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Effects of Peripheral Cannabinoid Receptor Ligands on Motility and Polarization in Neutrophil-like HL60 Cells and Human Neutrophils

2006; Elsevier BV; Volume: 281; Issue: 18 Linguagem: Inglês

10.1074/jbc.m510871200

1083-351X

Rina Kurihara, Yumi Tohyama, Satoshi Matsusaka, Hiromu Naruse, Emi Kinoshita, Takayuki Tsujioka, Yoshinao Katsumata, Hirohei Yamamura,

Neuroscience and Neuropharmacology Research

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration. The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration. The peripheral cannabinoid receptor (CB2) 2The abbreviations used are: CB2, the peripheral cannabinoid receptor; CB1, the central cannabinoid receptor; PI3K, phosphatidylinositol 3-kinase; fMLP, N-formyl-l-methionyl-l-leucyl-l-phenylalanine; 2-AG, 2-arachidonoylglycerol; ATRA, all-trans-retinoic acid; MLC, myosin light chain; GFP-PKB/Akt-PH domain, the pleckstrin homology domain of PKB/Akt tagged with green fluorescent protein; PMN, polymorphonuclear granulocyte; PI3P, phosphatidylinositol 3,4,5-triphosphate and other products of PI3K; p160-ROCK, Rho-dependent protein kinase; HRP, horseradish peroxidase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GST, glutathione S-transferase. 2The abbreviations used are: CB2, the peripheral cannabinoid receptor; CB1, the central cannabinoid receptor; PI3K, phosphatidylinositol 3-kinase; fMLP, N-formyl-l-methionyl-l-leucyl-l-phenylalanine; 2-AG, 2-arachidonoylglycerol; ATRA, all-trans-retinoic acid; MLC, myosin light chain; GFP-PKB/Akt-PH domain, the pleckstrin homology domain of PKB/Akt tagged with green fluorescent protein; PMN, polymorphonuclear granulocyte; PI3P, phosphatidylinositol 3,4,5-triphosphate and other products of PI3K; p160-ROCK, Rho-dependent protein kinase; HRP, horseradish peroxidase; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GST, glutathione S-transferase. was cloned in 1993 (1Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4048) Google Scholar) after cloning of the central cannabinoid receptor (CB1) in 1990 (2Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4137) Google Scholar). It has been suggested that the gene encoding CB2 is a protooncogene and that aberrant expression of CB2 in myeloid precursor cells results in the development of leukemia by blocking neutrophil differentiation (3Valk P.J.M. Hol S. Vankan Y. Ihle J.N. Askew D. Jenkins N.A. Gilbert D.J. Copeland N.G. De Both N.J. Löwenberg B. Delwel R. J. Virol. 1997; 71: 6796-6804Crossref PubMed Google Scholar, 4Jordà M.A. Rayman N. Tas M. Verbakel S.E. Battista N. Van Lom K. Löwen-berg B. Maccarrone M. Delwel R. Blood. 2004; 104: 526-534Crossref PubMed Scopus (58) Google Scholar). CB2 is expressed predominantly in immune cells (5Galiègue S. Mary S. Marchand J. Dussossoy D. Carrière D. Carayon P. Bouaboula M. 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Nature. 2005; 434: 782-786Crossref PubMed Scopus (360) Google Scholar) recently reported that doses of Δ9-tetrahydrocannabinol (the most psychoactive component of marijuana) too low to have psychotropic effects inhibit the progression of atherosclerosis via immunomodulatory effects on lymphoid and myeloid cells. This report indicates that CB2 may be involved in a wide range of physiologic phenomena related to immunity and that some CB2 ligands may have application in the treatment of inflammatory disease. However, research into CB2 is still in its early stages. In particular, the involvement of only a few molecules, Gαi/Gαo protein, phosphatidylinositol 3-kinase (PI3K), and members of the mitogen-activated protein kinase and nuclear factor-κB families, in the CB2 signaling pathways has been reported (6Berdyshev E.V. Chem. Phys. Lipids. 2000; 108: 169-190Crossref PubMed Scopus (228) Google Scholar, 7Klein T.W. Newton C. Larsen K. Lu L. Perkins I. Nong L. Friedman H. J. Leukocyte Biol. 2003; 74: 486-496Crossref PubMed Scopus (421) Google Scholar, 8Sugiura T. Oka S. Gokoh M. Kishimoto S. Waku K. J. Pharmacol. Sci. 2004; 96: 367-375Crossref PubMed Scopus (45) Google Scholar, 11Kaminski N.E. J. Neuroimmunol. 