Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0404252 on August 17, 2004

Published online before print August 17, 2004
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(Journal of Leukocyte Biology. 2004;76:1002-1009.)
© 2004 by Society for Leukocyte Biology

2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils

Saori Oka, Shinobu Ikeda, Seishi Kishimoto, Maiko Gokoh, Shin Yanagimoto, Keizo Waku and Takayuki Sugiura1

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa, Japan

1 Correspondence: Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan. E-mail: sugiurat{at}pharm.teikyo-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid receptor ligand. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, abundantly expressed in the brain, and the CB2 receptor, expressed in various lymphoid tissues such as the spleen. The CB1 receptor has been assumed to play an important role in the regulation of synaptic transmission, whereas the physiological roles of the CB2 receptor remain obscure. In this study, we examined whether the CB2 receptor is present in human eosinophils and found that the CB2 receptor is expressed in human peripheral blood eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB2 receptor. We then examined the effect of 2-AG on the motility of eosinophils. We found that 2-AG induces the migration of human eosinophilic leukemia EoL-1 cells. The migration evoked by 2-AG was abolished in the presence of SR144528, a CB2 receptor antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB2 receptor and Gi/o are involved in the 2-AG-induced migration. The migration of EoL-1 cells induced by 2-AG was suggested to be a result of chemotaxis. In contrast to 2-AG, neither anandamide nor free arachidonic acid elicited the migration. Finally, we examined the effect of 2-AG on human peripheral blood eosinophils and neutrophils and found that 2-AG induces migration of eosinophils but not neutrophils. These results suggest that the CB2 receptor and its endogenous ligand 2-AG may be closely involved in allergic inflammation accompanied by the infiltration of eosinophils.

Key Words: lipid mediators • monoacylglycerol • anandamide • chemotaxis • allergy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{Delta}9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors (cannabinoid receptors), thereby eliciting a variety of pharmacological responses in vitro and in vivo [1 ]. Two types of cannabinoid receptors have thus far been identified: the CB1 receptor, which is expressed abundantly in the nervous system, especially the brain [2 , 3 ], and the CB2 receptor, which is expressed predominantly in various lymphoid organs such as the spleen, tonsils, and lymph nodes [3 , 4 ]. The CB1 receptor has been considered to play an essential role in the attenuation of synaptic transmission [5 , 6 ], and the CB2 receptor is suggested to participate in the regulation of inflammatory reactions and immune responses [7 8 9 10 11 12 13 14 15 16 17 18 19 20 ], yet details still remain to be determined.

Two arachidonic acid (AA)-containing molecules have been identified as endogenous cannabinoid receptor ligands: anandamide [21 ] and 2-arachidonoylglycerol (2-AG) [22 , 23 ]. Anandamide and 2-AG exhibit various cannabimimetic activities. Notably, 2-AG acts as a full agonist toward both types of cannabinoid receptors (CB1 and CB2), whereas anandamide acts as a partial agonist toward these receptors [24 25 26 27 28 29 30 31 32 ]. Based on these experimental results, we have proposed that 2-AG, rather than anandamide, is the true, natural ligand for the cannabinoid receptors [25 26 27 , 33 , 34 ].

We then investigated in detail the biological activities and possible physiological significance of 2-AG, particularly in the immune system. We found that 2-AG induces the activation of p42/44 mitogen-activated protein kinase (MAPK) [35 ], p38 MAPK [36 ], and c-Jun N-terminal kinase in HL-60 cells [36 ]. The activation by 2-AG of p38 MAPK and c-Jun N-terminal kinase has also been demonstrated by several other investigators [37 , 38 ]. We also found that 2-AG induces the migration of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes [39 ]. Jorda et al. [40 ] also reported that 2-AG induced the migration of mouse splenocytes, and Walter et al. [41 ] demonstrated that 2-AG elicited the migration of mouse microglia cells. These observations strongly suggest that 2-AG is implicated in the regulation of several functions, such as the motility of certain types of leukocytes. There are marked differences in the amounts of CB2 receptor mRNA among the various types of leukocytes. The CB2 receptor is known to be expressed abundantly in B lymphocytes, natural killer (NK) cells, and macrophages/monocytes, whereas the levels of the CB2 receptor mRNA in polymorphonuclear leukocytes (mainly neutrophils) and T lymphocytes (T8 cells and T4 cells) were found to be rather low [42 ]. It is surprising that no information is available concerning the level of the CB2 receptor mRNA in eosinophils or the possible biological activities of 2-AG toward eosinophils, which are known to play crucial roles in the pathogenesis of a number of allergic diseases such as bronchial asthma. Therefore, it is essential to examine whether the CB2 receptor is expressed in eosinophils and whether its endogenous ligand 2-AG exerts some biological activity toward eosinophils.

