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Originally published online as doi:10.1189/jlb.0205111 on April 21, 2005

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(Journal of Leukocyte Biology. 2005;78:231-238.)
© 2005 by Society for Leukocyte Biology

Reduced endocannabinoid immune modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders

Jack C. Sipe1, Nathalie Arbour2, Alexandra Gerber and Ernest Beutler

Departments of Molecular and Experimental Medicine and Neuropharmacology, The Scripps Research Institute, La Jolla, California

1 Correspondence: Department of Molecular and Experimental Medicine, The Scripps Research Institute (MEM-215), 10555 North Torrey Pines Road, La Jolla, CA 92037. E-mail: jcsipe{at}scripps.edu


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ABSTRACT
 
Immune system responsiveness results from numerous factors, including endogenous cannabinoid signaling in immunocytes termed the "immunocannabinoid" system. This system can be an important signaling pathway for immune modulation. To assess the immunomodulating role of the cannabinoid 2 (CB2) receptor, we sought polymorphisms in the human gene, identified a common dinucleotide polymorphism, and investigated its effect on endocannabinoid-induced inhibition of T lymphocyte proliferation. The CB2 cDNA 188–189 GG/GG polymorphism predicts the substitution of glutamine at amino acid position 63 by arginine. T lymphocytes from CB2 188–189 GG/GG homozygotes had approximately twofold reduction of endocannabinoid-induced inhibition of proliferation compared with cells from CB2 188–189 AA/AA homozygotes. In GG/GG subjects, the reduced endocannabinoid inhibitory response was highly significant for N-arachidonylglycine and nearly significant for 2-arachidonylglycerol, and a specific CB2 receptor antagonist partially blocked these effects. Also, patients with autoimmune diseases had an increased prevalence of the homozygous GG/GG genotype. Collectively, these results demonstrate reduced endogenous fatty acid amide immunomodulatory responses in individuals with the CB2 188–189 GG/GG genotype and suggest that this CB2 gene variation may be a risk factor for autoimmunity. The results also support the proposition that the CB2 receptor may represent a novel pharmacological target for selective agonists designed to suppress autoreactive immune responses while avoiding CB1 receptor-mediated cannabinoid adverse effects.

Key Words: immunomodulation • endogenous cannabinoid • genetic variation • autoimmunity


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INTRODUCTION
 
Cannabinoids exhibit neurobehavioral and immunosuppressive effects in vivo that imply the existence of a mammalian endogenous cannabinoid system (ECS). The cannabinoid 1 (CB1) receptor is densely expressed in the central nervous system and mediates cannabinoid neurobehavioral effects [1 , 2 ], whereas the CB2 receptor is highly expressed on the surface of many types of immune cells, such as B and T lymphocytes and monocytes/macrophages, and is thought to be an important mediator of cannabinoid-induced, immunosuppressive effects [3 , 4 ]. Exogenous and endogenous cannabinoids have similar effects in mammals, and the components of the ECS that modulate the immune system have been referred to as the immunocannabinoid system [5 ]. This system is composed of the CB2 receptor, which is expressed at ten- to 100-fold greater levels on immune cells than the CB1 receptor [4 ], the naturally occurring, endogenous cannabinoid ligands (fatty acid amides), and the principal endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH) [5 ]. These components are present in human dendritic cells [6 ] and in most other effector cells of the immune system [7 ]. Recent reviews have detailed the functional complexity of the cannabinoid receptors, signaling pathways, and modulation of immune responses by cannabinoid receptor ligands [5 , 7 8 9 10 ].

The immunocannabinoid system is involved in immune regulation by suppression of cell activation, modulation of T helper cell types 1 and 2 (Th1 and Th2; balance) [11 ], inhibition of proinflammatory cytokine production [9 ], and nuclear factor-{kappa}B-dependent apoptosis [12 ]. Cannabinoid receptors are members of the G protein-coupled receptor family and are constituitively expressed in immunocytes [7 ]. In addition, their expression levels in immune cells can be modulated by activation factors [5 ]. CB2 receptors, which are expressed at higher levels than CB1 receptors on many types of immune cells, appear to have tonic activity [7 ] and have been shown to mediate suppressive effects on effector cells such as macrophages, natural killer (NK) cells, B and T lymphocytes [7 , 13 ], and Th1 proinflammatory cytokines [9 ]. Endogenous fatty acid amide CB2 receptor ligands or agonists are thus in a position to modulate innate and adaptive immune responses. Endocannabinoids could be important in regulating the amplitude and duration of self-recognition and immune responses in health and disease states such as autoimmune disorders. One example of a cannabinoid responsive autoimmune disorder is multiple sclerosis (MS), where cannabinoid administration may have some beneficial effects in MS patients [14 , 15 ] but may also be associated with CB1 receptor-mediated, adverse effects [16 ].

