2-Arachidonoylglycerol Induces the Migration of HL-60 Cells Differentiated into Macrophage-like Cells and Human Peripheral Blood Monocytes through the Cannabinoid CB2 Receptor-dependent Mechanism
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m301359200
ISSN1083-351X
AutoresSeishi Kishimoto, Maiko Gokoh, Saori Oka, Mayumi Muramatsu, Takashi Kajiwara, Keizo Waku, Takayuki Sugiura,
Tópico(s)Neuroscience of respiration and sleep
Resumo2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2) and has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we proposed that 2-arachidonoylglycerol is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB2) are primarily 2-arachidonoylglycerol receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of 2-arachidonoylglycerol on the motility of HL-60 cells differentiated into macrophage-like cells. We found that 2-arachidonoylglycerol induces the migration of differentiated HL-60 cells. The migration induced by 2-arachidonoylglycerol was blocked by treatment of the cells with either SR144528, a CB2 receptor antagonist, or pertussis toxin, suggesting that the CB2 receptor and Gi/Go are involved in the 2-arachidonoylglycerol-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, failed to induce the migration. The 2-arachidonoylglycerol-induced migration was also observed for two other types of macrophage-like cells, the U937 cells and THP-1 cells, as well as human peripheral blood monocytes. These results strongly suggest that 2-arachidonoylglycerol induces the migration of several types of leukocytes such as macrophages/monocytes through a CB2 receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses. 2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2) and has been shown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we proposed that 2-arachidonoylglycerol is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB2) are primarily 2-arachidonoylglycerol receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of 2-arachidonoylglycerol on the motility of HL-60 cells differentiated into macrophage-like cells. We found that 2-arachidonoylglycerol induces the migration of differentiated HL-60 cells. The migration induced by 2-arachidonoylglycerol was blocked by treatment of the cells with either SR144528, a CB2 receptor antagonist, or pertussis toxin, suggesting that the CB2 receptor and Gi/Go are involved in the 2-arachidonoylglycerol-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, failed to induce the migration. The 2-arachidonoylglycerol-induced migration was also observed for two other types of macrophage-like cells, the U937 cells and THP-1 cells, as well as human peripheral blood monocytes. These results strongly suggest that 2-arachidonoylglycerol induces the migration of several types of leukocytes such as macrophages/monocytes through a CB2 receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses. Δ9-Tetrahydrocannabinol (Δ9-THC) 1The abbreviations used are: Δ9-THC, Δ9-tetrahydrocannabinol; 2-AG, 2-arachidonoylglycerol; NBT, nitroblue tetrazolium; PTX, pertussis toxin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mitogen-activated protein; MEK, MAP kinase/extracellular signal-regulated kinase kinase. 1The abbreviations used are: Δ9-THC, Δ9-tetrahydrocannabinol; 2-AG, 2-arachidonoylglycerol; NBT, nitroblue tetrazolium; PTX, pertussis toxin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mitogen-activated protein; MEK, MAP kinase/extracellular signal-regulated kinase kinase. is a major psychoactive constituent of marijuana and is known to exert a variety of biological effects in experimental animals and human such as altered perception, inhibition of memory, immobility, analgesia, and the inhibition of immune response, although the mechanism of these actions of Δ9-THC remained elusive until the late 1980's. In 1988, Devane et al. (1Devane W.A. Dysarz III, F.A. Johnson M.R. Melvin L.S. Howlett A.C. Mol. Pharmacol. 1998; 34: 605-613Google Scholar) demonstrated the presence of a specific binding site for cannabinoids in rat brain synaptosomes. Later, Matsuda et al. (2Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Google Scholar) and Munro et al. (3Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Google Scholar) cloned the cDNAs for the cannabinoid receptors (CB1 and CB2). It has been assumed that the diverse actions of the cannabinoids are mediated in a large part through these receptors. In 1992, Devane et al. (4Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. Science. 1992; 258: 1946-1949Google Scholar) isolated N-arachidonoylethanolamine (anandamide) from pig brain as an endogenous cannabinoid receptor ligand. This compound has been shown to exhibit various cannabimimetic activities in vitro and in vivo (5Di Marzo V. Biochim. Biophys. Acta. 1998; 1392: 153-175Google Scholar, 6Piomelli D. Beltramo M. Giuffrida A. Stella N. Neurobiol. Dis. 1998; 5: 462-473Google Scholar, 7Mechoulam R. Fride E. Di Marzo V. Eur. J. Pharmacol. 