1998; 83: 124-132Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Among the possible roles of CB2 in immunity is the induction of leukocyte migration to sites of infection and inflammation, an important step in the host defense against pathogenic microorganisms. CB2 is a seven-transmembrane, Gαi/Gαo protein-coupled receptor, as are receptors for chemoattractants such as N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). It has been reported that several genes encoding chemotactic cytokines are up-regulated in response to CB2 stimulation (12Derocq J.-M. Jbilo O. Bouaboula M. Ségui M. Clère C. Casellas P. J. Biol. Chem. 2000; 275: 15621-15628Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Indeed, 2-arachidonoylglycerol (2-AG), a physiological ligand for both CB1 and CB2 (13Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. Biochem. Biophys. Res. Commun. 1995; 215: 89-97Crossref PubMed Scopus (1783) Google Scholar, 14Mechoulam R. Ben-Shabat S. Hanus L. Ligumsky M. Kaminski N.E. Schatz A.R. Gopher A. Almog S. Martin B.R. Compton D.R. Pertwee R.G. Griffin G. Bayewitch M. Barg J. Vogel Z. Biochem. Pharmacol. 1995; 50: 83-90Crossref PubMed Scopus (2302) Google Scholar), can induce migration in some subpopulations of hematopoietic cells (8Sugiura T. Oka S. Gokoh M. Kishimoto S. Waku K. J. Pharmacol. Sci. 2004; 96: 367-375Crossref PubMed Scopus (45) Google Scholar, 15Jordà M.A. Verbakel S.E. Valk P.J.M. Vankan-Berkhoudt Y.V. Maccarrone M. Finazzi-Agrò A. Löwenberg B. Delwel R. Blood. 2002; 99: 2786-2793Crossref PubMed Scopus (140) Google Scholar, 16Walter L. Franklin A. Witting A. Wade C. Xie Y. Kunos G. Mackie K. Stella N. J. Neurosci. 2003; 23: 1398-1405Crossref PubMed Google Scholar). However, contradictory findings have been reported by other researchers (10Steffens S. Veillard N.R. Arnaud C. Pelli G. Burger F. Staub C. Zimmer A. Frossard J.-L. Mach F. Nature. 2005; 434: 782-786Crossref PubMed Scopus (360) Google Scholar, 17Sacerdote P. Massi P. Panerai A.E. Parolaro D. J. Neuroimmunol. 2000; 109: 155-163Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 18Deusch E. Kraft B. Nahlik G. Weigl L. Hohenegger M. Kress H.G. J. Neuroimmunol. 2003; 141: 99-103Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Joseph J. Niggemann B. Zaenker K.S. Entschladen F. Cancer Immunol. Immunother. 2004; 53: 723-728Crossref PubMed Scopus (137) Google Scholar), in part due to differences in ligands and cells used. In particular, Steffens et al. (10Steffens S. Veillard N.R. Arnaud C. Pelli G. Burger F. Staub C. Zimmer A. Frossard J.-L. Mach F. Nature. 2005; 434: 782-786Crossref PubMed Scopus (360) Google Scholar) reported that Δ9-tetrahydrocannabinol inhibits monocyte chemoattractant protein 1-induced macrophage migration, a crucial step in the progression of atherosclerosis. In the present study, the possible role of CB2 in neutrophil migration was investigated. We constructed an in vitro model of neutrophil migration on blood vessels as described previously (20Miura Y. Tohyama Y. Hishita T. Lala A. De Nardin E. Yoshida Y. Yamaura H. Uchiyama T. Tohyama K. Blood. 2000; 96: 1733-1739Crossref PubMed Google Scholar) but with some modification: we studied human promyelocytic HL60 cells, differentiated by all-trans-retinoic acid (ATRA) into neutrophil-like cells, on plates coated with fibrinogen, an adhesive extracellular matrix glycoprotein. We show that two CB2 ligands, 2-AG and a synthetic CB2-specific ligand JWH015, induce increased motility of the cells but that the cells do not develop the front/rear polarity that migrating leukocytes typically exhibit, with a lamellipodium at the front and a retracting tail at the rear (21Sánchez-Madrid F. del Pozo M.A. EMBO J. 1999; 18: 501-511Crossref PubMed Scopus (518) Google Scholar, 22Bokoch G.M. Trends Cell Biol. 2005; 15: 163-171Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In addition, to investigate the possible physiological implication of the results, the effects of the CB2 ligands, alone and as pretreatment before fMLP stimulation, were analyzed in human neutrophils isolated from whole blood. We show that pretreatment of human neutrophils with the ligands disrupts fMLP-induced front/rear polarization and migration. Antibodies and Reagents—The rabbit polyclonal anti-CB2 anitibody was purchased from Calbiochem