To investigate the functional contribution of the CB2 receptor to immune cell regulation, we searched for naturally occurring genetic variations in the human CB2 gene that might cause impairment of immune modulation as a result of altered CB2 receptor function. A missense polymorphism was identified at CB2 cDNA position 188–189, which results in a dinucleotide conversion of AA to GG and predicts a nonconservative amino acid substitution of glutamine by arginine at position 63 (Q63R). A flow cytometry-based assay was developed to quantify inhibition of T lymphocyte proliferation by fatty acid amides in subjects homozygous for the CB2 188–189 AA/AA and GG/GG genotypes and in heterozygotes. To probe the association of this CB2 gene variation with autoimmunity, subjects with several autoimmune disorders were genotyped for this polymorphism. The results presented here provide a link between the CB2 188–189 GG/GG variant in humans, its increased prevalence in autoimmunity and decreased endocannabinoid inhibition of T lymphocyte proliferation in individuals with this genetic variation.


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MATERIALS AND METHODS
 
Human subjects
The human procedures used in this study were performed in compliance with relevant laws and institutional guidelines, and the Scripps Human Subjects Committee/Institutional Review Board (La Jolla, CA) approved this study. The subjects invited to participate were volunteers in a normal blood donor program in San Diego, California, where the medication history and demographic information of the healthy donors was known. All subjects’ samples were obtained with informed consent, and the samples and medical histories were made anonymous before being sent to the investigators. Blood obtained from 26 healthy donors, homozygous for the CB2 188–189 AA/AA or GG/GG genotypes, was used for the T lymphocyte proliferation assay. In addition, 519 Caucasian controls and 102 Caucasian autoimmune disease subjects from a local medical screening clinic consented to blood samples for CB2 genotyping. The 102 autoimmune disease subjects included 72 with MS, 20 with rheumatoid arthritis, four with systemic lupus, and six with myasthenia gravis. All Caucasian subjects were genotyped as described below.

CB2 genotyping
The unique coding exon of the CB2 gene was sequenced using DNA obtained from buffy coat of blood. The CB2 cDNA 188–189 AA -> GG polymorphism was identified as potentially, functionally significant because of the predicted substitution of glutamine at amino acid 63 by arginine, an amino acid structurally located near the nadir of the first intracellular CB2 signaling loop [17 ]. Genotyping at the CB2 cDNA 188–189 locus was performed by allele-specific oligonucleotide hybridization [18 ]. Linkage of the CB2 188–189 GG/GG genotype was investigated with three other synonymous polymorphisms in the CB2 coding region, including cDNA 465 C -> T, 660 G -> A, and 846 T -> C, which predict no amino acid substitutions (Fig. 1 ). All of these polymorphisms were in complete linkage disequilibrium with the CB2 188–189 polymorphisms in 36 subjects; 16 GG/GG homozygotes were all homozygous for mutant alleles T, A, and C, respectively.



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  		Figure 1. Schematic representation of CB2 gene polymorphisms in this study. The solid arrow points to the region of the CB2 188–89 (Q63R), the only nonsynonymous coding polymorphism. The other single nucleotide polymorphisms, indicated by dashed arrows, are synonymous polymorphisms that predict no amino acid change.

To evaluate whether the CB2 GG/GG genotype is associated with altered immune function and to maximize the chances of detecting a difference in CB2 function, cells obtained from healthy donors with one of the two CB2 188–189 homozygous genotypes, AA/AA or GG/GG, were used as the primary outcome measure, and heterozygotes were also studied. The blood from healthy donors was used for the T lymphocyte proliferation assay described below. To maximize statistical power and specificity, the donors of different CB2 genotypes were paired using stringent clinical criteria. We carefully matched ethnicity, gender, and age within 10 years and hormone status in the case of women to pair eight donor subjects with the CB2 AA/AA genotype with eight CB2 GG/GG subjects and two heterozygotes for analysis in the proliferation assays. Pairing of subjects with specific CB2 genotypes was assigned by a coworker with no knowledge of the assays and before the assays had been performed. Those performing the functional and flow cytometry assays were blinded to all clinical and genotype information. The subjects whose CD3-induced T lymphocyte proliferation was less than 10% were excluded from analysis, as inhibition of proliferation cannot be measured when there is no significant proliferation. Data from the remaining 10 subjects (out of the 26 subjects used for functional assays) without pairs were also included in a separate unpaired analysis.

Functional assay: inhibition of anti-CD3-induced T cell proliferation in peripheral blood mononuclear cells (PBMC)
To simulate activation of T lymphocytes by antigen-presenting cells and proliferation in vivo, total PBMC of 50 ml venous blood samples were obtained by Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Approximately 40 x 106 PBMC from each donor were labeled for 10 min with 2.5 µM 5,6-carboxyfluorescein succimidyl ester (CFSE; Molecular Probes, Eugene, OR) in RPMI medium (Gibco-Invitrogen Corp., Grand Island, NY) containing 0.1% human AB serum at 37°C. Labeling of cells was confirmed in each assay by fluorescence microscopy. CFSE-labeled PBMC were washed twice, resuspended in complete RPMI tissue-culture medium containing 10% human AB serum, and counted. During in vitro culture, CFSE-labeled PBMC lost 50% of the CFSE intensity at every cell division, which could be detected by flow cytometry.

