-four hours after the final administration, pM� from non-, vehicle-,and UA-treated female ICR mice were seeded onto a 96-well plate at adensity of 1 � 106 cells/dish, followed by incubation at 37°C for 24 hunder a humidified atmosphere of 5% CO2. After washing, serum-freemedium (200 �l) was added, and the cells were incubated for another 24 h,after which IL-1� production was examined by ELISA, as described inMaterials and Methods. �, p � 0.05; ��, p � 0.01 vs corn oil alone byStudent’s t test. Each value is shown as the mean � SD of six to nine mice.B, IL-1� production was increased in the mucosal layer of UA adminis-tration (i.p.) to ICR mice. Colonic mucosa samples were harvested fromnon-, vehicle-, and UA-treated ICR mice, as described in Materials andMethods, then homogenized (10 mg/300 �l) in ice-cold PBS to obtainsupernatants. The amounts of IL-1� protein were determined by ELISA, asdescribed in Materials and Methods. �, p � 0.01 vs corn oil alone byStudent’s t test. Each value is shown as the mean � SD of results from sixto seven mice. C, MPO activity was increased in the mucosal layer by UAadministration (i.p.) to ICR mice. Colonic mucosa samples were harvestedfrom non-, vehicle-, and UA-treated ICR mice, as described in Materialsand Methods, then homogenized (10 mg/500 �l) in 50 mM potassiumphosphate buffer containing 0.5% hexadecyltrimethylammonium bromideto obtain a supernatant. The MPO activities were measured, as described inMaterials and Methods. �, p � 0.01 vs corn oil alone by Student’s t test.Each value is shown as the mean � SD of results from six to seven mice.

FIGURE 9. Proposed molecular mechanisms by which UA inducesIL-1� production in murine pM�. UA, but not aggregated OA or others, isrecognized by CD36, and then ROS are intracellularly generated, presum-ably by NOX. This process triggers the activation of the Raf-1/MEK1/2/ERK1/2 and MKK3/6/p38 MAPK pathways for promoting transcription ofthe IL-1� gene, leading to IL-1� mRNA expression for intracellular pro-IL-1� production. Intracellular pro-IL-1� protein is then cleaved by ICE,and active IL-1� is released via an ABC1 transporter-dependent pathwayand exhibits its biological functions. The anti-CD36 Ab, NOX inhibitorDPI, antioxidant NAC, MEK1/2 inhibitor PD98059, p38 MAPK inhibitorSB203580, caspase-1 inhibitor YVAD-CHO, and ABC transporter inhib-itor glibenclamide each have potential to block or attenuate these UA-induced molecular events.

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and NF-�B-signaling pathways (34, 35), and MIF production wasinduced by ROS-activated ERK1/2 (36).

SRs, including SR-A, CD36, and CD68, have been reported torecognize negative-charged, high-m.w. substances, such as oxi-dized low-density lipoprotein (oxLDL), DSS, and amyloid � (33,37, 38). Nishimura et al. (39) showed that oxLDL binding to SRsmay generate intracellular ROS production through NOX activa-tion, thereby activating NF-�B. CD36, originally identified as gly-coprotein IV on platelets, is an integral membrane protein that hasmultiple ligands, including oxLDL, apoptotic cells, and long-chainfatty acids (40), and is expressed on monocytes/macrophages (41),platelets (42), and certain types of microvascular endothelia (43).In this study, the levels of CD36 mRNA and protein, both of whichwere expressed in a constitutive manner, did not change followingUA treatment (Fig. 5A), while a previous study found that ligand-binding induced oxidative stress up-regulated SR gene expression,thereby promoting the uptake of oxLDL (44).

SPR biosensors have been increasingly used for real-time anal-ysis of the binding between solubilized molecules and moleculesimmobilized on the surface of a biosensor chip without any label-ing based on changes in the refractive index of the biospecificsurface (45). Our analysis with the biosensor indicated that UAbound to pM� and that binding was inhibited by treatment with theanti-CD36 Ab (Fig. 5D). Furthermore, the production of IL-1�was suppressed by treatment with that Ab (Fig. 5B) and decreasedin pM� of CD36-deficient mice, as compared with wild-type mice(Fig. 5C). To the best of our knowledge, this is the first reportshowing that CD36 is one of the membrane receptors for a triter-penoid, while it was previously reported that UA binds to the hy-drophobic region of the dimeric interface of TGF-�1 (46). How-ever, it remains to be determined whether UA also binds with otherSRs or proteins because the anti-CD36 Ab and CD36 deficiencydid not abolish IL-1� protein release and UA binding (Fig. 5,B–D). Nevertheless, CD36 also partially mediates UA-, but notLPS-, induced IL-6 and MIF production (Fig. 5C).

Because UA is a low-m.w. substance, it is unlikely that it isrecognized by SRs, as they generally recognize anionic high-m.w.substances, as noted above. However, based on the hydrophobicityof this compound, it is reasonable to assume that some portions ofUA aggregates present in culture medium are recognized by CD36.In fact, 18–33% of the triterpenoids, at a concentration of 4 �M,were revealed to be aggregated in culture medium containing 0.1%DMSO (Fig. 7), which has been widely used as a vehicle in anumber of reports. Interestingly, there was a positive correlationbetween the aggregated UA concentration and IL-1� production(Fig. 6). Furthermore, the ability for inducing IL-1� was seen onlywith UA and not the other six triterpenoids, all of which demon-strated medium solubility similar to that of UA (Fig. 7). At present,there are no reasonable explanations for these puzzling outcomes;however, we speculate that aggregated UA, but not other triterpe-noids, may have a particular structure in which its anionic moietymay be revealed for CD36 recognition. We hope to address thisissue in the near future. Our results with the SPR biosensor dem-onstrated that UA binds to CD36 on not only pM� but alsoRAW264.7 macrophages (Fig. 5D). Recently, we reported that UApromoted MIF release via ERK2 activation in nonstimulatedRAW264.7 cells (28), whereas You et al. (27) presented similarfindings and noted that UA induced iNOS and TNF-� expressionvia NF-�B activation in the same cell lines. Because UA binds toRAW264.7 cells, in which CD36 is expressed in a constitutivemanner (data not shown), the effects of UA shown in our experi-ments with RAW264.7 cells may also be mediated by binding tothis SR.