Corp.. The mouse monoclonal anti-GFP antibody (sc-9996) and the rabbit polyclonal anti-RhoA (sc-179) and anti-Rac2 (sc-96) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse monoclonal anti-α-tubulin antibody was from Sigma. The mouse monoclonal anti-Rac1 and anti-Cdc42 antibodies were from BD Biosciences. The mouse monoclonal anti-phosphorylated (serine 473) PKB/Akt, the rabbit polyclonal anti-PKB/Akt, the mouse monoclonal anti-phosphorylated myosin light chain (MLC) II, and the rabbit polyclonal anti-MLC II antibodies were from Cell Signaling Technology (Beverly, MA). The Alexa Fluor 488-conjugated goat secondary anti-mouse and anti-rabbit antibodies were from Invitrogen Corp. The HRP-conjugated goat secondary anti-mouse and anti-rabbit antibodies were from Bio-Rad. Alexa Fluor 594-conjugated phalloidin was from Invitrogen Corp. Human fibrinogen was from Yoshitomiyakuhin Corp. (Osaka, Japan). JWH015 and 2-AG were from Biomol Research Laboratories (Plymouth Meeting, PA). CB2 inhibitor SR144528 was kindly donated by Sanofi Aventis (Paris, France). CB1 inhibitor AM251 was from Tocris Bioscience (Ellisville, MO). fMLP was from Sigma. Y27632 was from Calbiochem Corp. Cell Preparation—Human promyelocytic HL60 cells were cultured in RPMI 1640 medium (Sigma) supplemented with 8% fetal calf serum at 37 °C in an atmosphere of 95% air and 5% CO2. Some HL60 cells and HL60 cells transiently expressing the pleckstrin homology domain of PKB/Akt tagged with green fluorescent protein (GFP-PKB/Akt-PH domain) were induced to differentiate into neutrophil-like cells by treatment with 1 μm ATRA (Sigma) for 4 days. Human polymorphonuclear granulocytes (PMNs) were isolated from whole blood donated by healthy volunteers with Ficoll-Hypaque Polymorphprep™ solution (Axis-Shield PoC AS, Oslo, Norway). May-Gruenwald-Giemsa Staining—For the morphological assessment, the cell smears were prepared with cytospin centrifugation for 5 minat70 × g (Shandon Cytospin3; Thermo Electron Corp., Pittsburgh, PA) and stained with May-Gruenwald-Giemsa solution. Flow Cytometric Analysis—CB2 expression on the cell surface was examined by flow cytometric analysis. Cells were incubated for 45 min with anti-CB2 antibody in phosphate-buffered saline containing 1% bovine serum albumin (BSA-containing PBS) and washed twice with BSA-containing PBS. The cells were then incubated for 30 min with Alexa Fluor 488-conjugated secondary anti-rabbit antibody and washed with BSA-containing PBS before being subjected to flow cytometry with a FACSCalibur™ system (BD Biosciences). All of these procedures were conducted at 4 °C. Plasmid Constructs and DNA Transfection—The PH domain of human pkb/akt protein kinase (amino acids 1-167) was amplified from human spleen cDNAs (Ambion, Austin, TX) with the following primer pair: 5′-ATGAGCGACGTGGCTATTGTGAAGG-3′ and 5′-CACCAGGATCACCTTGCCGAAAGTG-3′. The second amplification reaction was performed with primers that contained EcoRI and BamHI restriction sites and the amplified products were cloned into the pEGFP-N1 plasmids (Clontech Laboratories, Inc.). After the sequences of both strands were verified with ABI PRISM Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems, Foster City, CA), the plasmids were transfected into COS-7 cells. Cell lysates were resolved by SDS-PAGE, and subjected to immunoblot analysis for the presence of GFP fusion proteins with anti-GFP antibody. GFP-PKB/Akt-PH domain was introduced into HL60 cells by electroporation. Cell Migration Assay—Migration of live cells was observed as described previously (23Matsusaka S. Tohyama Y. He J. Shi Y. Hazama R. Kadono T. Kurihara R. Tohyama K. Yamamura H. Biochem. Biophys. Res. Commun. 