1998; 359: 1-18Google Scholar, 8Di Marzo V. De Petrocellis L. Bisogno T. Berger A. Mechoulam R. Onaivi E.S. Biology of Marijuana. Taylor & Francis, London2002: 125-174Google Scholar). However, the levels of anandamide in various living tissues were very low (9Hansen H.S. Moesgaard B. Hansen H.H. Petersen G. Chem. Phys. Lipids. 2000; 108: 135-150Google Scholar, 10Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 173-192Google Scholar). Furthermore, anandamide was found to act as a partial agonist at the cannabinoid receptors (10Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 173-192Google Scholar). These observations strongly suggested the existence of another endogenous ligand in mammalian tissues. In 1995, we (11Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. Biochem. Biophys. Res. Commun. 1995; 215: 89-97Google Scholar) and Mechoulam et al. (12Mechoulam 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: 83-90Google Scholar) reported that 2-arachidonoylglycerol (2-AG) is the second endogenous ligand for the cannabinoid receptors. 2-AG has been shown to exhibit a strong binding activity toward the cannabinoid receptors (11Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. Biochem. Biophys. Res. Commun. 1995; 215: 89-97Google Scholar, 12Mechoulam 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: 83-90Google Scholar) and a variety of cannabimimetic activities (5Di Marzo V. Biochim. Biophys. Acta. 1998; 1392: 153-175Google Scholar, 6Piomelli D. Beltramo M. Giuffrida A. Stella N. Neurobiol. Dis. 1998; 5: 462-473Google Scholar, 7Mechoulam R. Fride E. Di Marzo V. Eur. J. Pharmacol. 1998; 359: 1-18Google Scholar, 8Di Marzo V. De Petrocellis L. Bisogno T. Berger A. Mechoulam R. Onaivi E.S. Biology of Marijuana. Taylor & Francis, London2002: 125-174Google Scholar, 10Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 173-192Google Scholar, 13Sugiura T. Waku K. Chem. Phys. Lipids. 2000; 108: 89-106Google Scholar, 14Sugiura T. Waku K. J. Biochem. 2002; 132: 7-12Google Scholar). Importantly, 2-AG was found to act as a full agonist at the cannabinoid receptors (15Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Google Scholar, 16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar, 17Hillard C.J. Prostaglandins Lipid Mediators. 2000; 61: 3-18Google Scholar, 18Gonsiorek W. Lunn C. Fan X. Narula S. Lundell D. Hipkin R.W. Mol. Pharmacol. 2000; 57: 1045-1050Google Scholar, 19Savinainen J.R. Jarvinen T. Laine K. Laitinen J.T. Br. J. Pharmacol. 2001; 134: 664-672Google Scholar). Moreover, 2-AG can be rapidly formed from arachidonic acid-containing phospholipids, such as inositol phospholipids, through the combined actions of phospholipase C and diacylglycerol lipase or the combined actions of phospholipase A1 and phospholipase C in various types of tissues and cells upon stimulation (20Bisogno T. Sepe N. Melck D. Maurelli S. De Petrocellis L. Di Marzo V. Biochem. J. 1997; 322: 671-677Google Scholar, 21Stella N. Schweitzer P. Piomelli D. Nature. 1997; 388: 773-778Google Scholar, 22Sugiura T. Kodaka T. Nakane S. Kishimoto S. Kondo S. Waku K. Biochem. Biophys. Res. Commun. 1998; 243: 838-843Google Scholar, 23Varga K. Wagner J.A. Bridgen D.T. Kunos G. FASEB J. 1998; 12: 1035-1044Google Scholar, 24Di Marzo V. Bisogno T. De Petrocellis L. Melck D. Orlando P. Wagner J.A. Kunos G. Eur. J. Biochem. 1999; 264: 258-267Google Scholar, 25Berdyshev E.V. Schmid P.C. Krebsbach R.J. Schmid H.H.O. FASEB J. 2001; 15: 2171-2178Google Scholar, 26Basavarajappa B.S. Saito M. Cooper T.B. Hungund B.L. Biochim. Biophys. Acta. 2000; 1535: 78-86Google Scholar). Noticeably, the levels of 2-AG in various mammalian tissues are markedly higher than that of anandamide. Based on these results, we proposed that 2-AG, and not anandamide, is the intrinsic natural ligand for the cannabinoid receptors (15Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Google Scholar, 16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar, 27Sugiura T. Kodaka T. Kondo S. Nakane S. Kondo H. Waku K. Ishima Y. Watanabe K. Yamamoto I. J. Biochem. 1997; 122: 890-895Google Scholar). Despite their potential physiological and pathophysiological importance, the exact functions of the CB1 and CB2 receptors and their endogenous ligand 2-AG have not yet been fully elucidated. As for the CB1 receptor, several lines of evidence strongly suggested that 2-AG suppresses the neurotransmission through acting at the CB1 receptor expressed predominantly in the presynapse (10Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 173-192Google Scholar, 13Sugiura T. Waku K. Chem. Phys. Lipids. 2000; 108: 89-106Google Scholar, 14Sugiura T. Waku K. J. Biochem. 