The endocannabinoids tested in the experimental assays included 2-arachidonlyglycerol (2-AG), a known CB1 and CB2 endogenous agonist, N-arachidonlyglycine (NAGly), an endogenous fatty acid amide synthesized in mammalian nervous system and lymphoid tissues [19 ], and a smaller number of studies using anandamide (n-arachidonylethanolamide; ANA), which showed results similar to 2-AG [20 ]. These fatty acid amides soluble in ethanol were assayed over a range of concentrations (see below) for percent inhibition of anti-CD3-induced T lymphocyte proliferation and were compared with untreated ethanol controls. PBMC, 1 x 106 per well, were incubated in a 12-well tissue-culture plate for 4 days with 500 ng/ml anti-CD3 antibody [clone HIT3a immunoglobulin G2a (IgG2a), BD PharMingen, San Diego, CA] in the presence or absence of NAGly (Cayman Chemicals, Ann Arbor, MI), 2-AG, or ANA (Cayman Chemicals or Sigma Chemical Co., St. Louis, MO) at concentrations 10–4 M, 10–5 M, 10–6 M, 10–7 M, and 10–8 M.

Some PBMC cultures treated with anti-CD3 and fatty acid amides were exposed to 5 µM and 1 µM SR144528 (CB2-specific antagonist) or SR141716A (CB1-specific antagonist) in dimethyl sulfoxide (DMSO) to evaluate blocking of the NAGly or 2-AG responses. Control cultures included PBMC without anti-CD3, PBMC with anti-CD3 alone, with an IgG2a isotype control, SR144528 alone, and SR141716A alone, and diluent controls—ethanol or DMSO—equivalent to fatty acid amide dilutions or antagonist dilutions. An investigator (A. G.) with no knowledge of the subjects’ genotypes performed all assays.

Flow cytometry method
After 4 days in tissue culture, cells were stained with anti-CD3 antibody conjugated to peridin-chlorophyll-protein with appropriate staining controls and then fixed in 1% (v/v) formaldehyde in phosphate-buffered saline. Cells were acquired on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and analyzed with Cell Quest Pro (Becton Dickinson) and FlowJo (Treestar, San Carlos, CA) software, as previously reported [21 ]. Gating on CD3-positive lymphocytes, the percent of proliferating cells was assessed as being CFSE-low compared with the negative control (no anti-CD3 antibody). The percent of inhibition of proliferation induced by NAGly and 2-AG was compared with ethanol control at each concentration listed above. To analyze inhibition of T lymphocyte proliferation as the primary outcome measure, only data from experiments with at least 10% anti-CD3-induced proliferation were used, as data analysis of inhibition could only be carried out on proliferating T lymphocytes.

Analysis of data
The percent inhibition of T lymphocyte proliferation compared with the ethanol control at day 4 in tissue culture was the primary outcome measure in all subjects and pairs. The mean percent inhibition of lymphocyte proliferation by NAGly and 2-AG at each concentration was analyzed using the Wilcoxin matched-pairs test, the paired t-test, and the Mann-Whitney test for the unpaired subjects analysis. To determine whether the prevalence of the homozygous genotype differed from controls in autoimmune diseases, the distribution of the CB2 188–189 alleles and CB2 AA/AA or GG/GG genotypes in a group of Caucasian autoimmune disease subjects and Caucasian European controls was compared using GraphPad InStat® software with the Fishers Exact test to determine two-sided P values reported throughout.


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RESULTS
 
Inhibition of lymphocyte proliferation by endocannabinoids is reduced in subjects with the CB2 188–189 GG/GG genotype
An example of the flow cytometry-based assay depicting T lymphocyte proliferation in response to anti-CD3 in the presence or absence of NAGly or 2-AG from one matched subject pair is shown in Figure 2 , where A, panels 1 and 4 show that the proliferation induced by anti-CD3 was well-detected in our assay compared with the nonstimulated cells for both donors regardless of their CB2 genotype. The mean percent T lymphocyte proliferation induced by anti-CD3 was similar in both CB2 genotype subject groups at 43.6% in AA/AA subjects and 51.7% in GG/GG subjects, a difference that was not statistically significant. To assess in total PBMC whether T lymphocytes from subjects with the CB2 188–189 AA/AA and GG/GG genotypes respond differently in vitro to endogenous fatty acid amide ligands, we compared each ligand’s percent inhibition of anti-CD3-induced T lymphocyte proliferation to diluent control PBMC cultures. For the subject pair depicted in Figure 2 , it is obvious in Figure 2A , panels 2 and 5 and 3 and 6, respectively, that 2-AG and NAGly, at a concentration of 10–7 M, were able to induce a significant inhibition of the anti-CD3-induced proliferation for the AA/AA donor (Fig. 2A , panels 2 and 3), whereas the percent inhibition was minimal for the paired GG/GG donor (Fig. 2A , panels 5 and 6). In Figure 2B , dose responses are shown for NAGly and 2-AG for one representative AA/AA donor.