In this study, we carefully investigated these issues. We found that the CB2 receptor is abundantly expressed in human eosinophils. We also found that 2-AG induces the migration of eosinophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
AA, essentially fatty, acid-free bovine serum albumin (BSA), nordihydroguaiaretic acid (NDGA), and sodium n-butyrate were purchased from Sigma Chemical Co. (St. Louis, MO). SR141716A was acquired from Biomol (Plymouth Meeting, PA). Pertussis toxin (PTX) was obtained from List Biological Laboratories (Campbell, CA). Anandamide was synthesized from AA and ethanolamine as described previously [24 ]. SR144528 was a generous gift from Sanofi (Montpellier, France). The rabbit anti-CB2 receptor polyclonal antibody and a CB2 receptor-blocking peptide (human CB2 receptor amino acids 20–33) were purchased from Cayman Chemical Co. (Ann Arbor, MI). The goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA). The enhanced chemiluminescence (ECL) reagent was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). 1,3-Benzylideneglycerol was synthesized from glycerol and benzaldehyde. 2-AG was prepared from 1,3-benzylideneglycerol and AA as described earlier [26 ]. An ether-linked, nonhydrolyzable analog of 2-AG (2-eicosa-5',8',11',14'-tetraenylglycerol; 2-AG ether) was synthesized from 1,3-benzylideneglycerol and eicosatetraenyl iodide as described previously [26 ].

Cells
Human eosinophilic leukemia EoL-1 cells were grown at 37°C in RPMI-1640 medium (Asahi Technoglass Co., Chiba, Japan) supplemented with 10% fetal bovine serum (Sigma Chemical Co.) in an atmosphere of 95% air and 5% CO2. The EoL-1 cells were differentiated by treatment with 0.5 mM sodium n-butyrate for 5 days before use. Human eosinophils and neutrophils were separated from the peripheral blood of healthy young donors as follows: A 1/4-vol 6% Dextran T-500 (Amersham Pharmacia Biotech) in saline was added to heparinized blood to sediment the erythrocytes. The supernatant (leukocyte-rich fraction) was aspirated and centrifuged at 400 g for 10 min. The cells were washed once with Ca2+, Mg2+-free Hanks’ balanced salt solution (HBSS; pH 7.4) containing 5 mM HEPES and 2 mM EDTA. The cells were then transferred onto LymphoprepTM (Axis Shield, Oslo, Norway) and centrifuged at 800 g for 20 min. The polymorphonuclear leukocyte fraction (the precipitate) was collected and washed with HBSS containing 5 mM HEPES and 2 mM EDTA. The eosinophils were purified by negative selection using magnetic cell sorter CD16 micro beads (Miltenyi Biotec Gmbh, Gladbach, Germany), and the neutrophils were purified by positive selection using the same separation kit. The purities of the eosinophils and neutrophils were 99%, as assessed by microscopic examination (Kimura staining) [43 ]. The viabilities of the eosinophils and neutrophils were above 99%, as assessed with the Trypan blue dye exclusion test. Usually, 4 x 106–8 x 106 eosinophils were obtained from 150 ml peripheral blood.