2002; 132: 7-12Google Scholar). It is becoming evident that 2-AG is a novel type of neuromodulator of profound physiological significance. On the other hand, the functions of the CB2 receptor, which is abundantly expressed in the immune system, still remain an enigma. Little is known concerning the biological activities of 2-AG toward inflammatory cells and immune competent cells. It is essential to investigate in detail the functions of the CB2 receptor and 2-AG to better understand the precise regulatory mechanisms of inflammatory reactions and immune responses. In this study, we investigated the possible biological activity of 2-AG toward HL-60 cells differentiated into macrophage-like cells. We found that 2-AG induces the migration of differentiated HL-60 cells through a cannabinoid CB2 receptor-dependent mechanism. A similar effect was also observed with human monocytes. The physiological and pathophysiological meanings of the 2-AG-induced migration of macrophages/monocytes are discussed. Chemicals—Arachidonic acid (20:4n-6), palmitic acid (16:0), oleic acid (18:1n-9), linoleic acid (18:2n-6), eicosa-5,8,11,14,17-pentaenoic acid (20:5n-3), docosa-4,7,10,13,16,19-hexaenoic acid (22:6n-3), essentially fatty acid-free bovine serum albumin, 1α,25-dihydroxyvitamin D3 (1,25-(OH)2vitamin D3), phorbol 12-myristate 13-acetate, and LY294002 were purchased from Sigma. Nitroblue tetrazolium (NBT), wortmannin, and herbimycin A were obtained from Wako Pure Chemicals (Osaka, Japan). Eicosa-5,8,11-trienoic acid (mead acid) (20:3n-9) was purchased from Cayman Chemical Co. (Ann Arbor, MI). SR141716A was acquired from Biomol (Plymouth Meeting, PA). CP55940 and Y-27632 were purchased from Tocris (Bristol, United Kingdom). WIN55212–2 was obtained from RBI (Natick, MA). PD98059 and SB203580 were acquired from Calbiochem. Pertussis toxin (PTX) was obtained from List Biological Laboratories (Campbell, CA). SR144528 was a generous gift from Sanofi (Montpellier, France). 1,3-Benzylideneglycerol was prepared as described in Ref. 15Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Google Scholar. 2-AG and the other monoacylglycerols were prepared from 1,3-benzylideneglycerol and respective fatty acids as described earlier (15Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Google Scholar). An ether-linked analog of 2-AG (2-AG ether) (2-eicosa-5′,8′,11′,14′-tetraenylglycerol) was synthesized from 1,3-benzylideneglycerol and eicosatetraenyl iodide as described previously (15Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Google Scholar). Cells—Human promyelocytic leukemia HL-60 cells, human monocytic leukemia U937 cells, and THP-1 cells were grown at 37 °C in RPMI 1640 medium (Asahi Technoglass Co., Chiba, Japan) supplemented with 10% fetal bovine serum in an atmosphere of 95% air and 5% CO2. HL-60 cells were differentiated into macrophage-like cells by treatment with 100 nm 1,25-(OH)2vitamin D3 for 5 days. U937 cells and THP-1 cells were also differentiated by treatment with 100 nm 1,25-(OH)2vitamin D3 for 5 days. Human monocytes were separated from the peripheral blood of young healthy donors as follows: ¼ volume of 6% dextran T-500 (Amersham Biosciences) in saline was added to heparinized blood to sediment the erythrocytes, the supernatant (leukocyterich fraction) was aspirated and centrifuged at 400 × g for 10 min, the cells were washed once with Ca2+, Mg2+-free Hanks' balanced salt solution containing 5 mm HEPES (pH 7.4), and then the cells were transferred onto Lymphoprep™ (Axis Shield, Oslo, Norway) and centrifuged at 800 × g for 20 min. The mononuclear leukocyte fraction (the interface layer) was collected and washed with Hanks' balanced salt solution. Monocytes were separated from other mononuclear leukocytes by negative selection using a MACS monocyte isolation kit (Miltenyi Biotec Gmbh, Gladbach, Germany). The purity of the monocytes was 93% as assessed by a nonspecific esterase assay described below. Migration Assay—The migration of the differentiated HL-60 cells, U937 cells, THP-1 cells, and human monocytes was assayed using Transwell™ inserts (pore size, 5 μm) and 24-well culture plates (Corning Costar, Cambridge, MA). Briefly, the cells (106 for the differentiated HL-60 cells, U937 cells, and THP-1 cells and 2.5 × 105 for human monocytes) suspended in 0.1 ml of RPMI 1640 medium containing 0.1% bovine serum albumin were transferred to the Transwell™ insert (the upper compartment). 