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  		Figure 2. Anti-CD3-induced proliferation of T cells in PBMC is inhibited by endogenous cannabinoids. PBMC, labeled with CFSE, a cytoplasmic fluorescent marker that allows analysis of cell proliferation, were cultured in the presence of anti-CD3 for 4 days and then labeled for CD3. CD3-gated cells are shown for CFSE analysis. Results from one pair of donors are shown. (A, 1–3) Donor AA/AA; (4–6), donor GG/GG; (1 and 4), comparison of anti-CD3 and negative control; shaded area, cells in culture without anti-CD3 antibody (negative control); solid line, cells in culture with anti-CD3 in the presence of ethanol (positive control). (2 and 5) Inhibition induced by 2-AG; solid line, cells in culture with anti-CD3 in the presence of ethanol, the diluent used for the cannabinoids (positive control); cross-hatched area, cells in culture with anti-CD3 in the presence of 2-AG 10–7 M. (3 and 6) Inhibition induced by NAGly; solid line, cells in culture with anti-CD3 in the presence of ethanol, the diluent used for the cannabinoids (positive control); dotted line, cells in culture with anti-CD3 in the presence of NAGly 10–7 M. (B) Dose responses for NAGly and 2-AG for one representative AA/AA donor. Solid line, Cells in culture with anti-CD3 in the presence of ethanol (positive control); shaded area, cells in culture with 10–4M 2-AG or 10–6 M NAGly; dotted line, culture with 10–7M 2-AG or 10–7 NAGly. The table beside the graph describes the percentage of T cells that has proliferated during the incubation period, such that they are CFSE-low, as depicted on the graphs. The percentage of inhibition is the percentage of proliferation that was inhibited by the treatment in comparison with the control (ethanol). n.a., Not applicable.

Results were compared amongst eight matched CB2 188–189 AA/AA and GG/GG subject genotype pairs. To express the results across all subject pairs consistently, the percent of inhibition obtained for the GG/GG individual of each pair was subtracted from the percent of inhibition obtained for the AA/AA individual of the same pair, as the latter genotype is listed in GenBank as "wild-type" or normal. Therefore, values above zero mean greater inhibition by the fatty acid amide ligands in T cells from AA/AA individuals than in T cells from GG/GG individuals, a value of 0 indicates no difference between both individuals within the same pair, and a negative value indicates that inhibition was greater in the GG/GG individuals (Fig. 3 ). In most subject pairs (Fig. 3) , at all concentrations of NAGly and 2-AG, the differences in the percent inhibition showed that inhibition was consistently less in the CB2 GG/GG subjects than in the matched AA/AA subjects. The differences observed were more significant for lower (10–7 M) and more physiological concentration of both ligands.



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  		Figure 3. Inhibition of lymphocyte proliferation by fatty acid amides in matched CB2 188–189 donor genotype pairs. (A) Percent difference inhibition of lymphocyte proliferation induced by NAGly, % AA/AA minus % GG/GG. (B) Percent difference inhibition of lymphocyte proliferation produced by 2-AG, % AA/AA minus % GG/GG. Note that the solid line connects data points from a single pair in which the AA/AA subject had an abnormally low starting total CD3 lymphocyte count.

The mean percent inhibition of anti-CD3-induced T lymphocyte proliferation was analyzed in 16 individuals (eight matched pairs) with CB2 188–189 AA/AA and GG/GG genotypes. At 10–7 M concentration of NAGly, the mean percent inhibition of T lymphocyte proliferation averaged 60.9% in AA/AA subjects compared with 31.2% in GG/GG subjects (Fig. 4A ; P=0.0078, Wilcoxin matched-pairs test), and with 2-AG at 10–7 M, the mean percent inhibition in GG/GG subjects was decreased less significantly (Fig. 4B ; P=0.07, Wilcoxin matched-pairs test). Two heterozygotes with matching criteria to two homozygote pairs showed intermediate inhibition of proliferation (data not shown). The consistently lower percent inhibition of proliferation in the paired analysis in GG/GG subjects by NAGly at 10–7 M (Fig. 4) was similar to the paired differences observed at other concentrations. The lower endogenous fatty acid amide-induced inhibition in GG/GG subjects compared with AA/AA subjects was still apparent when data from a larger group of 26 unpaired subjects (13 of each genotype) were analyzed (Fig. 5 ). In this group, the mean percent inhibition of lymphocyte proliferation by 10–7 M NAGly was significantly reduced in individuals with the CB2 GG/GG genotype compared with the AA/AA genotype (Fig. 5 ; P=0.0295, Mann-Whitney test).



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  		Figure 4. Mean percent inhibition of lymphocyte proliferation by fatty acid amides at 10–7 M; analysis of eight matched donor subject pairs with CB2 188–189 AA/AA and GG/GG genotypes. (A) Mean inhibition of lymphocyte proliferation produced by NAGly at 10–7 M, 60.9% in AA/AA subjects and 31.2% in GG/GG subjects (P=0.0078, Wilcoxin matched-pairs test). (B) Mean inhibition of lymphocyte proliferation produced by 2-AG, 55.7% in AA/AA subjects and 33.9% in GG/GG subjects (P=0.07, Wilcoxin matched-pairs test).