Migration assay
The migration of the EoL-1 cells and human peripheral blood eosinophils and neutrophils was assayed using TranswellTM inserts (pore size, 5 µm) and 24-well culture plates (Corning Costar, Cambridge, MA). Briefly, the cells (106 for the EoL-1 cells and 2x105 for human eosinophils and neutrophils), suspended in 0.1 ml RPMI-1640 medium containing 0.1% BSA were transferred to the TranswellTM insert (the upper compartment). 2-AG (1 µM) was dissolved in dimethyl sulfoxide (DMSO) and added to 0.6 ml RPMI-1640 medium containing 0.1% BSA, in the well of the culture plate (the lower compartment; the final concentration of DMSO was 0.2%). In the case of human eosinophils and neutrophils, NDGA (1 µM) was added to the upper and lower compartments to minimize possible generation of leukotrienes from 2-AG. After incubation at 37°C (4 h for EoL-1 cells and 1 h for human eosinophils and neutrophils) in an atmosphere of 95% air and 5% CO2, the number of cells that had migrated from the upper compartment to the lower compartment was counted using a hemocytometer.

Northern blot analysis
Total RNAs were obtained from EoL-1 cells using ISOGEN (Nippon Gene Co., Tokyo, Japan). Poly (A)+ RNA was isolated from the total RNA using Oligotex-dT30 Super (Takara Bio, Shiga, Japan). The poly (A)+ RNAs (5 µg) from the EoL-1 cells were electrophoresed in a 1.0% agarose-formaldehyde gel and transferred onto a Hybond-N+ nylon membrane (Amersham Biosciences, Little Chalfont, UK). The CB2 probe (human CB2 receptor cDNA SphI/SfiI digest, 524 base pairs), the CB1 probe (human CB1 receptor cDNA PstI digest, 699 base pairs), and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe (BD Biosciences, Palo Alto, CA) were labeled with [{alpha}-32P]deoxy-cytidine 5'-triphosphate (Perkin-Elmer Japan Co., Kanagawa) using the Megaprime DNA labeling system (Amersham Pharmacia Biotech). Hybridization was performed at 60°C for 16 h in QuikHyb solution (Stratagene, Cambridge, UK). The membrane was washed in 0.1x saline sodium citrate (SSC; 1xSSC=0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% sodium dodecyl sulfate (SDS) at 65°C and analyzed with a BAS 1500 bioimaging analyzer (Fuji Photo Film, Tokyo, Japan).

Reverse transcriptase-polymerase chain reaction (RT-PCR)
RT-PCR was performed as described previously [27 ]. Briefly, cDNA was synthesized from poly (A)+ RNA using superscript II RT and oligo(dT)18 as a primer. The primers used to amplify the CB2 receptor mRNA sequence were CB2-5 (sense: 5'-TGGCAGCGTGACTATGACCTT-3') and CB2-6 (antisense: 5'-ACCTCACATCCAGCCTCATTC-3'). The primers for the ß-actin mRNA sequence were ACT1 (sense: 5'-CAGAGCAAGAGAGGCATCCT-3') and ACT2 (antisense: 5'-AGGATCTTCATGAGGTAGTC-3'). PCR was carried out using 0.5 U Taq polymerase in 20 µl of the reaction mixture under the following conditions: Amplification of the human CB2 receptor gene was performed through 32 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and elongation at 75°C for 1 min; amplification of the ß-actin gene was performed through 24 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and elongation at 72°C for 1 min. Each gene was amplified logarithmically during the number of cycles used here. The samples were electrophoresed on a 2.0% agarose gel. Photographs were taken under ultraviolet light.