The predominant sources of ROS in sites of inflammation arephagocytes, including neutrophils, monocytes, and macrophages,with NOX responsible for O2

� generation (47). That enzyme isdormant in resident cells but becomes activated to generate ROSupon exposure to bacteria, chemical stimuli, or calcium influxthough a receptor-regulated channel (48). The present findingsdemonstrated that UA induced ROS generation for activatingERK1/2 and p38 MAPK and the resultant release of IL-1� (Figs.2 and 4). Of note, blockade of UA-induced ROS by an NOX in-hibitor, NAC (antioxidant), and EDTA (calcium chelator) resultedin a decrease in ERK1/2 and p38 MAPK activities along withIL-1� production (Fig. 4). Conversely, Schweyer et al. (49) pro-vided experimental evidence that both MEK1/2 and ERK1/2 acti-vation was mediated by ROS, and Fubini et al. (50) showed thatsilica-induced generation of ROS induced ERK1/2 activation andincreased the expression of IL-1� in cell culture models.

MAPKs control a number of cellular events, including differ-entiation, proliferation, and death (51). In this study, we used spe-cific inhibitors to identify which MAPKs are involved in IL-1�release. Our data obtained with Western blotting (Fig. 2) and phar-macological blockades (Fig. 3) strongly suggest that UA inducessecretion of IL-1� via the ERK1/2 and p38 MAPK pathways butnot that of JNK1/2. In support of these findings, both the ERK1/2and p38 MAPK pathways have been reported necessary for opti-mal cytokine gene expression in LPS-stimulated monocytes andmacrophages (52). Similarly, Hsu et al. (53) reported that LPS-induced ROS generation activated the ERK1/2 and p38 MAPKpathways and also regulated the release of IL-1� in mouse mac-rophages. The rate of inhibition of IL-1� production by the specificinhibitors of MEK1/2 and p38 MAPK in the present study rangedfrom 43 to 67% (Fig. 3C), whereas the expressions of IL-1�mRNA and protein were abolished by these inhibitors (Fig. 3A),suggesting that MAPKs do not contribute to the posttranslationalmechanism. Transcriptional factors, such as NF-�B, activator pro-tein-1, cAMP-response element-binding protein, and NF-IL6, areknown to be involved in the transcriptional regulation of IL-1�expression (54–56). Previous reports have also shown thatMAPK-mediated signal pathways contribute, in part, to the tran-scriptional activities of those factors (57–60). Thus, the abovementioned transcription factors may be crucial for UA-inducedIL-1� expression.

Some cytokines such as TNF-� and IL-6 have a secretory signalpeptide, which directs these proteins into the classical endoplasmicreticulum-to-Golgi secretory pathway for rapid secretion after syn-thesis (8, 61, 62). In contrast, IL-1� is a unique cytokine becauseit is not secreted via the classical exocytic pathway (63), and thelack of a secretory signal peptide hampers its targeting of theendoplasmic reticulum. ABC transporters are known to trans-port leaderless secretory protein (64). In this study, pretreat-ment with glibenclamide, an ABC transporter inhibitor, blockedUA-induced IL-1� release. This result is consistent with pre-vious reports by Zhou et al. (11) and Hamon et al. (12) whoshowed that the ABC transporter contributes to the secretion ofIL-1� from macrophages (11, 12).

Increasing evidence suggests that certain phytochemicals havemarked anti-inflammatory and cancer chemopreventive properties(65, 66). UA is one such compound that has attracted considerableinterest, based on its remarkable biological functions (17, 23, 24).For example, our group previously showed that UA has an inhib-itory effect on 12-O-tetradecanoylphorbol-13-acetate-induced skintumor promotion in mice (20). In addition, the compound attenu-ated LPS- or IFN-�-induced iNOS and cyclooxygenase-2 expres-sion through NF-�B abrogation in macrophages (26). In contrast,as noted above, UA-induced MIF release via activation of ERK2

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and iNOS and TNF-� expression via activation of NF-�B in restingmacrophages (27, 28). More recently, we reported that topical ap-plication of UA significantly increased proinflammatory gene ex-pression in mouse skin (67). Those findings along with the presentresults led us to hypothesize that the pro- and anti-inflammatoryeffects of UA are dependent on the biological status of cells andtissues.

In conclusion, our results showed that aggregated UA enhancedIL-1� secretion at transcriptional, translational, and posttransla-tional levels via its binding with CD36 for generating ROS,thereby activating the ERK1/2 and p38 MAPK pathways, andcaspase-1, and releasing IL-1� protein via the ABC transporter inresident murine pM� (Fig. 9). In addition, UA also dramaticallyinduced IL-1� secretion and MPO activity in our in vivo model(Fig. 8). Although UA has long been known as an effective anti-inflammatory and anticarcinogenic agent in vivo by its abilities tocounteract endogenous and exogenous stimuli, further extensiveevaluations regarding the effects of UA on nontreated tissues arenecessary to determine the risks and benefits of this triterpenoid.

DisclosuresThe authors have no financial conflict of interest.

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