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  		Figure 5. All subjects’ data, unpaired analysis. Mean percent inhibition of lymphocyte proliferation by NAGly at 10–7 M for all 26 donors (unpaired subjects), 52.2% in the CB2 188–189 AA/AA group and 31.7% in the GG/GG group (P=0.029, Mann-Whitney test).

CB2-specific antagonist partially blocks inhibition of lymphocyte proliferation by NAGly and 2-AG
To evaluate the contribution of the CB1 and CB2 receptors to NAGly- or 2-AG-induced inhibition of T lymphocyte proliferation, the effects of the specific CB2 receptor antagonist SR144528 (SR2) and CB1 receptor antagonist SR141716A (SR1) were tested at 5 µM and 1 µM concentrations, which were similar to those previously shown to prevent endocannabinoid effects [22 ]. The effect of each specific antagonist was expressed as percent blocking of inhibition of T lymphocyte proliferation compared with control lymphocyte cultures treated with endocannabinoids but without addition of the specific CB1 and CB2 antagonists. SR1, the CB1 antagonist, had no effect on blocking inhibition of T lymphocyte proliferation in any of the subjects tested (data not shown). In lymphocyte cultures of eight paired subjects of both genotypes treated with NAGly in the presence or absence of 5 µM SR2, the mean percent blocking of the inhibitory effect by SR2 was 15% (range 6–30%). This partial inhibition is consistent with studies, carried out under the same conditions when 2-AG served as the ligand, in which SR2 produced a mean blocking effect of 21.7% (range 5.1–39%).

CB2 188–189 GG/GG genotype and gene frequency are increased in autoimmune diseases
To determine whether the CB2 188–189 GG/GG genotype, discovered to be associated with decreased endocannabinoid inhibition of lymphocyte proliferation, could be a risk factor for autoimmune diseases, we assessed the frequency of the polymorphic GG allele and prevalence of the homozygous CB2 GG/GG genotype in a population of subjects with diverse autoimmune diseases (see Materials and Methods). The distribution of the GG allele (Fig. 6A ) and the homogenous GG/GG genotypes in the autoimmune disease study population of Caucasian ancestry was compared with healthy Caucasian donors (Fig. 6) . In 102 Caucasian subjects with autoimmune diseases, the frequency of the allele GG = 62% ± 3.4 (SE) was significantly higher than the frequency of the allele GG = 54% ± 1.5 in 519 Caucasian control subjects (P=0.03, Fishers Exact test; Fig. 6A ). As the impact of heterozygosity is uncertain, we compared the frequency of the CB2 homozygous GG/GG polymorphism in groups 519 Caucasian controls and 102 subjects and found a significant, increased prevalence of the homozygous GG/GG genotype of 41.2% ± 3.6% (SE) in autoimmune disease subjects compared with 32% ± 2.1 in normal, healthy controls (P=0.045, Fishers Exact test; Fig. 6B ). Table 1 shows the CB2 genotype data stratified for each clinical type of autoimmune disease within the entire autoimmune diseases group and control subjects group analyzed in Figure 6 .



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  		Figure 6. Allele and genotype frequencies in Caucasian autoimmune disease subjects compared with Caucasian controls. (A) CB2 188–189 AA and GG allele frequencies in 102 autoimmune disease subjects and 519 controls. (B) Percent incidence of the CB2 188–189 GG/GG homozygous polymorphism in 102 autoimmune disease subjects compared with 519 controls.


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  		Table 1. Stratification of Clinical Autoimmune Diseases by CB2 188–89 Genotype


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DISCUSSION
 
The term "immunocannabinoid system" refers to the ECS components that are present within the immune system [5 ]. These ECS components include several endogenously synthesized and degraded cannabinoid-like fatty acid amides and n-arachidonyl amino acids together with their receptors and primary inactivating enzyme FAAH. Earlier studies of exogenous cannabinoids such as {Delta}9-tetrahydrocannabinol and selected endogenous cannabinoids have demonstrated immune suppression with effects ranging from loss of resistance to infection [23 , 24 ] to inhibition of lymphocyte proliferation [20 ]. More recent studies of endocannabinoids in mammals have shown a complex range of immunomodulatory effects, primarily suppressive effects on leukocytes and immune functions [5 ], including modulation of Th cell development, chemotaxis, and cytokine secretion. As leukocytes express CB2 receptors at levels ten- to 100-fold greater than CB1 receptors, the CB2 receptor is thought to be the principal cannabinoid receptor that mediates immune modulation by endocannabinoids [13 ]. CB2 receptors are highly expressed in monocyte/macrophages, NK cells, neutrophils, and B and T lymphocytes [8 ]. Thus, endocannabinoids such as ANA and 2-AG inhibit macrophage functions [25 , 26 ], and cannabinoids inhibit activity of cytotoxic NK cells [24 ]. Moreover, the secretion of cytokines such as interleukin-2 from activated T cells was shown to be inhibited by 2-AG [27 ]. Several reviews have discussed the biology of the endocannabinoid system and its role in immune modulation [3 , 5 , 7 ]. ANA and 2-AG are intrinsic, natural ligands for the CB1 and CB2 receptors [8 ] and may play a role in physiological modulation of immune and inflammatory responses [10 ]. 2-AG is also a full agonist acting at the CB2 receptor [28 ]. Immunomodulation by cannabinoids is entirely absent in mice lacking the CB2 receptor [29 ]. Thus, the weight of recent evidence supports the notion of a prominent role for the CB2 receptor as the primary signaling pathway for endocannabinoid immune modulation.