Western blot analysis
Human peripheral blood eosinophils and neutrophils (2x106 cells each) were lysed with 100 µl ice-cold lysis buffer containing 20 mM HEPES (pH 7.3), 1% Triton X-100, 10% glycerol, 1 mM EDTA, 50 mM sodium fluoride, 2.5 mM p-nitrophenyl phosphate, 1 mM diisopropyl fluorophosphate, 1 mM sodium orthovanadate, and 10 µg/ml leupeptin. The cell lysate was centrifuged at 15,000 g for 20 min, and the supernatant was aspirated into another tube. The total protein concentration was assessed using a bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Protein extracts (50 µg) were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% gels and transferred to nitrocellulose membranes. The membranes were blocked with a 5% nonfat dry milk solution in Tris-buffered saline containing Tris-buffered saline/0.05% Tween 20 (TBST) for 1 h at room temperature. They were then incubated overnight with the anti-CB2 receptor polyclonal antibody (1:5000) in TBS containing 5% BSA at 4°C. The membranes were washed with TBST and incubated with the anti-rabbit IgG HRP-linked antibody (1:2000) in TBST containing 5% nonfat dry milk for 1 h at room temperature. After a further washing, the membranes were analyzed with the ECL reagent.

Statistical analysis
The statistical analysis was performed using Tukey’s (see Figs. 3 , 5 , and 8 ) or Dunnett’s test (see Fig. 4 ). A P value <0.05 was considered to be significant.



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  		Figure 3. Effects of the CB2 receptor antagonist SR144528, the CB1 receptor antagonist SR141716A, and PTX treatment on 2-AG-induced migration of EoL-1 cells. (A) 2-AG (1 µM) or the vehicle DMSO was added to the lower compartment and SR144528 (1 µM) or SR141716A (1 µM), to the upper and lower compartments. The migration of the cells from the upper to the lower compartment was examined as described in Materials and Methods. (B) Cells were pretreated with PTX (100 ng/ml) for 16 h and then added to the TranswellTM inserts. 2-AG (1 µM) or the vehicle DMSO was added to the lower compartment. The migration of PTX-treated cells from the upper to the lower compartment was determined as described in Materials and Methods. The data are the mean ± SD of four determinations. **, P < 0.01.

 


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  		Figure 5. Effects of coincubation of EoL-1 cells with 2-AG ether on the migration from the upper to the lower compartment. Cells were incubated with or without 2-AG ether (1 µM) in the upper compartment of the TranswellTM inserts. The migration of the cells from the upper to the lower compartment in response to 2-AG ether (1 µM; added to the lower compartment) was examined as described in Materials and Methods. The data are the mean ± SD of four determinations. **, P < 0.01.

 


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  		Figure 8. Effects of SR144528 on the abilities of 2-AG to induce the migration of human peripheral blood eosinophils and neutrophils. The migration of human eosinophils (A) and neutrophils (B) was examined using the TranswellTM, as described in Materials and Methods. The incubation was carried out for 1 h. NDGA (1 µM) and SR144528 (1 µM) or the vehicle DMSO were added to the upper and lower compartments. The data are the mean ± SD of four determinations. **, P < 0.01.

 


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  		Figure 4. Comparison of the abilities of 2-AG, 2-AG ether, anandamide, and free AA to induce the migration of EoL-1 cells. The migration of cells from the upper to the lower compartment in response to these compounds (1 µM each) or the vehicle DMSO (added to the lower compartment) was examined as described in Materials and Methods. The data are the mean ± SD of four determinations. *, P < 0.05; **, P < 0.01 [compared with the control (vehicle)].

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of the CB2 receptor mRNA in EoL-1 cells
First, we examined whether EoL-1 cells contain the CB2 receptor mRNA by Northern blot analysis. As shown in Figure 1 , EoL-1 cells contained a detectable amount of the CB2 receptor mRNA. We detected two major transcripts (4.4 kb and 2.5 kb) for CB2 receptor mRNA in EoL-1 cells, the result being consistent with previous reports on HL-60 cells [4 , 27 ]. In contrast to the CB2 receptor mRNA, the amount of CB1 receptor mRNA in EoL-1 cells was found to be negligible.



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  		Figure 1. The CB1 and CB2 mRNA levels in EoL-1 cells. Northern blot analysis of the CB1 and CB2 receptor mRNAs was performed using poly (A)+ RNA as described in Materials and Methods. CB1 receptor gene transcript, 6.2 kb; CB2 receptor gene transcripts, 4.4 and 2.5 kb; GAPDH gene transcript, 1.3 kb. The result is representative of two separate experiments.