2005; 328: 1163-1169Crossref PubMed Scopus (21) Google Scholar) but with some modification. In brief, cells were plated in culture medium onto a 100 μg/ml fibrinogen- or 10 μg/ml fibronectin-coated 35-mm culture dish, which was placed on the stage of a Power IX microscope (Olympus Corp., Tokyo, Japan) coupled to an MI-IBC CO2 incubator (Olympus Corp.) to allow for observations at 37 °C in 95% air and 5% CO2. Cells were stimulated with either uniform concentration of ligands or a point source of ligands from a micropipette (Femtotips®: Eppendorf AG, Hamburg, Germany). Cell morphology and motility alterations were monitored by obtaining single-frame images every 10 s for at least 20 min. Approximately 160-200 cells were present per 297.0 × 397.9-μm frame. Those cells which remained in the frame throughout the observation (∼90-95%) were examined. Cell lengths and migratory distances were measured with image analyzing software MacSCOPE Version 2.6 (Mitani Corp., Fukui, Japan). In particular, migratory distances were calculated by monitoring two-dimensional coordinates of each cell center at 30-s intervals. Other morphologic alterations were examined by observing individual cells during stop-time playback of time-lapse images. Immunofluorescence Microscopic Analysis—In preparation for staining, cells were plated in culture medium onto 100 μg/ml fibrinogencoated FALCON™ culture slides (BD Biosciences), fixed with 3% paraformaldehyde for 15 min, washed twice with PBS containing 2 mm MgCl2 and 0.5% BSA (staining buffer), and permeabilized with 0.2% Triton X-100 in staining buffer for 3 min. Nonspecific binding to Fc receptors was blocked by incubation for 15 min with PBS containing 2 mm MgCl2 and 5% BSA. To confirm CB2 expression, samples thus prepared were incubated with Alexa Fluor 488-conjugated anti-CB2 antibody for 45 min. This antibody was produced with the use of a Zenon™ rabbit IgG labeling kit (Invitrogen Corp.) according to the manufacturer's instructions. After three washes with staining buffer, chromosomes were stained with the DNA-specific fluorescent dye Hoechst 33342 (Wako Pure Chemical Industries, Osaka, Japan). To examine cytoskeletal organization, prepared cells were incubated with anti-α-tubulin antibody for 45 min. After three washes with staining buffer, cells were incubated with Alexa Fluor 488-conjugated secondary anti-mouse antibody and Alexa Fluor 594-conjugated phalloidin (to visualize F-actin) for 30 min. To examine localization of the active form of MLC II, cells were incubated with anti-phosphrylated MLC II antibody. The following procedures were identical to those for staining of α-tubulin. All of these procedures were conducted at room temperature. Stained cells were washed twice with staining buffer, mounted in antifade mounting medium (ProLong™ Antifade kit; Invitrogen Corp.), and analyzed by confocal microscopy with an LSM 510 laser scanning unit and an Axiovert 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany) run with imaging software LSM 510 META Version 3.0 (Olympus Corp.). Rho-GTPase Pull-down Assay—The GTP-bound (active) forms of four Rho-GTPases, Rac1, Rac2, Cdc42, and RhoA, were isolated by pull-down assay. Recombinant protein PAK1-PBD-GST, which binds specifically to the GTP-bound forms of Rac and Cdc42, was produced as described previously (24Miah S.M.S. Hatani T. Qu X. Yamamura H. Sada K. Genes Cells. 2004; 9: 993-1004Crossref PubMed Scopus (47) Google Scholar). In brief, cDNA of the Rac- and Cdc42-binding domain of human PAK1 (PAK1-PBD; amino acids 67-150) was cloned into expression vector pGEX4T-3 as a fusion protein with glutathione S-transferase (GST) and was expressed in Escherichia coli DH5α cells treated with isopropyl β-d-thiogalactopyranoside (Nacalai Tesque, Kyoto, Japan). The fusion protein was purified with gluthathione-Sepharose 4B beads (Amersham Biosciences, Uppsala, Sweden). For production of recombinant protein rhotekin-RBD-GST, which binds specifically to the GTP-bound forms of Rho (-A, -B, and -C), cDNA of the Rho-binding domain of mouse rhotekin (rhotekin-RBD; amino acids 7-89) was cloned according to the procedure of Ren et al. (25Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1350) Google Scholar). Other procedures were identical to those for PAK1-PBD-GST. Neutrophil-like HL60 cells and human neutrophils (1 × 107) were stimulated with 100 nm JWH015, 300 nm 2-AG, or 100 nm fMLP. Because cell adhesion to extracellular matrix affects Rho-GTPase activity (25Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1350) Google Scholar) and because cells are known to polarize in suspension culture (26Eddy R.J. Pierini L.M. Maxfield F.R. Mol. Biol. Cell. 2002; 13: 4470-4483Crossref PubMed Scopus (66) Google Scholar), we stimulated cells in suspension