CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined...

CB2 cannabinoid receptor agonist JWH-015 modulates human monocytemigration through defined intracellular signaling pathways

Fabrizio Montecucco, Fabienne Burger, Francois Mach, and Sabine SteffensDivision of Cardiology, Foundation for Medical Researches, University Hospital, Geneva, Switzerland

Submitted 12 November 2007; accepted in final form 2 January 2008

Montecucco F, Burger F, Mach F, Steffens S. CB2 cannabinoidreceptor agonist JWH-015 modulates human monocyte migrationthrough defined intracellular signaling pathways. Am J Physiol HeartCirc Physiol 294: H1145–H1155, 2008. First published January 4,2008; doi:10.1152/ajpheart.01328.2007.—Recruitment of leukocytesto inflammatory sites is crucial in the pathogenesis of chronic inflam-matory diseases. The aim of this study was to investigate if activationof CB2 cannabinoid receptors would modulate the chemotactic re-sponse of human monocytes. Human monocytes treated with the CB2

agonist JWH-015 for 12–18 h showed significantly reduced migrationto chemokines CCL2 and CCL3, associated with reduced mRNA andsurface expression of their receptors CCR2 and CCR1. The inductionof ICAM-1 in response to IFN-� was inhibited by JWH-015. More-over, JWH-015 cross-desensitized human monocytes for migration inresponse to CCL2 and CCL3 by its own chemoattractant properties.The CB2-selective antagonist SR-144528, but not the CB1 antagonistSR-147778, reversed JWH-015-induced actions, whereas the CB2

agonist JWH-133 mimicked the effects of JWH-015. The investiga-tion of underlying pathways revealed the involvement of phosphati-dylinositol 3-kinase/Akt and ERK1/2 but not p38 MAPK. In conclu-sion, selective activation of CB2 receptors modulates chemotaxis ofhuman monocytes, which might have crucial effects in chronic in-flammatory disorders such as atherosclerosis or rheumatoid arthritis.

chemokines; chemokine receptors; intracellular adhesion molecule-1;chronic inflammation; atherosclerosis

CANNABINOIDS modulate immune functions and therefore have atherapeutic potential for the treatment of inflammatory diseases(28, 29, 40). It is thought that the immunomodulatory effects ofcannabinoids are mediated by the CB2 cannabinoid receptorexpressed on immune cells. A growing body of evidencesuggests that endocannabinoid signaling plays a critical role inpathophysiological conditions such as hemorrhagic or endo-toxic shock (53, 56), liver cirrhosis (4), fibrosis (24, 52),ischemia-reperfusion (I/R) injury (5, 44), acute heart failure(36), osteoporosis (25, 39, 51), and cutaneous contact hyper-sensitivity (26). We (49) have recently provided the firstexperimental evidence for a possible role of CB2 receptors inatherosclerosis progression.

The recruitment of inflammatory cells into the intima iscrucial for the development and progression of atherosclerosis(19, 20, 57). The tethering, rolling, adhesion, and transendo-thelial migration of leukocytes are triggered by the localproduction of chemokines and chemokine receptors as well asadhesion molecules (8). Cannabinoids have been reported tomodulate the migration of various cell types. Thus, it has beenreported that treatment of rat macrophages with the synthetic

cannabinoid CP-55,940 reduced both spontaneous and formyl-metionyl-leucine-phenylalanine-induced chemotaxis (48). Thenonpsychoactive marijuana component cannabidiol has beenshown to inhibit murine macrophage chemotaxis in vitro and invivo in a CB2 receptor-dependent manner (47). Both endoge-nous as well as synthetic agonists 2-AG, CP-55,940 andWIN-55,212-2, as well as the CB2-selective agonists JWH-015and JWH-133, caused a significant inhibition of the chemokinestromal cell-derived factor-1/CXCL12-induced and CXCR4-mediated chemotaxis of Jurkat or primary human T cells (11,15). In addition, in an experimental autoimmune encephalomy-elitis mouse model, WIN-55,212-2 attenuated leukocyte rollingand adhesion on endothelial cells through the activation of CB2

receptors (38). Furthermore, the novel CB2-selective inverse ag-onist Sch.336 potently inhibited leukocyte chemotaxis to 2-AG,HU-210, or monocyte chemoattractant protein-1/CCL2 in vitroand in vivo (33). In a mouse model of liver I/R injury, theCB2-selective agonist JWH-133 protected against I/R damage bydecreasing inflammatory cell infiltration, tissue and serum TNF-�,macrophage inflammatory protein (MIP)-1�/CCL3 and MIP-2/CXCL2 levels, and expression of adhesion molecule ICAM-1 (5).In vitro, JWH-133 has been shown to attenuate TNF-�-inducedICAM-1 and VCAM-1 expression in human liver sinusoidalendothelial cells (HLSECs) and the adhesion of human neutro-phils to HLSECs. In a different study, anandamide dose depen-dently attenuated TNF-�-induced ICAM-1 and VCAM-1 expres-sion in human coronary artery endothelial cells (HCAECs) andthe adhesion of THP-1 monocytes to HCAECs in a CB1- andCB2-dependent manner (6). The role of the CB2 receptor in thisprocess could be confirmed by additional data obtained with CB2

agonists, which reduced ICAM-1 and VCAM-1 expression inHCAECs as well as the adhesion and transendothelial migrationof THP-1 monocytes (43). Finally, we (49) have shown thattetrahydrocannabinol inhibited murine peritoneal macrophagechemotaxis in response to CCL2 and reduced expression of thechemokine receptor CCR2 on splenocytes. These effects wereblocked by the CB2 receptor antagonist SR-144528 or when cellsfrom CB2 knockout mice were used.