 
2-AG induces the migration of EoL-1 cells through a CB2 receptor-dependent and PTX-sensitive mechanism
We then investigated whether 2-AG induces the migration of EoL-1 cells. We found that the addition of 2-AG to the lower compartment of the TranswellTM induces migration from the upper to the lower compartment. The number of EoL-1 cells that migrated in response to 1 µM 2-AG added to the lower compartment increased with time (Fig. 2A ). The number 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 approximately 160 nM.



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  		Figure 2. 2-AG induces the migration of EoL-1 cells. (A) EoL-1 cells were added to TranswellTM inserts (the upper compartment), and 2-AG (1 µM) or the vehicle DMSO was added to the well of the culture plate (the lower compartment). The incubation was carried out for the indicated periods of time. •, 2-AG; {circ}, vehicle DMSO alone. (B) EoL-1 cells were added to the TranswellTM inserts (the upper compartment) and various concentrations of 2-AG or the vehicle DMSO were added to the well of the culture plate (the lower compartment). The incubation was carried out for 4 h. The migration of cells from the upper to the lower compartment was determined as described in Materials and Methods. The data are the mean ± SD of four determinations.

 
Whether the cannabinoid receptors are involved in the 2-AG-induced migration of EoL-1 cells was examined next. We found that the addition of SR144528 (1 µM), a CB2 receptor-specific antagonist, to the upper and lower compartments markedly reduced the migration of EoL-1 cells induced by 2-AG (Fig. 3A ). Conversely, only a slight effect was observed in the case of SR141716A (1 µM), a CB1 receptor-specific antagonist. It is apparent, therefore, that the 2-AG-induced migration is primarily mediated via the CB2 receptor. We then examined the effect of PTX treatment on the 2-AG-induced migration of EoL-1 cells. As demonstrated in Figure 3B , pretreatment of the cells with PTX abolished the migration triggered by 2-AG, indicating that Gi/o is involved in the 2-AG-induced migration.

We then compared the activity of 2-AG with that of 2-AG ether (a nonhydrolyzable analog of 2-AG), anandamide, and free AA. As shown in Figure 4 , 2-AG (1 µM) induced the migration of EoL-1 cells. 2-AG ether (1 µM) also evoked the migration of EoL-1 cells. However, its activity was apparently lower than that of 2-AG. In contrast to 2-AG and 2-AG ether, anandamide (1 µM) failed to induce the migration of EoL-1 cells. Free AA (1 µM) did not induce the migration of EoL-1 cells either, indicating that free AA, possibly generated from 2-AG during the incubation, is not involved in the 2-AG-induced migration of cells.

2-AG ether induces chemotaxis of EoL-1 cells
We then investigated whether the 2-AG-induced migration is a result of chemotaxis (directional movement along a concentration gradient) or chemokinesis (stimulated movement in no specific direction). In this experiment, we used 2-AG ether instead of 2-AG, as 2-AG has been shown to be rapidly degraded when incubated with cells in the upper compartment of the TranswellTM inserts [39 ]. As shown in Figure 5 , 4.9% of the EoL-1 cells migrated from the upper compartment to the lower compartment when 2-AG ether (1 µM) was added only to the lower compartment. Conversely, the proportion of migrating cells upon the addition of 2-AG ether (1 µM) to the upper compartment (with cells) and the lower compartment was low (0.8%), the proportion being almost the same as the control level (0.7%). 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 (0.7%). These results clearly indicate that 2-AG ether induced mainly chemotaxis.

Comparison of the expression of the CB2 receptor in human peripheral blood eosinophils and neutrophils
We then examined whether human peripheral blood eosinophils contain the CB2 receptor mRNA by RT-PCR. As shown in Figure 6A , human eosinophils contained an appreciable amount of the CB2 receptor mRNA. Conversely, the amount of the CB2 receptor mRNA in neutrophils was negligible.