culture in a BSA-coated microtube. The cells were then lysed for 15 min at 4 °C in Mg2+ lysis buffer (MLB) containing 100 mm NaCl, 50 mm Tris-HCl (pH 7.5), 1% Triton X-100, 10 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 1.5 μm aprotinin, and 1 mm dithiothreitol. Cell lysates were centrifuged at 12,000 × g for 15 min at 4 °C. A proportion of each supernatant was diluted in Laemmli sample buffer at 100 °C for 5 min for detection of total (both GTP- and GDP-bound) Rho-GTPases. The remaining supernatants were incubated with either PAK1-PBD-GST or rhotekin-RBD-GST for 1 h at 4°C followed by three washes with MLB. Proteins bound to the beads were eluted by being heated in Laemmli sample buffer at 100 °C for 5 min and, along with samples for detecting total Rho-GTPases, were subjected to Western blot analysis. Western Blot Analysis—To detect activity of the four Rho-GTPases, we used samples lysed in MLB. To detect PKB/Akt and MLC II activity, cells were lysed in a solution containing 100 mm NaCl, 50 mm Tris-HCl (pH 7.2), 1 mm EDTA, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 1.5 μm aprotinin. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane (Millipore Corp., Bedford, MA). The membranes were then blocked with 1% skim milk in PBS for 30 min at room temperature, incubated with antibodies to Rac1, Rac2, Cdc42, RhoA, phosphorylated PKB/Akt, or phosphorylated MLC II for 1 h at room temperature, and washed three times with a solution containing 150 mm NaCl, 25 mm Tris-HCl (pH 8.0), and 0.1% Tween 20 (TBS-T). The membranes were then incubated with HRP-conjugated secondary anti-mouse antibody (to detect Rac1, Cdc42, phosphorylated PKB/Akt, or phosphorylated MLC II) or HRP-conjugated secondary anti-rabbit antibody (to detect RhoA and Rac2) for 30 min at room temperature and washed twice with TBS-T and once with PBS. Immune complexes were visualized with Western Lightning™ chemiluminescence reagent (PerkinElmer Life Sciences) and a lumino-image analyzer (LAS-1000; Fuji Photo Film, Tokyo, Japan). For quantification, the density of each band was measured with image analyzing software NIH Image Version 1.63 f. To detect total (both phosphorylated and unphosphorylated) PKB/Akt and MLC II, immune complex of HRP-conjugated secondary anti-mouse antibody with anti-phosphorylated PKB/Akt and that with anti-phosphorylated MLC II antibody were stripped by bathing the membranes in a solution containing 62.5 mm Tris-HCl (pH 6.8), 7 mm SDS, and 95 mm 2-mercaptoethanol for 30 min at 50 °C, and the membranes were reprobed with either general anti-PKB/Akt or anti-MLC II antibody. Statistical Analysis—Statistical significance was determined by Student's t test, with a p value of 95% of the PMNs were mature neutrophils (data not shown). Moreover, CB2 expression in these cells was confirmed by immunofluorescence microscopic analysis (Fig. 1B, panels d-f). We also observed nuclear morphology with Hoechst 33342 and confirmed that ATRA-treated HL60 cells were successfully induced to differentiate into neutrophil-like cells (Fig. 1B, panel b), and that most of the PMNs were mature neutrophils (Fig. 1B, panel c). Because similar levels of CB2 expression in neutrophil-like HL60 cells and in human neutrophils were confirmed, we used neutrophil-like HL60 cells as an in vitro model of human neutrophils in further experiments. JWH015 and 2-AG Induced Motility with No Front/Rear Polarization in Neutrophil-like HL60 Cells—Alterations in motility and morphology of neutrophil-like HL60 cells following stimulation with either 100 nm JWH015 (a synthetic CB2-specific ligand) or 300 nm 2-AG (a physiological ligand for both CB1 and CB2) were monitored by video microscopy. Most unstimulated cells were spherical, with occasional small spike-like projections (Fig. 2A, upper left image). Approximately 3 min after JWH015 or 2-AG stimulation, 60% of the cells elongated (i.e. polarized), rapidly extending and retracting one or more pseudopods in different directions (Fig. 2A (JWH015); data not shown for 2-AG). These cells did not develop the front/rear polarity that migrating leukocytes typically exhibit and displayed almost no migratory activity during observations of up to 60 min (data not shown). A