In this study, we investigated if CB2-selective agonistswould modulate the migration of human monocytes and thusmay have a therapeutic potential in inflammatory conditionssuch as atherosclerosis.

MATERIALS AND METHODS

Reagents. The CB2-selective agonist JWH-015 was obtained fromCayman Chemical and JWH-133 was obtained from Tocris Bio-science. The CB1 antagonist SR-147778 and the CB2 antagonist

Address for reprint requests and other correspondence: S. Steffens, Div. ofCardiology, Dept. of Internal Medicine, Geneva Univ. Hospital, Foundationfor Medical Researches, 64 Avenue Roseraie, 1211 Geneva, Switzerland(e-mail: Sabine.Steffens@medecine.unige.ch).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Heart Circ Physiol 294: H1145–H1155, 2008.First published January 4, 2008; doi:10.1152/ajpheart.01328.2007.

0363-6135/08 $8.00 Copyright © 2008 the American Physiological Societyhttp://www.ajpheart.org H1145

SR-144528 were kindly provided from Sanofi-Synthelabo (France)(45, 46). Stock solutions (10–20 mM) of cannabinoids and antago-nists were prepared in DMSO. Appropriate dilutions of DMSO wereadded as vehicle controls in all experiments. BSA was from Sigma-Aldrich. CCL2, CCL3, IFN-�, anti-phosphorylated ERK1/2 (T202/Y204, AF1018) polyclonal antibody (Ab), anti-phosphorylated p38MAPK (T180/Y182, AF869) polyclonal Ab, anti-ERK1/2 polyclonalAb (AF1576), phycoerythrin (PE)-conjugated anti-human CCR1 Ab(FAB145P), and PE-conjugated anti-human CCR2 Ab (FAB151P)were all purchased from R&D Systems Europe. PE-conjugated anti-human CD54/ICAM-1 Ab (no. 555511) and FITC-conjugated anti-human CD14 Ab (no. 555397) were obtained from BD Pharmingen.Anti-human CB2 receptor Ab was obtained from Abcam (ab3561) andPE-conjugated goat anti-rabbit IgG antibody was obtained fromMolecular Probes. The kinase inhibitor LY-294002 [phosphatidylino-sitol 3-kinase (PI3K) inhibitor] was obtained from Sigma-Aldrich, andPD-98059 (MEK inhibitor) and SB-203580 (p38 MAPK inhibitor)were obtained from BioMol Research Laboratories. Anti-p38 (H-147,sc-7149) polyclonal Ab, anti-Akt1/2/3 (H-136, sc-8312) polyclonalAb, and anti-phosphorylated Akt1/2/3 (Thr308, sc-16646-R) poly-clonal Ab were obtained from Santa Cruz Biotechnology.

Isolation of human monocytes. Human monocytes were isolatedfrom buffy coats of healthy volunteers under a protocol approved by thelocal Ethics Committee. All donors provided written, informed consent tothe procedure and use of the cells. After centrifugation on a Ficoll-Hypaque density gradient, mononuclear cells were collected from theinterface and washed with PBS. Monocytes were then purified from theupper interface of a hypotonic Percoll density gradient (1.129 g/ml).Purified monocytes were resuspended in RPMI 1640 with 25 mMHEPES and 500 ng/ml polymyxin B. The purity of monocytes wasdetermined by flow cytometric analysis (CD14 staining), confirming thatat least 85% purity was achieved in all experiments.

Modified Boyden chamber migration assays and checkerboardanalysis. To study the effect of cannabinoid treatment on migration,cells were incubated in RPMI medium containing 25 mM HEPES and

500 ng/ml polymyxin B in the presence or absence of different dosesof JWH-015 (5, 10, and 20 �M) for 12 h at 37°C in a humidifiedatmosphere with 5% CO2. Cells were then washed three times in PBSand resuspended in chemotaxis medium (RPMI medium containing25 mM HEPES and 1% BSA) to test their locomotory responses tomedium alone, 10 nM CCL2, or 10 nM CCL3. Monocyte chemotaxiswas assessed in a 48-well microchemotaxis chamber (NeuroProbe)using a 5-�m pore size, 5-�m-thick polyvinylpyrrolidone-free poly-carbonate filter (NeuroProbe). Cells were seeded in the upper wellswhile medium or chemoattractant solutions were added to the lowerwells.

To test the chemotactic properties of CB2-selective cannabinoids,untreated monocytes were tested for their ability to migrate in re-sponse to medium alone, JWH-015 (5, 10, and 20 �M), or JWH-133(10 �M). In some experiments, cells were pretreated for 10 min withthe receptor antagonists SR-147778 (1 �M) or SR-144528 (1 �M) orfor 60 min with the kinase inhibitors LY-294002 (50 �M), PD-98059(50 �M), or SB-203580 (1 �M). After treatment with the kinaseinhibitors, cells were washed three times in chemotaxis mediumbefore their locomotory abilities were tested. In a different set ofexperiments, cells and different doses of JWH-015 (5, 10, and 20 �M)were seeded in the upper well while medium alone or 10 nM CCL2 or10 nM CCL3 were added to the lower well. Checkerboard analysiswas performed by the addition of different doses of JWH-015 (0, 5,10, and 20 �M) in both the upper (with cells) and lower wells.