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  		Figure 6. RT-PCR and Western blot analyses of the CB2 receptor in human peripheral blood eosinophils and neutrophils. (A) RT-PCR analysis. RT-PCR was performed on poly (A)+ RNAs as described in Materials and Methods. Eos, Eosinophil; Neu, neutrophil. The result is representative of two separate experiments. (B) Western blot analysis. Protein extracts (50 µg) were fractionated by SDS-PAGE on 12.5% gels, transferred to nitrocellulose membranes, and analyzed with the ECL reagent as described in Materials and Methods. The predicted molecular mass of the CB2 receptor is 39 kDa, based on its amino acid sequence. The band at 47 kDa is a possible glycosylated form of the CB2 receptor molecule. The result is representative of five separate experiments.

 
The expression of the CB2 receptor molecule was further examined by Western blot analysis using an anti-CB2 receptor antibody. As depicted in Figure 6B , eosinophils contained a major immunoreactive protein of 39 kDa and a minor immunoreactive protein of 47 kDa. The immunoreactive band at 39 kDa is the nonglycosylated form of the CB2 receptor, and the band at 47 kD is possibly a glycosylated form of the CB2 receptor. We confirmed that these immunoreactive bands were abolished when the antibody was preabsorbed with a CB2 receptor-blocking peptide (data not shown). In contrast to eosinophils, neutrophils contained only negligible amounts of immunoreactive proteins.

2-AG induces the migration of human eosinophils but not neutrophils
Next, we examined the effect of 2-AG on the human peripheral blood eosinophils and neutrophils. In this study, we added NDGA (1 µM), a lipoxygenase inhibitor, to minimize possible generation of leukotrienes. As shown in Figure 7A , 2-AG induced the migration of human eosinophils in a dose-dependent manner, as in the case of EoL-1 cells. In contrast to eosinophils, however, 2-AG did not induce the migration of human neutrophils (Fig. 7B) .



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  		Figure 7. Comparison of the abilities of 2-AG to induce the migration of human peripheral blood eosinophils and neutrophils. The activities of various concentrations of 2-AG to induce the migration of human eosinophils (A) and neutrophils (B) were examined using the TranswellTM, as described in Materials and Methods. The incubation was carried out for 1 h. NDGA (1 µM) was added to the upper and lower compartments. The data are the mean ± SD of four determinations.

 
We then examined the effects of SR144528, a CB2 receptor antagonist. We found that the migration of eosinophils induced by 2-AG (1 µM) was completely blocked by the addition of SR144528 (1 µM; Fig. 8A ). Conversely, migration was not observed with neutrophils that were exposed to 2-AG (1 µM) added to the lower compartment of the TranswellTM in the presence or absence of SR144528 (1 µM; Fig. 8B ). We confirmed that free AA (1 µM) did not induce the migration of human eosinophils under the present experimental conditions (data not shown). These results clearly indicate that the 2-AG-induced migration of human eosinophils is mediated via the CB2 receptor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Eosinophils are crucially involved in the pathogenesis of a variety of allergic diseases including bronchial asthma. The infiltration of eosinophils into airway tissues as well as bronchial hyper-responsiveness and airway edema are recognized as central features of bronchial asthma [44 , 45 ]. Eosinophils produce and release several types of unique, bioactive molecules such as major basic protein, eosinophil cationic protein, eosinophil peroxidase, and eosinophil-derived neurotoxin [44 , 45 ]. These cationic proteins are cytotoxic and have been assumed to be closely implicated in the pathogenesis of allergic reactions, such as bronchial hyper-reactivity, through damage to surrounding tissues and cells. Eosinophils are also known to generate several types of lipid mediators such as the platelet-activating factor (PAF) and leukotriene C4 [45 ]. PAF and leukotriene C4 possess a variety of potent biological activities and exert profound effects on target tissues and cells, such as bronchoconstriction; these lipid mediators have also been suggested to be involved in several steps of allergic reactions [45 ]. Detailed studies of the pathophysiological roles of eosinophils are thus essential for a better understanding of the pathogenesis of various allergic diseases, including bronchial asthma.