2-AG was dissolved in Me2SO and added to 0.6 ml of the RPMI 1640 medium containing 0.1% bovine serum albumin in the well of the culture plate (the lower compartment) (the final concentration of Me2SO was 0.2%). After the incubation at 37 °C (for 4 h for HL-60 cells, U937 cells, and THP-1 cells and 2 h for human monocytes) in an atmosphere of 95% air and 5% CO2, the number of cells that migrated from the upper compartment to the lower compartment was counted using a hemocytometer. NBT Reduction Assay—The NBT reduction assay was performed as described previously (28Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. J. Exp. Med. 1979; 149: 969-974Google Scholar) with some modifications. Cells were suspended in 25 mm HEPES-Tyrode's solution (pH 7.4) containing 0.05% NBT and incubated at 37 °C for 7 min. Phorbol 12-myristate 13-acetate was added to the cell suspension at a concentration of 4 μm, and the suspension was incubated for 30 min at 37 °C. After the addition of 10 mm EDTA to stop the reaction, the percentage of positive cells (blue-stained cells) was determined using a hemocytometer. Nonspecific Esterase Assay—Nonspecific esterase activity was assayed using an α-naphtyl acetate esterase assay kit (Sigma). Northern Blot Analysis—poly(A)+ RNAs (5 μg) from undifferentiated and differentiated HL-60 cells were electrophoresed in a 1.0% agarose-formaldehyde gel and transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences). The CB2 probe (human CB2 receptor cDNA SphI/SfiI digest; 524 bp) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (BD Biosciences) were labeled with [α-32P]dCTP (PerkinElmer Life Sciences) using the Megaprime DNA labeling system (Amersham Biosciences). Hybridization was performed at 60 °C for 16 h in QuikHyb solution (Stratagene). The filter was washed in 0.1× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate) containing 0.1% SDS at 65 °C and analyzed by a bioimaging analyzer BAS 1500 (Fuji Photo Film, Tokyo, Japan). Estimation of the Amount of Remaining 2-AG Following the Incubation with the Cells—HL-60 cells differentiated into macrophage-like cells (4 × 106) were suspended in 0.4 ml of RPMI 1640 medium. The cells were then incubated with 1 μm 2-AG for 30 min, 1 h, 2 h, and 4 h. Following the incubation, the supernatant was aspirated, and the lipids were extracted by the method of Bligh and Dyer (29Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). Butylated hydroxytoluene (final, 0.05%) was added to avoid lipid peroxidation, and 2-heptadecanoylglycerol was added as an internal standard. The lipids were fractionated by TLC using development with petroleum ether: diethyl ether:acetic acid (20:80:1, v/v) in a tank sealed with N2 gas. The area corresponding to standard monoacylglycerol was scraped off the TLC plate, followed by extraction from the silica gel by the method of Bligh and Dyer (29Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). The extraction was conducted in the presence of butylated hydroxytoluene (0.001%) in an N2 gas-sealed tube. The purified monoacylglycerols were converted to their 1-anthroyl derivatives and then analyzed with a high pressure liquid chromatography system equipped with a reverse phase column (CAPCELL PAK C18 SG120, 4.6 mm × 250 mm; Shiseido Co., Tokyo, Japan) and a fluorescence detector (excitation at 370 nm, emission at 470 nm). The mobile phase was acetonitrile:2-propanol:water (90:4:6, v/v), and the flow rate was 1.4 ml/min as described previously (30Kondo S. Kondo H. Nakane S. Kodaka T. Tokumura A. Waku K. Sugiura T. FEBS Lett. 1998; 429: 152-156Google Scholar). Statistical Analysis—Statistical analysis was performed using the Student's t test. 1,25-(OH)2vitamin D3 is known to induce the differentiation of HL-60 cells into macrophage-like cells (31Munker R. Norman A. Koeffler H.P. J. Clin. Invest. 1986; 78: 424-430Google Scholar). We first examined the effect of 1,25-(OH)2vitamin D3-treatment on several cellular markers of differentiation and the CB2 receptor mRNA level in HL-60 cells. As shown in Fig. 1A, the percentage of NBT reduction assay-positive cells in undifferentiated HL-60 cells was low (10.0%). On the other hand, the proportion of NBT reduction assay-positive cells was elevated to 65.7% following the differentiation with 1,25-(OH)2vitamin D3. A similar result was observed for nonspecific esterase-positive cells (data not shown). We next examined whether differentiated HL-60 cells actually possess the CB2 receptor mRNA. As shown in Fig. 1B, differentiated HL-60 cells were found to contain a substantial amount of the CB2 receptor mRNA. The ratio of the CB2 receptor mRNA/GAPDH mRNA in the differentiated HL-60 cells was almost the same as that in the undifferentiated HL-60 cells (the ratio of the CB2 receptor mRNA/GAPDH mRNA in the differentiated cells was 0.9 of that in the undifferentiated cells). We then investigated the effect of 2-AG on the motility of HL-60 cells. The addition of 1 μm 2-AG enhanced the migration of undifferentiated HL-60 cells to some extent, although the number of migrated cells was low; the percentages of the migrated cells were 0.05 and 0.30% for the control and 2-AG-stimulated cells, respectively. On the other hand, 1 μm 2-AG exerted dramatic effects on the migration of the HL-60 cells differentiated into macrophage-like cells by treatment with 1,25-(OH)2vitamin D3 for 5 days. The proportion of migrated cells was 3.4% for the control and 26.7% for the 2-AG-stimulated cells. The number of migrated HL-60 cells differentiated into macrophage-like cells