The CB2 receptor gene [30 ] is located on chromosome 1 (1p36.11) and consists of a single translated exon flanked by 5' and 3' untranslated regions. In mammalian phylogeny, the sequence in most regions of the CB2 gene is highly conserved but at amino acid position 63, an amino acid sequence alignment shows glutamine in human CB2 and arginine in mouse and rat [31 ], and there is an arginine at position 63 in baboon and chimpanzee CB2 (Table 2 ). Thus, the mammalian ancestral CB2 genotype at cDNA position 188–189 appears to be a GG/GG-encoding arginine, and humans appear to be the only species sequenced so far in which more than 50% of individuals have AA alleles that encode glutamine at amino acid position 63. The CB2 gene sequence, amino acid sequence, and tertiary structure have been elucidated [17 , 24 , 31 ]. We identified within the CB2 gene the 188–189 AA/AA -> GG/GG tandem polymorphism as potentially significant, as it results in a predicted amino acid substitution of a polar, uncharged glutamine at position 63 to a positively charged arginine that could change the CB2 tertiary structure in the region of the first intracellular cytoplasmic signaling loop. As we hypothesized that the CB2 188–189 GG/GG could result in a change of function in the mature expressed protein, we investigated the CB2-mediated endocannabinoid inhibitory effect by CB2 agonists in a system of mitogen-induced T lymphocyte proliferation. The endogenous fatty acid amides evaluated in this study included 2-AG, an agonist for CB1 and CB2 receptors, and NAGly, known to be a pain-inhibiting n-arachidonyl amino acid, which does not bind to CB1 receptors or vanilloid receptors [19 ] and may have CB2 agonist activity. The fact that SR144528 at 5 µM partially blocked NAGly-mediated inhibition of T lymphocyte proliferation in our experiments could suggest that NAGly is a partial CB2 agonist in lymphocytes similar to 2-AG.


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  		Table 2. Alignment of Mammalian CB2 Amino Acid Sequence of the First Intracytoplasmic Loop, aa 57–76

Our preliminary study of the prevalence of the CB2 188–189 GG genotype showed a significantly increased incidence of the homozygous CB2 188–189 GG/GG polymorphism in a group of Caucasian autoimmune disease subjects compared with similar healthy controls, P = 0.029 (Fig. 6) . This finding is of interest because of the need to identify genetic susceptibility risk factors in autoimmune diseases. Although it is clear that in most autoimmune disorders, many genetic variants may play a partial role, the finding here of significantly increased prevalence of the CB2 188–189 polymorphism suggests this variant may be one risk factor for autoimmunity. For example, recent studies show no single major genetic locus confers susceptibility in MS [32 ], but a number of chromosomal regions with modest linkage and risk effects have been identified. It is interesting that the CB2 188–189 GG/GG mis-sense polymorphism we have studied is near a chromosomal region, 1p34, previously identified as a promising candidate risk factor in MS with a lod score of >0.90 [33 ].

The results presented here are consistent with the findings of Schwarz et al. [20 ] that endocannabinoid inhibition of mitogen antibody-induced lymphocyte proliferation in total PBMC is a function of the immunocannabinoid system. Using a similar PBMC assay, we found a significant, approximately twofold reduction of inhibition of anti-CD3 antibody-induced T lymphocyte proliferation in CB2 188–189 GG/GG subjects compared with AA/AA subjects. We also investigated the prevalence of this polymorphism in a population of subjects with several autoimmune diseases, and the initial results indicate a significant over-representation of the CB2 188–189 GG/GG genotype. These findings provide evidence that this CB2 receptor missense polymorphism results in impairment of endocannabinoid inhibition of immune system function and further suggests that the CB2 188–189 GG/GG variant may be one potential genetic risk factor for some autoimmune disorders. Further studies are now needed to define the functionality of the CB2 188–189 variant in different human immune cells including CD4+, CD8+, B cells, and monocytes, as well as cell transfection studies to investigate the mechanism of decreased endocannabinoid signaling in this polymorphism. Here, these studies demonstrate that the CB2 cDNA 188–189 GG/GG missense polymorphism is associated with reduced endogenous fatty acid amide modulation of T lymphocyte proliferation within PBMC in vitro and suggest a link between this polymorphism and autoimmunity. The present results also support the corollary concept that the CB2 receptor on immune effector cells may represent a potential molecular target for selective CB2 agonist therapies that could suppress autoreactive, proinflammatory innate and adaptive immune responses without the risk of the adverse, neurobehavioral effects of most cannabinoid compounds that are CB1 receptor ligands.


