Using two distinct anti-CB2 receptor Abs, we investigated the expression patterns of the peripheral cannabinoid receptor CB2 in human secondary lymphoid organs. Immunohistochemical analysis using an N-terminal specific anti-CB2 Ab revealed high protein expression in the germinal centers (GCs) of secondary follicles. A C-terminal specific anti-CB2 Ab, which only recognizes a nonphosphorylated inactive receptor, showed positivity in the mantle zones (MZs) and marginal zones (MGZs) of the secondary follicles where resting cells reside, and in the primary follicles. In contrast, no positivity was observed in GCs using the C-terminal Ab, suggesting that active CB2 receptors are mainly present on cells in the GCs. Dual immunohistochemical analysis revealed that B lymphocytes express the CB2 protein abundantly. In contrast to B cells in the MZ or MGZ, CB2-expressing cells in the GCs coexpress the costimulatory membrane protein CD40, which is mainly expressed in the GCs and at very low levels in the MZs and MGZs and the proliferation marker Ki-67. Using the human Raji B cell line as a model, we demonstrate in a transwell assay that moderate migration occurs upon stimulation of the CB2 receptor with the endocannabinoid 2-arachidonoylglycerol, which is enhanced by CD40 costimulation. Our findings, that GC-related cells express active CB2 and that CB2-dependent migration requires CD40 costimulation, suggest that CB2 is involved in B cell activation.

Two cannabinoid receptors, which are expressed in distinct organ systems, have previously been identified. The central cannabinoid receptor CB1 (1) is primarily expressed in brain, whereas the peripheral cannabinoid receptor CB2 (2) is mainly present in immune tissues. CB2 mRNA has been detected in human hemopoietic cells, with B cells expressing the highest transcript levels (3, 4). In fact, using a polyclonal Ab raised against the C terminus of the human CB2 receptor, Carayon et al. (5) demonstrated expression of the CB2 protein in the mantle zones (MZs) 3 of secondary follicles of tonsils. These investigators reported that there was no CB2 staining on B lymphocytes in the germinal centers (GCs) of the secondary follicles. Importantly, the Ab used recognized CB2 receptors in a nonphosphorylated inactive state (5, 6). The observation that no expression was evident on B cells in the GCs could mean that the cells in the GCs did not express CB2 receptors at all or that CB2 receptors were present on these cells, but in an active form undetectable with this Ab. Insight into the distribution of active vs inactive CB2 receptors on cells of the immune system may provide understanding of the functional aspects of this receptor in its physiological environment. Using another anti-CB2 Ab raised against the N terminus of the CB2 receptor in combination with the previously reported C-terminal specific anti-CB2 Ab, we therefore have investigated the distribution of CB2 receptors within normal lymphoid organs. We have demonstrated different staining patterns depending on the CB2 Ab used, implying distinct distribution of active vs nonactive receptors. The inactive receptors were found in the MZs of secondary follicles, whereas active peripheral cannabinoid receptors were present in the GCs. Next, dual immunohistochemistry was applied to investigate the immunophenotype of the CB2-expressing cells. The data show that whether present in an active or inactive form, CB2 was mainly expressed on B lymphocytes. Moreover, in the GCs of secondary follicles, the active form of CB2 was observed in cells expressing both the costimulatory molecule CD40 and the proliferation marker Ki-67, suggesting an immunomodulatory function of the CB2 receptor in the GCs. We therefore assessed the functional relation between CB2 and CD40. We have demonstrated that the major function of the CB2 receptor on splenic B cells is stimulation of migration, upon exposure to the endocannabinoid 2-arachidonoylglycerol (2-AG) (7). Using the Raji B lymphoma cell line as a model, we investigated the involvement of CD40 on CB2-mediated effects on B cells. We have found that migration is a major function of the CB2 receptor upon stimulation with 2-AG, which is significantly augmented following CD40 stimulation, suggesting cross talk between the two receptors. Our investigations suggest a critical role for CB2 in migration of B cells and the GC response.