Under all conditions, the modified Boyden chamber was incubatedfor 60 min at 37°C in a humidified atmosphere with 5% CO2. Filterswere then removed from the chambers and stained with Diff-Quick(Baxter). Each condition was performed in duplicate. Cells of fiverandom oil-immersion fields were counted at �1,000 magnification(by a blinded observer), and the chemotaxis index was calculated fromthe number of cells migrated to the test sample divided by the numberof cells migrated to the medium.

Flow cytometry. Monocytes were cultured at a concentration of 5 �106 cells/ml in serum-free RPMI medium containing 25 mM HEPES

Fig. 1. CB2 cannabinoid receptor expression inprimary human monocytes. A: representativehistogram of flow cytometric analysis, demon-strating high CB2 surface expression on mono-cytes with (solid line) or without CD32 block-ade (dashed line) versus the isotype control(shaded peak). B: representative RT-PCR re-sults for CB2 mRNA expression of 2 differentmonocyte donors (donors 1 and 2). No PCRproduct was detected in the control (ctrl) with-out reverse transcriptase (no RT control), con-firming that the amplification product wascDNA specific. C: quantification of CB2 surfaceexpression (flow cytometric analysis) on humanmonocytes cultured for the indicated timepoints in the presence or absence of 20 �MJWH-015 (n � 4). *P � 0.05 for medium alone(w/o) at 24 h vs. time 0 or 18 h; #P � 0.05 forJWH-015 at 12 h vs. time 0; ##P � 0.05 forJWH-015 at 24h vs. time 0 and 18 h.

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and 500 ng/ml polymyxin B in the presence or in absence of JWH-015(5, 10, and 20 �M) or JWH-133 (10 �M) with or without receptorantagonists SR-147778 (1 �M) and SR-144528 (1 �M) or kinaseinhibitors LY-294002 (50 �M), PD-98059 (50 �M), and SB-203580(1 �M) for 18 or 24 h at 37°C in a humidified atmosphere with 5%CO2. In some experiments, cells were stimulated with IFN-� (100U/ml). FITC- or PE-labeled anti-human CCR1, CCR2, CD54, andCD14 Ab as well as corresponding isotype controls were used.Surface CB2 receptor staining was performed by Fc blocking withhuman IgG and a further incubation with a rabbit polyclonal antibodyto the CB2 receptor followed by an incubation with PE-conjugatedgoat anti-rabbit IgG Ab. CellQuest software was used for acquisitionand analysis on a FACSCalibur (BD Biosciences). Data are expressedas mean fluorescence intensities (MFIs) compared with baselineexpression (defined as 100%).

Cytotoxicity assay. Cell death was determined by the quantificationof lactate dehydrogenase (LDH) release (BioVision) in cell culturesupernatants after 12, 18, and 24 h of incubation in the presence orabsence of JWH-015 (20 �M), JWH-133 (10 �M), SR-147778 (1�M), or SR-144528 (1 �M) and counts of trypan blue-positive cells.

Apoptosis assay. Apoptosis rates of monocytes after 0, 18, and 24 hof treatment with JWH-015 (20 �M), JWH-133 (10 �M), SR-147778(1 �M), or SR-144528 (1 �M) were determined via the analysis ofphosphatidylserine externalization using an annexin V-FITC apopto-sis detection kit (MBL).

Immunoblot analysis. Monocytes were cultured at a concentrationof 5 � 106 cells/ml in serum-free RPMI medium containing 25 mMHEPES in the presence or absence of JWH-015 (20 �M), 10 nMCCL2, or DMSO [0.1% for various time points (between 2.5 and 30min)]. The reaction was stopped on ice, and cells were centrifuged at4°C to remove culture supernatants. Total protein was extracted inlysis buffer containing 20 mM Tris�HCl (pH 7.5), 150 mM NaCl, 10mM NaF, 1% Nonidet P-40, 10% glycerol, 1 mM PMSF, 10 �g/mlaprotinin, 10 �g/ml leupeptin, and 0.5 mM Na3VO4. Proteins (40 �g)were electrophoresed through polyacrylamide-SDS gels and trans-ferred by electroblotting onto nitrocellulose membranes. Membraneswere blocked for 1 h in 5% (wt/vol) nonfat milk before beingincubated with appropriate dilutions of primary phospho-specific Abto ERK1/2, Akt, or p38 MAPK as well as corresponding secondaryAb. Blots were developed using the ECL system (Immobilion, West-

Fig. 2. Analysis of cell viability and theapoptosis rate in cultured human monocytes.Monocytes were cultured in serum-free me-dium with or without 0.1% DMSO [vehicle(v)], 20 �M JWH-015, 10 �M JWH-133, theCB1 antagonist (aCB1) 1 �M SR-147778, orthe CB2 antagonist (aCB2) 1 �M SR-144528for the indicated time points. A and B: celldeath was determined as the percentage oftrypan blue-positive cells in a total of 500counted cells (n � 2). C and D: the apoptosisrate was determined by flow cytometric anal-ysis of annexin V-FITC and propidium iodide(PI) staining. Early apoptotic cells areannexin V single-positive cells, whereas lateapoptotic/necrotic cells are annexin V/PIdouble-positive cells (n � 4). E: representa-tive histograms of flow cytometric analysis ofannexin V staining after 12 h (cells weregated to exclude PI-positive cells). CTL, con-trol.