In this study, we examined whether the CB2 receptor, which is known to be expressed in various lymphoid tissues, is present in eosinophils. We found that a detectable amount of the CB2 receptor mRNA is present in EoL-1 cells (Fig. 1) . We also found that the CB2 receptor is expressed in human peripheral blood eosinophils (Fig. 6) . In contrast to eosinophils, neutrophils did not contain an appreciable amount of the CB2 receptor (Fig. 6) . This is in agreement with the observation by Galiegue et al. [42 ] that the level of CB2 receptor mRNA is markedly lower in polymorphonuclear leukocytes (mainly neutrophils) than in B lymphocytes, NK cells, and macrophage/monocytes, although they did not examine the level in eosinophils. To date, no information has been available as to whether the CB2 receptor is present in eosinophils. This is the first study showing that the CB2 receptor is expressed in eosinophils, which are involved in various allergic reactions, whereas neutrophils are involved in acute inflammation; it is tempting, therefore, to postulate that the CB2 receptor participates in allergic inflammation rather than acute inflammation involving the infiltration of neutrophils. In relation to this, it is interesting to note that several investigators have demonstrated that cannabinoids bias the immune response toward T helper cell type 2 (Th2) and away from Th1 [11 ].

So far, little is known concerning the biological activities of endogenous CB2 receptor ligands toward eosinophils. In this study, we found that 2-AG evoked the migration of EoL-1 cells (Fig. 2) . The migration of EoL-1 cells induced by 2-AG was mediated via the CB2 receptor and Gi/o; the addition of the CB2 receptor antagonist SR144528 or pretreatment of the cells with PTX abolished the migration induced by 2-AG (Fig. 3) . A remarkable observation was that 2-AG and 2-AG ether, a nonhydrolyzable analog of 2-AG, induced the migration of EoL-1 cells, and anandamide was found to be less potent (Fig. 4) . The very low ability of anandamide to induce the migration of EoL-1 cells is not surprising, as anandamide was found to act as a weak, partial agonist toward the CB2 receptor [27 , 29 , 31 , 35 , 39 ]. In contrast to anandamide, 2-AG was found to act as a full agonist in many cases [27 , 30 31 32 , 35 , 39 ]. We have previously proposed that 2-AG rather than anandamide is the true, natural ligand for the CB2 receptor [27 ]. The result of the present investigation, that 2-AG exhibited strong activity toward eosinophils, whereas anandamide did not (Fig. 4) , is consistent with this notion. It is noticeable that 2-AG induced the migration of not only EoL-1 cells but also human peripheral blood eosinophils (but not neutrophils; Figs. 7 and 8 ). Previously, several investigators have demonstrated that 2-AG induced the migration of 32D/granulocyte-colony stimulating factor receptor (G-CSF-R) cells transfected with the CB2 receptor gene, mouse splenocytes [40 ], and microglia cells [41 ]. We also found that 2-AG induced the migration of HL-60 cells that had been differentiated into macrophage-like cells and human peripheral blood monocytes through a CB2 receptor-dependent mechanism [39 ]. However, there have been no data concerning eosinophils. The present study has provided the first evidence that 2-AG induces the migration of eosinophils.

2-AG has been shown to be metabolized rapidly when incubated with cells [39 , 46 , 47 ]; it is thus rather difficult to determine whether 2-AG induces chemotaxis or chemokinesis using 2-AG itself as a ligand in a migration assay. In this study, we used 2-AG ether, a nonhydrolyzable analog of 2-AG, instead of 2-AG. We found that 2-AG ether induced the chemotaxis of EoL-1 cells (Fig. 5) . This strongly suggests that the 2-AG-induced migration of EoL-1 cells is primarily attributable to chemotaxis. Similar results have been obtained for HL-60 cells differentiated into macrophage-like cells [39 ]. Conversely, Jorda et al. [40 ] reported that 2-AG induced the chemotaxis and chemokinesis of 32D/G-CSF-R cells transfected with the CB2 receptor gene.