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ACKNOWLEDGEMENTS
 
This work was supported by the TSRI Skaggs Clinical Scholars Program and by National Institutes of Health Grants DK5305-02, RR00833, DA13173, DA15197, and M01 RR00833. This is manuscript number MEM16751from The Scripps Research Institute. N. A. was supported by a postdoctoral fellowship from the Canadian Institutes of Health Research and a Shiley Scholarship from the Scripps Clinic.


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FOOTNOTES
 
2 Current address: Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4. Back

Received February 23, 2005; revised March 21, 2005; accepted March 27, 2005.


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REFERENCES
 
    1
  1. Elphick, M. R., Egertova, M. (2001) The neurobiology and evolution of cannabinoid signaling Philos. Trans. R. Soc. Lond. B Biol. Sci. 356,381-408[Abstract/Free Full Text]
    2. 2
  2. Iversen, L. (2003) Cannabis and the brain Brain 126,1252-1270[Abstract/Free Full Text]
    3. 3
  3. Salzet, M., Breton, C., Bisogno, T., Di Marzo, V. (2000) Comparative biology of the endocannabinoid system possible role in the immune response Eur. J. Biochem. 267,4917-4927[Medline]
    4. 4
  4. Galiegue, S., Mary, S., Marchand, J., Dussossoy, D., Carriere, D., Carayon, P., Bouaboula, M., Shire, D., Le Fur, G., Casellas, P. (1995) Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations Eur. J. Biochem. 232,54-61[Medline]
    5. 5
  5. Klein, T. W., Newton, C., Larsen, K., Lu, L., Perkins, I., Nong, L. (2003) The cannabinoid system and immune modulation J. Leukoc. Biol. 74,486-496[Abstract/Free Full Text]
    6. 6
  6. Matias, I., Pochard, P., Orlando, P., Salzet, M., Pestel, J., Di Marzo, V. (2002) Presence and regulation of the endocannabinoid system in human dendritic cells Eur. J. Biochem. 269,3771-3778[Medline]
    7. 7
  7. Berdyshev, E. V. (2000) Cannabinoid receptors and the regulation of immune response Chem. Phys. Lipids 108,169-190[CrossRef][Medline]
    8. 8
  8. Howlett, A. C., Barth, F., Bonner, T. I., Cabral, G., Casellas, P., Devane, W. A., Felder, C. C., Herkenham, M., Mackie, K., Martin, B. R., Mechoulam, R., Pertwee, R. G. (2002) International Union of Pharmacology. XXVII. Classification of cannabinoid receptors 1 Pharmacol. Rev. 54,161-202[Abstract/Free Full Text]
    9. 9
  9. Klein, T. W., Lane, B., Newton, C. A., Friedman, H. (2000) The cannabinoid system and cytokine network Proc. Soc. Exp. Biol. Med. 225,1-8[Abstract/Free Full Text]
    10. 10
  10. Sugiura, T., Keizo, W. (2002) Cannabinoid receptors and their endogenous ligands J. Biochem. (Tokyo) 132,7-12[Abstract/Free Full Text]
    11. 11
  11. Yuan, M., Kiertscher, S. M., Cheng, Q., Zoumalan, R., Tashkin, D. P., Roth, M. D. (2002) {Delta}9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells J. Neuroimmunol. 133,124-131[CrossRef][Medline]
    12. 12
  12. Do, Y., McKallip, R., Nagarkatti, M., Nagarkatti, P. (2004) Activation through cannabinoid receptors 1 and 2 on dendritic cells triggers NF-{kappa}B-dependent apoptosis: novel role for endogenous and exogneous cannabinoids in immunoregulation J. Immunol. 173,2373-2382[Abstract/Free Full Text]
    13. 13
  13. Parolaro, D., Massi, P., Rubino, T., Monti, E. (2002) Endocannabinoids in the immune system and cancer Prostaglandins Leukot. Essent. Fatty Acids 66,319-332[CrossRef][Medline]
    14. 14
  14. Zajicek, J., Fox, P., Sanders, H., Wright, D., Vickery, J., Nunn, A., Thompson, A. (2003) Cannabinoids for treatment of spasticity and other symptoms related to multiple sclerosis (CAMS study): multicentre randomized placebo-controlled trial Lancet 362,1517-1526[CrossRef][Medline]
    15. 15
  15. Svendsen, K., Jensen, T., Bach, F. (2004) Does the cannabinoid dronabinol reduce central pain in multiple sclerosis? Randomized double blind placebo controlled crossover trial BMJ 329,253[Abstract/Free Full Text]
    16. 16
  16. Carlini, E. (2004) The good and the bad effects of (-)trans-{Delta}-9-tetrahydrocannabinol ({Delta}(9)-THC) on humans Toxicon 44,461-467[Medline]
    17. 17