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ern, Millipore). Membranes were then stripped, reblocked, and re-probed to detect total ERK1/2, Akt, or p38 MAPK. Immunoblots werescanned, and quantification was carried out by Image Quant softwareversion 3.3 (Molecular Dynamics). Values were normalized to totalamounts of ERK1/2, Akt, or p38, respectively, and expressed aspercentages of medium control (defined as 100%).

RT-PCR. Total RNA from 5 � 106 human monocytes was ex-tracted using TRI Reagent (Molecular Research Center). RT and PCRwere carried out with the OneStep RT-PCR Kit (Qiagen) using thefollowing primer pairs for the human CB2 receptor as previouslydescribed (34): forward 5�-CGCCGGAAGCCCTCATACC-3� andreverse 5�-CCTCATTCGGGCCATTCCTG-3� (PCR product of 522bp). Amplification of GAPDH was used as an endogenous controlusing forward primer 5�-ACCACAGTCCATGCCATCAC-3� and re-verse primer 5�-TCCACCACCCTGTTGCTGTA-3� (PCR product of452 bp).

Real-time RT-PCR. Monocytes were cultured at the concentrationof 5 � 106 cells/ml in serum-free RPMI medium containing 25 mMHEPES and 500 ng/ml polymyxin B in the presence or absence ofJWH-015 (5, 10, and 20 �M) for 12 h at 37°C in a humidifiedatmosphere with 5% CO2. Total RNA was extracted using TRIReagent (MRC) and reverse transcribed using the Quantitect kit(Qiagen) according to the manufacturer’s instructions. Real-time PCRwas performed with the ABI Prism 7000 Sequence Detection System(Applied Biosystems, Foster City, CA). Each measurement was done

in triplicate using hypoxanthine guanine phosphoribosyl transferase(HPRT) as an endogenous control (run in separate tubes on the sameplate), and relative quantification was performed with the comparativemethod. The following human CCR2 primers and probe were de-signed with Primer Express software (Applied Biosystems): forward5�-GCGTTTAATCACATTCGAGTGTTT-3�, reverse 5�-CCACTG-GCAAATTAGGGAACAA-3�, and probe 5�-FAM-AGTGCTTCG-CAGATGTCCTTGATGCTC-TAMRA-3�. We used human CCR1and HPRT primers and probes as previously described (23, 32).

Statistical analysis. All data are expressed as means � SE. One-way ANOVA with Bonferroni’s post test was performed using Graph-Pad InStat version 3.05 (GraphPad Software, San Diego, CA). Dif-ferences between P values of �0.05 were considered significant.

RESULTS

CB2 cannabinoid receptors are expressed on primary humanmonocytes. To obtain nonactivated primary human monocytes,we performed Ficoll-Percoll density gradient centrifugation.We first confirmed the presence of CB2 cannabinoid receptorson freshly isolated monocytes and found high expression levelson the cell surface (Fig. 1A). In addition, CB2 mRNA expres-sion in monocytes was detected by RT-PCR (Fig. 1B). Inter-estingly, we observed a significant increase of CB2 surface

Fig. 3. Pretreatment with JWH-015 reducesmonocyte migration to CCL2 and CCL3 viainhibition of CCR1 and CCR2 expression in aCB2-dependent manner. A: monocytes weretreated with JWH-015 for 12 h, washed, andtested for their ability to migrate to 10 nMCCL2 or CCL3 (n � 6). **P � 0.01 and***P � 0.001 vs. CCL2 alone; #P � 0.05 and##P � 0.01 vs. CCL3 alone. B: FACS analysisof CCR2 and CCR1 expression on monocytesafter 18 h of JWH-015 treatment. **P � 0.01and ***P � 0.001 for CCR2 vs. untreated(n � 3); #P � 0.05 for CCR1 vs. untreated(n � 4). C: real-time RT-PCR analysis ofCCR2 and CCR1 mRNA levels (normalized tothe hypoxanthine guanine phosphoribosyltransferase control) in monocytes after 12 h ofJWH-015 treatment. ***P � 0.001 for CCR2vs. untreated (n � 5); CCR1 was not signifi-cant (n � 4). D: FACS analysis after 18 h withor without 20 �M JWH-015 alone (n � 11) orin the presence of aCB1 SR-147778 (1 �M) oraCB2 SR-144528 (1 �M) (n � 9 for antago-nists JWH-015 and n � 2 for antagonistsalone), or the CB2 agonist JWH-133 (10 �M)alone (n � 5). ***P � 0.001 vs. untreated;‡P � 0.001 vs. JWH-015; #P � 0.05 vs.untreated; ##P � 0.01 vs. JWH-015. E andF: representative histograms of CCR2 andCCR1 expression of untreated cells (shadedpeaks) or treated with 20 �M JWH-015(solid lines).

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expression when monocytes were cultured for up to 24 h (Fig.1C). The presence of CB2 agonist JWH-015 (20 �M) did notchange the observed modulation. To validate our experimentalconditions, we tested the cell viability and extent of apoptosisin monocytes treated with cannabinoids or antagonists for up to24 h. We found an increasing percentage of dead and apoptoticcells in monocyte cultures (Fig. 2), comparable with magni-tudes previously reported (21, 37). The increase of cell deathwas due to the culture conditions in serum-free medium, whichwas performed to avoid the interference with serum factors.Importantly, treatment with cannabinoids and/or antagonistsdid not affect cell viability and apoptosis rates (Fig. 2). Undernone of the experimental conditions did we observe increasedLDH release compared with untreated cells after 12, 18, and24 h of incubation (data not shown).