was augmented with time (FIG. 2A). The addition of 1 μm 2-AG markedly accelerated the migration (Fig. 2A). The number of migrated cells also increased dose-dependently (Fig. 2B). The effect was observed from 10 nm 2-AG and reached a peak at 10 μm. The EC50 was around 430 nm. We next examined whether the cannabinoid receptors are involved in the 2-AG-induced migration of differentiated HL-60 cells. We found that the addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells markedly reduced the migration induced by 2-AG (Fig. 3A). On the other hand, the treatment of the cells with SR141716A, a cannabinoid CB1 receptor-specific antagonist, exerted only a slight effect on the 2-AG-evoked migration. These results indicate that the 2-AG-induced migration is mediated mainly via the CB2 receptor. The effect of PTX treatment on the 2-AG-induced migration of the differentiated HL-60 cells was next examined. As shown in Fig. 3B, pretreatment of the cells with PTX abolished the migration triggered by 2-AG, indicating that Gi/Go is involved in the 2-AG-induced migration. We further examined the effects of various inhibitors of intracellular signaling pathways on 2-AG-induced cell migration. As shown in Fig. 4, PD98059 (a p42/44 MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor; 20 μm), SB203580 (a p38 MAP kinase inhibitor; 20 μm), and Y-27632 (a Rho kinase inhibitor; 20 μm) suppressed the migration of differentiated HL-60 cells induced by 2-AG. On the other hand, LY294002 (a phosphatidylinositol 3-kinase inhibitor; 20 μm) and herbimycin A (a tyrosine kinase inhibitor; 20 μm) did not affect the migration markedly. We also confirmed that wortmannin (a phosphatidylinositol 3-kinase inhibitor; 200 nm) did not influence the migration at all (data not shown).Fig. 4Effects of various inhibitors on 2-AG-induced migration of HL-60 cells differentiated into macrophage-like cells by treatment with 1,25-(OH)2vitamin D3. HL-60 cells differentiated into macrophage-like cells were treated with various inhibitors (20 μm) at 37 °C for 1 h and then transferred to the Transwell™ (the upper compartment). A, vehicle (Me2SO) was added to the lower compartment. B, 2-AG (1 μm) was added to the lower compartment. The migration of the cells from the upper compartment to the lower compartment was examined as described under “Experimental Procedures.” The data are the means ± S.D. of four determinations. ***, p < 0.001 (compared with the control (open bar)).View Large Image Figure ViewerDownload (PPT) We then compared the activities of the various cannabinoid receptor ligands to induce migration. As shown in Fig. 5, the activity of 2-AG (1 μm) was the highest among those of the various cannabinoid receptor ligands examined in the present study. 2-AG ether (1 μm), a metabolically stable ether-linked analog of 2-AG, exhibited appreciable migration-inducing activity, although its activity was much lower than that of 2-AG. CP55940 (1 μm) and WIN55212–2 (1 μm) were also found to possess weak activities. However, anandamide (1 μm) was almost totally inactive. The activities of the various species of the 2-monoacylglycerols to induce migration were next compared (Fig. 6). The highest activity was observed with 2-AG (1 μm). Appreciable activities were also observed with 2-eicosa-5′,8′,11′-trienoylglycerol (1 μm) and 2-eicosa-5′,8′,11′,14′,17′-pentaenoylglycerol (1 μm). However, the activities of the other species such as 2-palmitoylglycerol, 2-oleoylglycerol, 2-linoleoylglycerol, and 2-docosa-4′,7′,10′,13′,16′,19′-hexaenoylglycerol were almost negligible. We then examined whether the 2-AG-induced migration is because of chemotaxis (the directional movement along a concentration gradient) or chemokinesis (stimulated movement in no specific direction). The migration of the cells from the upper compartment to the lower compartment in the absence of 2-AG was 4.4% (Fig. 7A). The proportion of migrated cells was elevated to 21.5% when 2-AG (1 μm) was added to the lower compartment. The migration was slightly reduced when 2-AG (1 μm) was added to both the upper compartment (with cells) and the lower compartment (18.5%). On the other hand, the presence of 2-AG (1 μm) in the upper compartment alone (with cells) did not evoke cell migration (5.2%). Because 2-AG is known to be rapidly metabolized by a variety of cells (10Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 173-192Google Scholar, 13Sugiura T. Waku K. Chem. Phys. Lipids. 