The polyclonal human anti-C-terminal CB2 receptor Ab and the synthetic peptide derived from the predicted amino acid sequence of the C terminus of the receptor (Y-P-D-S-R-D-L-D-L-S-D-C) were kindly provided by P. Casellas (Sanofi-Synthelabo Recherche, Montpellier, France). The anti-CB2 receptor polyclonal Ab raised against the first 33 aa residues of the N terminus of the receptor was purchased from Affinity BioReagents (Golden, CO). The nonstimulatory/noninhibitory CD40 control Ab (nCD40), the anti-CD40 stimulatory Ab (sCD40/clone 7), and the anti-CD40 inhibitory Ab (iCD40/5D12) were kindly donated by L. Boon (PanGenetics BV, Amsterdam, The Netherlands). Other Abs used for immunohistochemical analysis are listed in Table I. The Raji cell line, donated by I. Touw (Department of Hematology, Erasmus Medical Centre, Rotterdam, The Netherlands), was cultured in RPMI 1640 medium (Life Technologies, Breda, The Netherlands) and supplements: penicillin (100 U/ml), streptomycin (100 ng/ml), and 10% FCS (Life Technologies). The Chinese hamster ovary (CHO) cell line and CHO cell line transfected with CB2 (CHO-CB2), kindly donated by P. Casellas, were cultured in MEM-α medium (Life Technologies) and supplements. The CB2 cannabinoid ligand 2-AG was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). The CB1-specific inverse agonist SR141716 and CB2-specific inverse agonist SR 144528 were kindly donated by P. Casellas.

Table I.

Abs and conjugates used for dual immunohistochemical analysis

Primary AbsSpecificityDilution/Epitope RetrievalaSource
CD3 (polyclonal) Pan-T cell 1/600/yes DAKO 
CD20 (monoclonal) Pan B cell 1/400/no DAKO 
CD79a (monoclonal) Pan B cell; plasma cells 1/100/yes DAKO 
IgD (polyclonal) Naive B cells 1/200/yes DAKO 
CD40 (monoclonal) B cell subset (GC related) 1/100/yes Donated 
Ki-67 (monoclonal) Proliferating cells 1/100/yes Immunotechb 
Secondary Abs and Conjugates    
Swarbio Swine anti-rabbit 1/200 DAKO 
RaMbio Rabbit anti-mouse 1/200 DAKO 
S-ABC AP-kit Streptavidin-biotin alkaline phosphatase 1/100 DAKO 
S-ABC HRP-kit Streptavidin biotin HRP 1/100 DAKO 
Primary AbsSpecificityDilution/Epitope RetrievalaSource
CD3 (polyclonal) Pan-T cell 1/600/yes DAKO 
CD20 (monoclonal) Pan B cell 1/400/no DAKO 
CD79a (monoclonal) Pan B cell; plasma cells 1/100/yes DAKO 
IgD (polyclonal) Naive B cells 1/200/yes DAKO 
CD40 (monoclonal) B cell subset (GC related) 1/100/yes Donated 
Ki-67 (monoclonal) Proliferating cells 1/100/yes Immunotechb 
Secondary Abs and Conjugates    
Swarbio Swine anti-rabbit 1/200 DAKO 
RaMbio Rabbit anti-mouse 1/200 DAKO 
S-ABC AP-kit Streptavidin-biotin alkaline phosphatase 1/100 DAKO 
S-ABC HRP-kit Streptavidin biotin HRP 1/100 DAKO 
a

Ag retrieval was performed by pretreating sections in a microwave oven (see Materials and Methods).

b

Immunotech, Prague, Czech Republic.

Single staining.