Treatment with the CB2 agonist JWH-015 reduces monocytemigration to CCL2 and CCL3 by reducing chemokine receptorexpression. We assessed monocyte migration in response toCCL2 and CCL3 with the modified Boyden chamber system.Pretreatment with 5–20 �M of the CB2 agonist JWH-015 for12 h significantly reduced the chemotactic response to CCL2and CCL3 in a dose-dependent manner (Fig. 3A). Maximuminhibition was observed at 10–20 �M JWH-015. We nextanalyzed the effect of JWH-015 on the expression of thechemokine receptors that bind CCL2 and CCL3. Treatment ofmonocytes with JWH-015 for 18 h inhibited surface expression

of chemokine receptor CCR2 and, to a lesser extend, CCR1 ata concentration of 10–20 �M (Fig. 3, B, E, and F). This effectwas associated with significantly reduced CCR2 mRNA levels,as determined after 12 h of JWH-015 treatment (Fig. 3C). We alsoobserved a marked, but not significant, reduction of CCR1 mRNAlevels. Pretreatment with the CB2 antagonist SR-144528 reversedthe effect of JWH-015 on surface CCR2 and CCR1 expression,whereas the CB1 antagonist SR-147778 had no effect (Fig.3D). It is of note that cotreatment of cells with JWH-015 andthe CB2 antagonist SR-144528 induced a marked, but notsignificant, increase of chemokine receptor expression com-pared with untreated cells. Treatment with the CB1 antagonistSR-147778 or the CB2 antagonist SR-144528 alone did nothave a significant effect. Similar to JWH-015, incubation withthe CB2 agonist JWH-133 at 10 �M also reduced CCR2 andCCR1 surface expression (Fig. 3D), whereas the vehicleDMSO alone did not affect mRNA or surface expression ofchemokine receptors (data not shown).

Treatment with JWH-015 modulates ICAM-1 expression onhuman monocytes. We further investigated the effect of JWH-015 on the expression of ICAM-1, which is implicated inmonocyte recruitment. The basal expression of ICAM-1, whichwas strongly expressed on all monocytes, was significantly(1.44-fold) enhanced after 24 h of incubation with IFN-�(Fig. 4, A and B). When JWH-015 (20 �M) or JWH-133 (10 �M)was added during stimulation, the IFN-�-induced activation

Fig. 4. A: JWH-015 inhibits IFN-�-induced ICAM-1 ex-pression on monocytes. Monocytes were stimulated with100 U/ml IFN-� for 24 h alone or in the presence of 20 �MJWH-015 or 10 �M JWH-133 with or without aCB1 SR-147778 (1 �M) or aCB2 SR-144528 (1 �M) (n � 2 foraCB1 and aCB2 alone, n � 4 for aCB1 and aCB2 IFN-�with or without JWH-015, and n � 6 for all other condi-tions). *P � 0.05; ***P � 0.001. B: representative histo-gram of ICAM-1 expression (flow cytometric analysis) ofunstimulated cells (shaded peak) and cells stimulated withIFN-� alone (solid line) or in the presence of 20 �MJWH-015 (dashed line).

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was significantly inhibited. The CB2 antagonist SR-144528reversed the effect of JWH-015, whereas the CB1 antagonistSR-147778 did not inhibit the effect of JWH-015. Coincuba-tion with JWH-015 and the CB2 antagonist SR-144528 induceda marked, but not significant, increase of IFN-�-inducedICAM-1 expression compared with IFN-�-treated cells. Simi-larly, the antagonists alone had a marked, but not significant,effect on the IFN-�-induced activation of ICAM-1. Neithertreatment with JWH-015 nor receptor antagonists alone re-duced the basal ICAM-1 expression on monocytes (Fig. 4A anddata not shown).

JWH-015 induces chemotaxis of human monocytes. Becausevarious cannabinoids have been reported to chemoattract dif-ferent cell types, we next tested the ability of JWH-015 andJWH-133 to induce migration of freshly isolated monocytes.The number of cells migrating to the respective cannabinoidwas increased compared with the medium control, with asignificant effect observed at 20 �M JWH-015 (chemotaxisindex of 1.98 � 0.26) or 10 �M JWH-133, respectively(chemotaxis index of 1.98 � 0.2; Fig. 5A). Pretreatment withthe CB2 antagonist SR-144528 inhibited the effect of JWH-015and JWH-133, whereas the CB1 antagonist SR-147778 had noeffect (Fig. 5B).

To distinguish if the migration was due to the induction ofchemotaxis or chemokinesis, we performed a checkerboardanalysis with increasing concentrations of JWH-015 above andbelow the filter. We found a directed migration versus agradient across the filter, indicating that JWH-015 inducedchemotaxis rather than random chemokinesis (Fig. 5C).

JWH-015 cross-desensitizes monocytes for migration toCCL2 and CCL3. To investigate the chemotactic effect ofJWH-015 in the presence of a chemokine gradient, freshly

isolated monocytes were seeded with increasing concentrationsof JWH-015 to the upper well and chemoattracted with CCL2or CCL3, respectively. We found that the migration to bothCCL2 and CCL3 was significantly reduced in the presence of10–20 �M JWH-015, suggesting that JWH-015 cross-desen-sitizes monocytes for migration to these chemokines (Fig. 5D).