point source of 10 μm JWH015 by micropipette also induced polarization accompanied by the extension of one or more pseudopods in different directions (Fig. 2B). Quantitative analysis of image sequences of samples unstimulated or stimulated with uniform concentration of the CB2 ligands is shown in Fig. 2, C-E. To ensure consistency in distinguishing polarized cells from non-responding cells, cells with a value of X greater than 2 were counted as polarized (X = L/W, where L is the longest distance across the cell, and W is the greatest width perpendicular to L as shown in the illustration next to Fig. 2C). The percentage of polarized cells increased significantly after stimulation with the CB2 ligands (p < 0.01). This increase was almost completely inhibited by pretreatment with 1 μm CB2 inhibitor SR144528 but not by 1 μm CB1 inhibitor AM251 (Fig. 2C). In addition, the percentage of cells that extended multiple pseudopods increased significantly after CB2 stimulation (p < 0.01, Fig. 2D). Approximately 90% of the cells that polarized in response to the CB2 ligands were devoid of a retracting tail of the sort observed at the rear of migrating leukocytes (Fig. 2E). In contrast, >90% of the cells responding to uniform concentration (100 nm) of fMLP exhibited front/rear polarity, with a single pseudopod (lamellipodium) at the front and a retracting tail at the rear (Fig. 2, D and E; an image of a typical fMLP-stimulated cell is shown in the illustration next to Fig. 2E). F-actin Formed in the Pseudopods in CB2-stimulated Cells—The cytoskeletal reorganization underlying the morphologic alterations accompanying CB2 stimulation was investigated by immunofluorescence microscopy. Fig. 3A, panels a-d, shows fluorescence images of F-actin (red) and microtubules (green). In a typical unstimulated cell (panel a), only faint F-actin staining was observed at the periphery, and from a single point, an array of microtubule filaments extended radially to the cell periphery. Three types of JWH015-stimulated cells are shown. In one type with a single pseudopod (panel b), a large accumulation of F-actin was observed in the pseudopod, toward which microtubule filaments emanated from a single point. In the other types, F-actin was found in two (panel c) or 3 (panel d) pseudopods, and microtubule filaments emanated from a single point to each pseudopod. GFP-PKB/Akt-PH Domain Accumulated in One or More Regions in Neutrophil-like HL60 Cells—HL60 cells transiently expressing GFP-PKB/Akt-PH domain, a fluorescence probe for phosphatidylinositol 3,4,5-triphosphate and other products of PI3K (PI3Ps) (28Várnai P. Balla T. J. Cell Biol. 1998; 143: 501-510Crossref PubMed Scopus (657) Google Scholar, 29Xu J. Wang F. Van Keymeulen A. Herzmark P. Straight A. Kelly K. Takuwa Y. Sugimoto N. Mitchison T. Bourne H.R. Cell. 2003; 114: 201-214Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar), were induced to differentiate into neutrophil-like cells by treatment with ATRA and stimulated with JWH015. Distribution of GFP-PKB/Akt-PH domain was examined by immunofluorescence microscopy. In cells that exhibited typical morphologic alterations in response to CB2 stimulation, GFP-PKB/Akt-PH domain accumulated in one or more regions, mostly in pseudopods (Fig. 3B, panels b and c). JWH015 Decreased RhoA Activity in Neutrophil-Like HL60 Cells—Among numerous molecules implicated in cytoskeletal reorganization and cell migration, four Rho-GTPases, RhoA, Rac1, Rac2, and Cdc42, play particularly important roles. The effects of CB2 stimulation on the activities of these four Rho-GTPases were assessed by Rho-GTP pull-down assay, where active (GTP-bound) forms of Rho-GTPases can be detected. The time courses of RhoA, Rac1, Rac2, and Cdc42 activities are shown in Fig. 4, A-D. Notably, RhoA activity decreased 2 min after JWH015 stimulation (A). In contrast, Rac1 activity increased rapidly and peaked at 30 s at ∼20-fold the basal level (B). Rac2 activity also increased although more gradually and to a lower peak of 3-fold basal (C). Cdc42 activity increased even more gradually (D). We also observed that activity of PI3K-dependent kinase PKB/Akt increased rapidly in response to JWH015 stimulation, peaking at a high level (Fig. 4E).