2000; 108: 89-106Google Scholar), we examined whether exogenously added 2-AG exists as an intact molecule following the co-incubation with the cells. We found that more than 99% of the exogenously added 2-AG (1 μm) was metabolized within 30 min of incubation. Thus, it is rather difficult to determine whether the migration is because of chemotaxis or chemokinesis under the present experimental conditions using 2-AG as a stimulant. To settle this problem, we added 2-AG ether, a metabolically stable analog of 2-AG, instead of 2-AG (Fig. 7B). We found that the percentage of migrated cells when 2-AG ether (1 μm) was added to both the upper compartment (with cells) and the lower compartment (5.8%) was significantly lower than that observed when 2-AG ether (1 μm) was added only to the lower compartment (8.3%), the proportion of migrated cells in the former case being close to the level of the control (4.6%). We also found that the addition of 2-AG ether (1 μm) to the upper compartment alone (with cells) did not enhance the migration of cells from the upper compartment to the lower compartment (4.9%). These results strongly suggest that 2-AG ether elicited mainly chemotaxis rather than chemokinesis. We next investigated whether 2-AG induces the migration of other types of macrophage-like cells. In this study, we employed two types of human monocytic leukemic cells, U937 cells and THP-1 cells, which were differentiated by treatment with 1,25-(OH)2vitamin D3 before use as in the case of the HL-60 cells. We found that 2-AG (1 μm) significantly enhanced the migration of the differentiated U937 cells and THP-1 cells (Fig. 8), although the magnitude of augmentation was rather small compared with the case of the differentiated HL-60 cells. Finally, we examined whether human peripheral blood monocytes respond to 2-AG. As shown in Fig. 9, 2-AG (1 μm) markedly accelerated the migration of human monocytes. The effect of 2-AG was abolished by treatment of the cells with SR144528 (1 μm), a CB2 receptor-specific antagonist, as in the case of differentiated HL-60 cells. The cannabinoid CB2 receptor is a seven transmembrane, G protein-coupled receptor and is expressed abundantly in various types of inflammatory cells and immune competent cells such as macrophages, natural killer cells, and B lymphocytes (32Berdyshev E.V. Chem. Phys. Lipids. 2000; 108: 169-190Google Scholar, 33Parolaro D. Massi P. Rubino T. Monti E. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 319-332Google Scholar, 34Cabral G.A. Onaivi E.S. Biology of Marijuana. Taylor & Francis, London2002: 282-307Google Scholar). Previously (16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar), we examined in detail the structure-activity relationship of a number of CB2 receptor ligands using HL-60 cells, which express the CB2 receptor and exhibit a Ca2+ response when challenged with the CB2 receptor ligands. We found that the structure of 2-AG is strictly recognized by the CB2 receptor (16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar). The agonistic activity of 2-AG was most potent among various structural analogs. Noticeably, 2-AG acted as a full agonist at the CB2 receptor whereas anandamide acted as a weak partial agonist. Gonsiorek et al. (18Gonsiorek W. Lunn C. Fan X. Narula S. Lundell D. Hipkin R.W. Mol. Pharmacol. 2000; 57: 1045-1050Google Scholar) also demonstrated that 2-AG is a full agonist, and anandamide is a partial agonist using the membrane fraction of Sf9 cells transfected with the human CB2 receptor cDNA. We proposed that 2-AG, but not anandamide, is the intrinsic natural ligand for the cannabinoid CB2 receptor, and the CB2 receptor is primarily a 2-AG receptor (16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar). Not much information is currently available concerning the biological activities of 2-AG toward inflammatory cells and immune competent cells. Previously, Kaminski and co-workers (35Lee M. Yang K.H. Kaminski N.E. J. Pharmacol. Exp. Ther. 1995; 275: 529-536Google Scholar) reported that 2-AG affects lymphocyte proliferation. They also demonstrated that 2-AG suppresses the interleukin 2 gene expression in murine T lymphocytes through down-regulation of the nuclear factor (36Ouyang Y. Hwang S.G. Han S.H. Kaminski N.E. Mol. Pharmacol. 1998; 53: 676-683Google Scholar). In addition, Chang et al. (37Chang Y.H. Lee S.T. Lin W.W. J. Cell. Biochem. 2001; 81: 715-723Google Scholar) demonstrated recently that 2-AG inhibited the production of interleukin 6 in J774 macrophage-like cells. It remains unclear, however, whether these effects of 2-AG are mediated through the cannabinoid receptor. Recently, we found that 2-AG induces rapid phosphorylation and activation of the p42/44 MAP kinase in HL-60 cells (38Kobayashi Y. Arai S. Waku K. Sugiura T. J. Biochem. 2001; 129: 665-669Google Scholar). 2-AG-induced activation of the p42/44 MAP kinase was abolished when the cells were pretreated with either SR144528 or PTX, indicating that the response was mediated through the CB2 receptor and Gi/Go. We also found that rapid phosphorylation of the p38 MAP kinase and c-Jun N-terminal kinase takes place in 2-AG-stimulated HL-60 cells. 2T. Sugiura and Y. Kobayashi, unpublished results. The 2-AG-induced activation of the p38 MAP kinase and c-Jun N-terminal kinase has also been reported by several investigators (39Derkinderen P. Ledent C. Parmentier M. Girault J.A. J. Neurochem. 