JWH-015-mediated modulation of monocyte migration in-volves PI3K/Akt and ERK1/2 but not p38 MAPK signalingpathways. To investigate if the JWH-015-mediated effects onchemokine receptor expression and migration were dependenton distinct kinase signaling pathways, we performed experi-ments with selective kinase inhibitors for PI3K and MEK1/2(the activator of ERK1/2) as well as p38 MAPK. Pretreatmentwith the PI3K inhibitor LY-2940002 or the MEK1/2 inhibitorPD-98059 abolished the inhibition of CCR2 expression inresponse to JWH-015 (Fig. 6A). Conversely, pretreatment withthe p38 MAPK inhibitor SB-203580 had no effect. Similareffects of kinase inhibitors were observed on CCR1 expres-sion; however, the differences were not significant. To study ifJWH-015-induced migration was dependent on the samedownstream pathways as the above-described effects on che-mokine receptor expression, we performed migration assaysafter pretreatment with kinase inhibitors. We found that boththe PI3K inhibitor LY-2940002 and the MEK1/2 inhibitorPD-98059 inhibited the chemotactic response to JWH-015,whereas the p38 MAPK inhibitor SB-203580 had no effect(Fig. 6B). To confirm these findings, we determined the effectof JWH-015 on kinase activation by Western blot analysis(Fig. 6, C and D). CCL2 was used as a positive control forkinase activation in parallel experiments. We found increasedphosphorylation of the direct downstream kinases of PI3K andMEK1/2, namely, Akt and ERK1/2. However, we also deter-

Fig. 5. JWH-015 and JWH-133 induce chemo-taxis of human monocytes in a CB2-dependentmanner. A: untreated monocytes were testedfor their ability to migrate to increasing con-centrations of JWH-015 (n � 6) or JWH-133(n � 3). **P � 0.01 and *P � 0.05 vs. withoutJWH-015. B: migration versus 20 �M JWH-015 or 10 �M JWH-133 in the presence ofaCB1 SR-147778 (1 �M) or aCB2 SR-144528(1 �M) (n � 8 for JWH-015 and n � 4 forJWH-133). ***P � 0.001 vs. JWH-015;**P � 0.01 vs. JWH-133. C: checkerboardanalysis showing that monocyte migration de-pended on a JWH-015 gradient across the fil-ter. Untreated monocytes were seeded withincreasing concentrations of JWH-015 to theupper well, and increasing concentrations ofJWH-015 were added to the lower well. Barsrepresent means; n � 10. D: JWH-015 desen-sitizes monocytes for migration to CCL2 andCCL3 gradients. Untreated monocytes wereseeded with increasing concentrations of JWH-015 to the upper well, and 10 nM CCL2 orCCL3 was added to the lower well (n � 6).*P � 0.05 vs. without JWH-015; #P � 0.05 vs.without JWH-015.

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mined a significant activation of p38 MAPK, as shown in therepresentative blots in Fig. 6C and the densitometric quantifi-cation of four to five individual experiments in Fig. 6D.

DISCUSSION

Monocytes are circulating precursors of tissue macrophagesand dendritic cells (30). Monocyte-derived macrophages anddendritic cells fulfill critical functions in innate and adaptiveimmunity during inflammation and play a crucial role invarious chronic diseases, such as rheumatoid arthritis andatherosclerosis (20, 50). Recruitment of monocytes from thebloodstream into tissues is a complex process, in which adhe-sion molecules, chemokines, chemokine receptors, and alsointracellular proteins are involved (17, 54). Because cannabi-noids have been reported to modulate the migration of variouscell types, including immune cells, we aimed to investigate ifselective activation of CB2 cannabinoid receptors would mod-ulate human monocyte migration.

The major findings of our study are that CB2 receptoractivation via JWH-015 modulates the recruitment of humanmonocytes by various mechanisms: 1) JWH-015 reduces theexpression of CCR2 and CCR1, which results in reducedchemotaxis to CCL2 and CCL3; 2) JWH-015 blocks the IFN-�induced upregulation of adhesion molecule ICAM-1; and 3)JWH-015 induces monocyte migration by its own chemotacticproperties and is able to cross-desensitize the migration inresponse to CCL2 and CCL3.

Our data confirming high levels of CB2 receptor expressionon human monocytes (using flow cytometry analysis andRT-PCR) suggest an important role for the CB2 receptor indifferent monocyte functions. We first studied the effect ofJWH-015 on CCL2- and CCL3-induced migration, two majorchemokines expressed at inflammatory sites that trigger mono-cyte recruitment (10). The observed reduction of monocytemigration after 12 h of treatment with JWH-015 is explainedby the reduced expression of CCR2 and, to a lesser extent,

Fig. 6. JWH-015-induced Akt and ERK1/2,but not p38 MAPK, signaling pathways areinvolved in the modulation of chemokine re-ceptors and migration. A: FACS analysis ofmonocyte CCR1 and CCR2 expression after18 h of treatment with JWH-015 (20 �M) inthe presence of LY-294002 [LY; 50 �M,inhibitor of phosphatidylinositol 3-kinase(PI3K), a kinase directly activating Akt], PD-98059 (PD; 50 �M, inhibitor of MEK1/2, akinase directly activating ERK1/2), and SB-203580 (SB; 1 �M, inhibitor of p38 MAPK).n � 5 for CCR2 and n � 6 for CCR1. **P �0.01 vs. control; ##P � 0.01 and #P � 0.05vs. JWH-015. B: untreated monocytes weretested for their ability to migrate to JWH-015(20 �M) in the presence of LY (50 �M), PD(50 �M), and SB (1 �M) kinase inhibitors(n � 4). *P � 0.05 and **P � 0.01 vs.JWH-015. C and D: monocytes were treatedwith 20 �M JWH-015, 10 nM CCL2 (positivecontrol), or vehicle (0.1% DMSO) for theindicated time points, and whole cell lysateswere subjected to Western blot analysis forthe detection of phosphorylated (P) and totalAkt, ERK1/2, and p38 MAPK. RepresentativeWestern blots and quantification of 5 (Akt andp38) or 4 (ERK1/2) different experiments areshown. *P � 0.05 vs. untreated cells.