  17. Xie, X. Q., Chen, J. Z., Billings, E. M. (2003) 3D structural model of the G-protein-coupled cannabinoid CB2 receptor Proteins 53,307-319[CrossRef][Medline]
    18. 18
  18. Beutler, E., Gelbart, T. (2000) Large-scale screening for HFE mutations: methodology and cost Genet. Test. 4,131-142[CrossRef][Medline]
    19. 19
  19. Huang, S. M., Bisognos, T., Petros, T. J., Chang, S. Y., Zavitsanos, P. A., Zipkin, R. E., Sivakumar, R., Coop, A., Maeda, D. Y., De Petrocellis, L., Burstein, S., Di Marzo, V., Walker, J. M. (2001) Identification of a new class of molecules, the arachidonyl amino acids, and characterization of one member that inhibits pain J. Biol. Chem. 276,42639-42644[Abstract/Free Full Text]
    20. 20
  20. Schwarz, H., Blanco, F. J., Lotz, M. (1994) Anadamide, an endogenous cannabinoid receptor agonist inhibits lymphocyte proliferation and induces apoptosis J. Neuroimmunol. 55,107-115[CrossRef][Medline]
    21. 21
  21. Arbour, N., Holz, A., Sipe, J., Naniche, D., Romine, J., Zyroff, J., Oldstone, M. B. A. (2003) A new approach for evaluating antigen-specific T cell responses to myelin antigens during the course of multiple sclerosis J. Neuroimmunol. 137,197-209[CrossRef][Medline]
    22. 22
  22. Rao, G., Zhang, W., Kaminski, N. (2004) Cannabinoid receptor-mediated regulation of intracellular calcium by {Delta}(9)-tetrahydrocannabinol in resting T cells J. Leukoc. Biol. 75,884-892[Abstract/Free Full Text]
    23. 23
  23. Morahan, P. S., Klykken, P. C., Smith, S. H., Harris, L. S., Munson, A. E. (1979) Effects of cannabinoids on host resistance to Listeria monocytogenes and herpes simplex virus Infect. Immun. 23,670-674[Abstract/Free Full Text]
    24. 24
  24. Klein, T. W., Newton, C., Friedman, H. (1998) Cannabinoid receptors and immunity Immunol. Today 19,373-381[CrossRef][Medline]
    25. 25
  25. Cabral, G., Toney, D. M., Fischer-Stenger, K., Harrison, M. P., Marciano-Cabral, F. (1995) Anandamide inhibits macrophage-mediated killing of tumor necrosis factor-sensitive cells Life Sci. 56,2065-2072[CrossRef][Medline]
    26. 26
  26. Chang, Y. H., Lee, S. T., Lin, W. W. (2001) Effects of cannabinoids on LPS-stimulated inflammatory mediator release from macrophages: involvement of eicosanoids J. Cell. Biochem. 81,715-723[CrossRef][Medline]
    27. 27
  27. Ouyang, Y., Hwang, S., Han, S., Kaminski, N. (1998) Suppression of interleukin-2 by the putative endogenous cannabinoid 2-arachidonlyglycerol is mediated through down-regulation of the nuclear factor of activated T cells Mol. Pharmacol. 53,676-683[Abstract/Free Full Text]
    28. 28
  28. Gonsiorek, W., Lunn, C., Fan, X., Narula, S., Lundell, D., Hipkin, R. W. (2000) Endocannabinoid 2-arachidonyl glycerol is a full agonist through human type 2 cannabinoid receptor: antagonism by anandamide Mol. Pharmacol. 57,1045-1050[Abstract/Free Full Text]
    29. 29
  29. Buckley, N. E., McCoy, K. L., Mezey, E., Bonner, T., Zimmer, A., Felder, C. C., Glass, M., Zimmer, A. (2000) Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor Eur. J. Pharmacol. 396,141-149[CrossRef][Medline]
    30. 30
  30. Munro, S., Thomas, K. L., Abu-Shaar, M. (1993) Molecular characterization of a peripheral receptor for cannabinoids Nature 365,61-65[CrossRef][Medline]
    31. 31
  31. Brown, S. M., Wager-Miller, J., Mackie, K. (2002) Cloning and molecular characterization of the rat CB2 cannabinoid receptor Biochim. Biophys. Acta 1576,255-264[Medline]
    32. 32
  32. Barcellos, L. F., Thomson, G. (2003) Genetic analysis of multiple sclerosis in Europeans J. Neuroimmunol. 143,1-6[CrossRef][Medline]
    33. 33
  33. Pericak-Vance, M. A., Rimmler, J. B., Haines, J. L., Garcia, M. E., Oksenberg, J. R., Barcellos, L. F., Lincoln, R., Hauser, S. L., Cournu-Rebeix, I., Azoulay-Cayla, A., Lyon-Caen, O., Fontaine, B., Duhamel, E., Coppin, H., Brassat, D., Roth, M. P., Clanet, M., Alizadeh, M., Yaouanq, J., Quelvennec, E., Semana, G., Edan, G., Babron, M. C., Genin, E., Clerget-Darpoux, F. (2004) Investigation of seven proposed regions of linkage in multiple sclerosis: an American and French collaborative study Neurogenetics 5,45-48[CrossRef][Medline]



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