2001; 77: 957-960Google Scholar, 40Rueda D. Galve-Roperh I. Haro A. Guzman M. Mol. Pharmacol. 2000; 58: 814-820Google Scholar). These results strongly suggest that 2-AG plays some essential role in the inflammation and immune responses, although the exact physiological functions of 2-AG in inflammatory cells and immune competent cells still remain unclear. In this study, we explored the effect of 2-AG on the motility of HL-60 cells. We found that 2-AG induces the migration of HL-60 cells differentiated into macrophage-like cells (see Figs. 2, 3, 4, 5, 6, 7). Similar effects were observed with other macrophage-like cells of human origin such as U937 cells and THP-1 cells and human peripheral blood monocytes (see Figs. 8 and 9), suggesting that 2-AG-induced migration is a common event in human macrophages/monocytes. The 2-AG-induced migration of differentiated HL-60 cells was markedly reduced when the cells were pretreated with either SR144528 or PTX (Fig. 3), suggesting that the migration was mediated through the CB2 receptor and Gi/Go. Arachidonic acid and its metabolites did not participate in the 2-AG-induced migration, because free arachidonic acid was not capable of inducing the migration (data not shown). This was also confirmed by the fact that 2-AG ether was able to induce the migration (Fig. 5), although its activity was rather weak compared with that of 2-AG. On the other hand, the Rho kinase, MEK and p38 MAP kinase, were suggested to be involved in the 2-AG-induced migration of differentiated HL-60 cells, because Y-27632 (a Rho kinase inhibitor), PD98059 (a MEK inhibitor), and SB203580 (a p38 MAP kinase inhibitor) suppressed the migration (Fig. 4). The inhibition of cell migration by Y-27632 (1–100 μm) (41Adachi T. Vita R. Sannohe S. Stafford S. Alam R. Kayaba H. Chiara J. J. Immunol. 2001; 167: 4609-4615Google Scholar, 42Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Google Scholar), PD98059 (10–20 μm) (43Kampen G.T. Stafford S. Adachi T. Jinquan T. Quan S. Grant J.A. Skov P.S. Poulsen L.K. Alam R. Blood. 2000; 95: 1911-1917Google Scholar, 44Grimshaw M.J. Wilson J.L. Balkwill F.R. Eur. J. Immunol. 2002; 32: 2393-2400Google Scholar, 45Boehme S.A. Sullivan S.K. Crowe P.D. Santos M. Conlon P.J. Sriramarao P. Bacon K.B. J. Immunol. 1999; 163: 1611-1618Google Scholar), and SB203580 (10–50 μm) (42Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Google Scholar, 46Heuertz R.M. Tricomi S.M. Ezekiel U.R. Webster R.O. J. Biol. Chem. 1999; 274: 17968-17974Google Scholar) has already been reported for several types of cells stimulated with various chemoattractants, although there are conflicting results as to the inhibition by PD98059 (42Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Google Scholar, 46Heuertz R.M. Tricomi S.M. Ezekiel U.R. Webster R.O. J. Biol. Chem. 1999; 274: 17968-17974Google Scholar). The relationship between Rho kinase and p38 MAP kinase, as well as MEK, is known to be complicated. Ashida et al. (42Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Google Scholar) reported that Rho kinase is upstream of p38 MAP kinase in monocyte chemoattractant protein-1-stimulated THP-1 cells. Details of the intracellular signaling pathways for 2-AG-induced migration of HL-60 cells will be clarified in the future. The activity of 2-AG was highest among those of the various cannabinoid receptor ligands (Fig. 5). This is reasonable in view of the fact that 2-AG is the true endogenous ligand of the cannabinoid CB2 receptor. It has already been shown that 2-AG is present in appreciable amounts in various mammalian tissues (10Sugiura T. Kobayashi Y. Oka S. Waku K. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 173-192Google Scholar, 13Sugiura T. Waku K. Chem. Phys. Lipids. 2000; 108: 89-106Google Scholar). 2-Monoacylglycerols containing saturated, monoenoic, dienoic, and hexaenoic fatty acids did not exhibit any appreciable activity, whereas 2-eicosa-5′,8′,11′-trienoylglycerol and 2-eicosa-5′,8′,11′,14′,17′-pentaenoylglycerol induced the migration to some extent (Fig. 6). These results are in general agreement with the results of the Ca2+ transient experiments reported previously (16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar). We have found that the presence of the double bond at the Δ5-position is important for some characteristic conformation of the agonistic molecules (15Sugiura T. Kodaka T. Nakane S. Miyashita T. Kondo S. Suhara Y. Takayama H. Waku K. Seki C. Baba N. Ishima Y. J. Biol. Chem. 1999; 274: 2794-2801Google Scholar, 16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar). The migration of HL-60 cells induced by 2-AG was assumed to mainly involve chemotaxis rather than chemokinesis, because 2-AG ether elicited mainly chemotaxis (Fig. 7). This was also confirmed by the fact that the migration of HL-60 cells observed in the presence of 2-AG in both the upper and lower compartments was markedly reduced compared with the case of the presence of 2-AG only in the lower compartment, when 0.5 mm diisopropylfluorophosphate, a monoacylglycerol lipase inhibitor, was added to block the hydrolysis of 2-AG: 9.3 + 0.4, 26.7 + 2.1, and 19.5 + 3.3% for vehicle alone, 2-AG present only in the lower compartment, and 2-AG present in both compartments, respectively (the means ± S.D. of four determinations). 