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CCR1. We focused on the principal receptors involved inmonocyte locomotion, such as CCR2, the corresponding re-ceptor for CCL2, and CCR1, the receptor for CCL3. AlthoughCCL3 is also capable of binding CCR5, this receptor is ex-pressed at low levels on human monocytes and is consideredless important for monocyte migration to CCL3 (58). We alsotested the effect of JWH-015 on ICAM-1 expression. ICAM-1is a member of the IgG superfamily and represents a specificligand for integrins, such as CD11a/CD18 (lymphocyte func-tion-associated Ag-1) and CD11b/CD18 (Mac-1) (41).ICAM-1 is expressed on both leukocytes and endothelial cellsand is crucial in monocyte rolling and transendothelial migra-tion (14). Furthermore, ICAM-1 also interacts with its ligandson monocytes, thus generating homotypic aggregation ofmonocytes (14). In our experiments, JWH-015 was capable ofinhibiting the IFN-�-induced upregulation of ICAM-1. This isin agreement with previously published data demonstratingthat CB2 agonists as well as the endocannabinoid anandamideare able to attenuate TNF-�-induced ICAM-1 and VCAM-1expression in HLSECs or HCAECs(5, 6, 43). However, ourdata provide the first evidence for the modulation of ICAM-1expression on monocytes, suggesting a possible double actionto reduce monocyte-endothelial cell adherence and monocytetransendothelial migration.

In our experiments, JWH-015 was capable of inhibitingchemokine-induced migration at doses of 5–20 �M. Thesemicromolar concentrations have been well demonstrated aspharmacological concentrations for obtaining a functional ac-tivity of the response to JWH-015-mediated CB2 receptoractivation in various monocytic cells (15, 16, 27, 31, 34).JWH-015 is considered as a CB2-selective agonist because thebinding affinity to CB1 receptors is 30-fold lower comparedwith CB2 receptors (Ki values of 13.8 and 383 nM for CB2 andCB1 receptors, respectively) (42). However, the effective dosesfor inhibition were much higher than the Ki value for CB2

receptors, which may also lead to activation of CB1 receptors,

which are also expressed at considerable levels on monocytes(13). To investigate the implication of CB1 and CB2 receptorsin the observed modulation of migration and downregulation ofchemokine receptor and adhesion molecule expression, weused selective receptor antagonists as well as a more selectiveCB2 agonist, JWH-133 (Ki values of 3.4 and 680 nM for CB2

and CB1 receptors, respectively) (22). Only the CB2 antagonistSR-144528 reversed the effect of JWH-015, whereas the CB1

antagonist SR-147778 had no effect. In accordance with this,the CB2 agonist JWH-133, at a concentration of 10 �M,exhibited similar effects as JWH-015. These findings suggestthat the effects on migration and chemokine receptor andICAM-1 expression were all dependent on CB2 activation.Interestingly, our experiments performed with the CB2 antag-onist indicate that it may not only reverse the effect of JWH-015 on chemokine receptor and ICAM-1 expression but furtherenhance their expression. However, neither the increase ofchemokine receptor expression compared with untreated cellsnor the increase of ICAM-1 compared with IFN-�-treated cellswere not significant. A possible underlying mechanism for theobserved effects has been previously described by Bouaboulaand co-workers (7). In a stably CB2 receptor-transfected rodentcell line, the CB2 antagonist SR-144528 has been shown toblock not only the activity of CB2 receptors but also the MAPKactivity of other G protein-coupled receptors. Sustained treat-ment with SR-144528 induced an upregulation of the cellularG protein level, which was associated with a concomitant lossof the ability of SR-144528 to inhibit MAPK activation. It isconceivable that altered G protein levels in response to antag-onist treatment might directly affect the expression of chemo-kine receptors that are G protein-coupled receptors or down-stream signals of ICAM-1 and/or chemokine receptors, whichmay, in turn, alter their expression levels.

In addition to the long-term effects (after 12–24 h) ofJWH-015 on monocyte migration, we also investigated short-term actions (after a few minutes) of JWH-015 on monocytes.

Fig. 7. Schematic representation of JWH-015-induced modulation of monocyte migra-tion (left) and the underlying molecular mech-anisms (right). The immediate action of JWH-015 binding to the CB2 receptor is tochemoattract monocytes, which is dependenton PI3K/Akt and ERK1/2 kinase phosphory-lation (1). The delayed effect of JWH-015binding to the CB2 receptor is inhibition ofCCR2 and CCR1 mRNA and, consequently,surface expression (2). This effect, which isalso dependent on PI3K/Akt and ERK1/2 sig-naling, leads to reduced chemotaxis versusCCL2 and CCL3, the corresponding chemo-kines that bind to CCR2 and CCR1. Con-versely, p38 MAPK, which is also phosphor-ylated by JWH-015, is neither required fordirect chemoattraction nor for inhibition ofchemokine receptor expression.