The mechanisms underlying the 2-AG-induced migration of eosinophils are not yet fully elucidated. Previously, we found that 2-AG induced the activation of p42/44 MAPK [35 ]. The activation of p42/44 MAPK has been assumed to be involved in the migration of HL-60 cells differentiated into macrophage-like cells, as the addition of PD98059, a p42/44 MAPK kinase inhibitor, to HL-60 cells markedly reduced the migration induced by 2-AG [39 ]. It is conceivable that p42/44 MAPK is involved in the 2-AG-induced migration of eosinophils as well; recently, we obtained evidence that a rapid phosphorylation of p42/44 MAPK takes place in 2-AG-stimulated EoL-1 cells (S. Oka, S. Ikeda, and T. Sugiura, unpublished results). The involvement of MAPK in eotaxin-induced eosinophil migration has already been reported by several investigators [48 , 49 ]. We also found that 2-AG induced rapid actin polymerization in EoL-1 cells (S. Oka and M. Gokoh, unpublished results). Such a 2-AG-induced actin polymerization appears to be closely involved in the 2-AG-induced migration of EoL-1 cells, as actin rearrangement is indispensable for cell migration. Further studies are required for a thorough elucidation of the intracellular mechanisms underlying the 2-AG-induced migration of eosinophils.

A number of endogenous substances have been demonstrated to act as chemotactic agents for eosinophils: e.g., eotaxin, regulated on activation, normal T expressed and secreted (RANTES), monocyte chemotactic protein-2 (MCP-2), MCP-3, MCP-4, macrophage inflammatory protein-1{alpha}, C5a, and several lipid mediators such as PAF and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) [44 , 50 51 52 53 ]. These chemoattractants are considered to play important roles in the infiltration by eosinophils that have migrated through the endothelium to allergic sites. Some of these chemoattractants are rather selective for eosinophils, and others are not. For example, 5-oxo-ETE is a selective chemoattractant for eosinophils [50 51 52 ], whereas PAF is not selective and induces the migration of eosinophils and neutrophils [53 ]. It is apparent that the chemoattractants selective for eosinophils, rather than the nonselective ones, play central roles in various allergic reactions, as the accumulation of eosinophils, rather than neutrophils, is a common feature of allergic inflammation. In this regard, 2-AG is a quite notable molecule, as its intrinsic receptor, i.e., the CB2 receptor, is expressed in eosinophils but not in neutrophils (Fig. 6) . Moreover, recently, we found that the level of 2-AG was markedly elevated in a mouse ear following a challenge with oxazolone, which induces allergic inflammation in sensitized mice (S. Oka, S. Ikeda, and S. Yanagimoto, unpublished results). As for the generation of 2-AG, several investigators have demonstrated that 2-AG was produced and released from several types of inflammatory cells such as macrophages following stimulation [46 , 54 55 56 ]. It is possible that 2-AG released from inflammatory cells upon stimulation plays an important role in the accumulation of eosinophils at allergic sites. Probably, 2-AG and other chemoattractants, such as 5-oxo-ETE, eotaxin, and RANTES, work in concert to induce the infiltration of eosinophils. In addition to this, 2-AG may exert other unknown effects on eosinophils. Detailed studies of the pathophysiological roles of 2-AG and the CB2 receptor in allergic reactions are thus essential for a better understanding of the pathogenesis of various allergic diseases such as bronchial asthma.

In conclusion, we found that the cannabinoid CB2 receptor is abundantly expressed in human eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB2 receptor. We also found that 2-AG, the endogenous, natural ligand for the CB2 receptor, induces the migration of eosinophils but not neutrophils. These results strongly suggest a close involvement of 2-AG and the CB2 receptor in allergic inflammation involving the infiltration of eosinophils.

Received April 23, 2004; revised July 7, 2004; accepted July 18, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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