3S. Kishimoto, M. Gokoh, and T. Sugiura, unpublished results. We confirmed that 2-AG was rapidly metabolized during the co-incubation with the cells when the monoacylglycerol lipase inhibitor was not included in the incubation mixture as mentioned before. It cannot be ruled out, however, that some part of the migration induced by 2-AG was because of chemokinesis. Whatever the mode and the mechanism of action, the fact that 2-AG induces the migration of macrophage-like cells and monocytes is quite noticeable, because various types of proinflammatory molecules are known to induce the migration and recruitment of inflammatory cells. Very recently, Jorda et al. (47Jorda M.A. Verbakel S.E. Valk P.J. Vankan-Berkhoudt V. Maccarrone M. Finazzi-Agro A. Lowenberg B. Delwel R. Blood. 2002; 99: 2786-2793Google Scholar) also demonstrated that 2-AG induces the migration of mouse splenocytes and myeloid cells, yet the elucidation of the detailed mechanism of 2-AG-induced migration of these cells awaits further investigations. Previously, Gallily et al. (48Gallily R. Breuer A. Mechoulam R. Eur. J. Pharmacol. 2000; 406: R5-R7Google Scholar) reported that 2-AG suppresses the production of tumor necrosis factor α in lipopolysaccharide-stimulated mouse macrophages in vitro and in lipopolysaccharide-administered mice in vivo, although whether these effects of 2-AG are mediated through the CB2 receptor is uncertain. On the other hand, we found that the addition of 2-AG to HL-60 cells enhanced the production of chemokines such as interleukin 8 and MCP-1 through a CB2 receptor- and Gi/Go-dependent mechanism. 4S. Kishimoto and T. Sugiura, unpublished results. Based on the results of a previous investigation on chemokine production and the present study on cell migration, we assume that 2-AG acts as a stimulator or accelerator, rather than as a suppressor, of inflammation reactions and immune responses. As for Δ9-THC, it has been reported that Δ9-THC suppresses inflammation and immune responses in vivo (32Berdyshev E.V. Chem. Phys. Lipids. 2000; 108: 169-190Google Scholar, 33Parolaro D. Massi P. Rubino T. Monti E. Prostaglandins Leukotrienes Essent. Fatty Acids. 2002; 66: 319-332Google Scholar, 34Cabral G.A. Onaivi E.S. Biology of Marijuana. Taylor & Francis, London2002: 282-307Google Scholar). The mechanism by which Δ9-THC suppresses the inflammatory reactions and immune response has long remained obscure. Previously, we demonstrated that Δ9-THC is a weak partial agonist of the cannabinoid CB2 receptor (16Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Google Scholar). Bayewitch et al. (49Bayewitch M. Rhee M.H. Avidor-Reiss T. Breuer A. Mechoulam R. J. Biol. Chem. 1996; 271: 9902-9905Google Scholar) also reported that Δ9-THC acted as an antagonist toward the CB2 receptor. Noticeably, SR144528 and JTE-907, CB2 receptor antagonists/inverse agonists, inhibited inflammation in vivo (50Iwamura H. Suzuki H. Ueda Y. Kaya T. Inaba T. J. Pharmacol. Exp. Ther. 2001; 296: 420-425Google Scholar). It is possible, therefore, that Δ9-THC blocks the action of the endogenous natural ligand of the CB2 receptor, that is, 2-AG, thereby inducing the suppression of inflammatory reactions and immune responses. In conclusion, we found that 2-AG induces the migration of HL-60 cells differentiated into macrophage-like cells through the CB2 receptor-, Gi/Go-, and several other signaling molecule-dependent mechanisms. Similar effects were observed with other macrophage-like cells and human monocytes. The migration induced by 2-AG was mainly attributed to chemotaxis rather than chemokinesis. 2-AG is known to be generated from stimulated inflammatory cells and immune competent cells such as macrophages upon stimulation (20Bisogno T. Sepe N. Melck D. Maurelli S. De Petrocellis L. Di Marzo V. Biochem. J. 1997; 322: 671-677Google Scholar, 23Varga K. Wagner J.A. Bridgen D.T. Kunos G. FASEB J. 1998; 12: 1035-1044Google Scholar, 24Di Marzo V. Bisogno T. De Petrocellis L. Melck D. Orlando P. Wagner J.A. Kunos G. Eur. J. Biochem. 1999; 264: 258-267Google Scholar, 25Berdyshev E.V. Schmid P.C. Krebsbach R.J. Schmid H.H.O. FASEB J. 2001; 15: 2171-2178Google Scholar) through an increased phospholipid metabolism such as inositol phospholipid turnover. It is possible that 2-AG, derived from a variety of stimulated tissues and cells, plays physiologically and pathophysiologically essential roles during the course of inflammatory reactions and immune responses.
Referência(s)