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We found that JWH-015 itself is chemoattractant for mono-cytes, an effect that has been previously reported for variouscannabinoids on other cell types (11, 15, 33, 38, 47–49). Themigration versus 20 �M JWH-015 was dependent on CB2

receptors, as verified by experiments with selective receptorantagonists and the use of the CB2 agonist JWH-133, whichwas also chemoattractant at a concentration of 10 �M. More-over, checkerboard analysis confirmed that the locomotion wasrather not due to induction of random cellular movement butmore likely a directed migration versus a JWH-015 gradient. Inan attempt to understand the effect of systemic administrationof the CB2 agonist in an inflammatory condition, we furtherinvestigated the effect of JWH-015 on monocyte migration inthe presence of a chemokine gradient. Increasing concentra-tions of JWH-015 (10–20 �M) added to the cells were able toinhibit the chemotactic response versus CCL2 and CCL3,indicating that the CB2 agonist desensitizes monocytes formigration to other chemoattractants. A similar action has beenpreviously reported for other chemoattractants. Indeed, certainchemoattractants desensitize the cells toward further stimula-tion with other chemoattractants (1). Consequently, the sys-temic administration of sufficient amounts of JWH-015 maydesensitize cells toward chemokines such as CCL2 and CCL3and, thus, reduce recruitment of monocytes to inflammatorysites. Likewise, opioids have been shown to inhibit chemokine-induced migration through desensitization of chemokine recep-tors (18). The underlying mechanisms involve in opiate-in-duced phosphorylation of chemokine receptors but not receptorinternalization or an altered chemokine binding capacity. It hasbeen hypothesized that receptor dimers may exist between CB2

receptors and chemokine receptors, which may explain, at leastin part, the observed pleiotropic effects on monocyte migration(35). It is conceivable that binding of CB2 ligands could affectthe ability of chemokine receptors to signal properly.

Various studies have investigated the role of CB2 receptoractivation on inflammatory cell migration in the past, with bothincreases and decreases of cell migration being reported, de-pending on the agonist and cell type used (35). Thus, underdifferent pathogenic conditions, cannabinoids may exhibit ei-ther pro- or anti-inflammatory effects, which does not allowgeneral conclusions to be drawn. Our data on both long- andshort-term effects of the two CB2 agonists JWH-015 andJWH-133 suggest an anti-inflammatory action in chronic in-flammatory conditions where monocyte recruitment plays aprominent role. While reducing chemokine receptor and adhe-sion molecule expression, and consequently migration versusCCL2 and CCL3, JWH-015 may also recruit monocytes by itsintrinsic chemotactic properties. Thus, enhanced plasma can-nabinoid levels might counterbalance recruitment to local che-mokine gradients at inflammatory sites.

Finally, to study the intracellular signaling pathways trig-gered by JWH-015, we used selective inhibitors of variouskinases involved in cell migration, i.e., PI3K/Akt (LY-294002)and MEK1/2 (PD-98059), which is a kinase activatingERK1/2, as well as p38 MAPK (SB-203580). Inhibitors ofPI3K/Akt and MEK1/2 reversed JWH-015-mediated inhibitionof chemokine receptor expression (long-term effect) and mi-gration versus JWH-015 (short-term effect), whereas the in-hibitor of p38 MAPK was ineffective. Conversely, Westernblot analysis revealed that JWH-015 not only induced theactivation of Akt and ERK1/2 but also of p38 MAPK.

Similar discrepancies between kinase activation and theirinvolvement in cellular functions have been previously de-scribed. Indeed, it has been shown that PI3K/Akt and MEK1/2pathways were not involved or only partially involved inmonocyte chemotaxis in response to CCL2 and CCL3, al-though these two chemokines were capable of activating bothPI3K/Akt and ERK1/2 (2, 3, 12, 55). On the other hand, CCL2has been shown to activate p38 MAPK, and inhibitors of p38MAPK induced a substantial inhibition of CCL2-induced mi-gration (2, 3, 9). Our data suggest two potential targets,namely, PI3K/Akt and ERK1/2, selectively involved in JWH-015-mediated short-term and long-term effects on monocytemigration, without the involvement of classical p38 MAPK-dependent intracellular pathways (Fig. 7). However, whileJWH-015 induces p38 MAPK phosphorylation, the potentialdownstream targets of p38 signaling in our model remainunclear.

In conclusion, we have shown that the cannabinoid JWH-015 modulates the recruitment of human monocytes by variousimmediate and delayed effects in a CB2-dependent manner.Both short- and long-term effects depend on PI3K/Akt andERK1/2 signaling: the immediate effect is the induction ofmonocyte migration by its own potent chemotactic properties,which might inhibit the recruitment to local inflammatory sitesby desensitizing cells to chemokine gradients; the delayedeffects are reduced monocyte migration versus CCL2 andCCL3 via downregulation of CCR2 and CCR1 and inhibitionof IFN-�-induced ICAM-1 upregulation. Taken together, theseanti-inflammatory properties might have crucial effects inchronic inflammatory disorders such as atherosclerosis andrheumatoid arthritis.

ACKNOWLEDGMENTS

We thank Ana Da Costa and Charlotte Berlier for excellent technicalassistance.

GRANTS

This work was supported by Swiss National Science Foundation Grants310000-116324 (to S. Steffens) and 3200B0-105896/1 (to F. Mach). Theauthors belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community.

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