Formalin-fixed, paraffin-embedded normal lymph node, spleen, and thymus were retrieved from the files of the Department of Pathology at the Erasmus Medical Centre. Thick sections (5 μm) were deparaffinized and rehydrated, and endogenous peroxidase was blocked with 3% H2O2 in methanol for 20 min at room temperature (RT). Heat-induced Ag retrieval (required for staining with the C-terminal anti-CB2 Ab) was achieved by boiling the sections for 20 min at 100°C in citrate buffer (10 mM, pH 6.0) in a Micromed T/T Mega microwave oven (Salm and Kipp, Breukelen, The Netherlands). Sections were cooled down, washed, and incubated with the first panel of primary Abs for 1 h at RT (C-terminal anti-CB2 1/400, or N-terminal anti-CB2 1/600), and subsequently incubated for 10 min with biotinylated secondary Abs (anti-mouse and rabbit Igs; Labvision, Freemont, CA), followed by a 10-min incubation with streptavidin-conjugated (S-ABC) HRP (Labvision). Visualization was achieved using 3-amino-9-ethylcarbazole (Sigma-Aldrich) in NaAc buffer (0.2 M, pH 4.6) for 30 min in dark, which yielded in a red signal. Finally, sections were counterstained with hematoxylin, according to Harris (Klini-path, Duiven, The Netherlands), dehydrated, and covered by imsol (Klini-path) and by pertex (Histolab, Göteborg, Sweden). Standard H&E staining and negative controls (omission of primary Ab) of all examined tissues were included. Specificity controls were performed by preincubating the C-terminal anti-CB2 receptor Ab for 1 h with the synthetic peptide at 10 μg/μl.

Dual staining.

A panel of B and T cell markers, a proliferation marker (Ki-67), and biotin-conjugated secondary Abs were used for dual immunohistochemistry. The list of the Abs, their Ag retrieval, and their sources and dilutions are listed in Table I. Sections were first incubated with the CB2 Abs, and stained with 3-amino-9-ethylcarbazole, as described for the single staining procedure. Sections were individually incubated with the primary Abs listed in Table I for 30 min, washed, and incubated with the biotin-conjugated secondary Abs for 30 min at RT. An S-ABC-conjugated alkaline phosphatase kit (DAKO, Glostrup, Denmark) was used to enhance the signal. Visualization of the signal was achieved using a solution containing Fast Blue BB salt (4-benzoylamino-2, 5-diethoxylbenzene diazoniumchloride; Sigma-Aldrich), naphtol-AS-MX phosphate (Sigma-Aldrich), and levamisole hydroxychloride (Acros Organics, Geel, Belgium) in Tris-HCl buffer (0.2 M, pH 8.0). Sections were incubated for 30 min in dark with this solution, which finally resulted in blue staining of cells. In case of coexpression/costaining, the mixture of red and blue signals resulted in a dark purple staining of cells. Cells were counterstained with hematoxylin, according to Harris (Klinipath), and covered by pertex.

Flow cytometric analysis was conducted using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). In brief, cells were washed with HBSS (Invitrogen, Paisley, U.K.) and PBS, incubated on ice with the polyclonal N-terminal anti-CB2 Ab (1/50) or the nCD40 mAb (1/100) for 1 h, washed twice with PBS, and incubated with the FITC-conjugated secondary rabbit (1/200 goat anti-rabbit-FITC/IgG; Nordic Immunological Labs, Tilburg, The Netherlands) or mouse Ab (1/200 goat anti-mouse FITC; ITK Diagnostics, Uithoorn, The Netherlands) for 30 min. Finally, cells were washed twice with PBS and resuspended in 500 μl of PBS and analyzed.

After flow cytometric analysis, cytospins of cells stained with the N-terminal CB2 were made and analyzed for membrane expression. To investigate cytoplasmatic CB2 expression, cells were fixed in 4% paraformaldehyde at 4°C for 20 min, washed twice in PBS, and incubated for 1 h at RT with the N-terminal CB2 Ab in a permeabilizing PBG-T solution (0.1 M phosphate buffer; 0.5% BSA; 0.2% gelatin; 0.05% Triton X-100). Then cells were washed twice in PBG-T and incubated with the FITC-conjugated secondary rabbit Ab (1/200) for 1 h at RT. Cells were washed, embedded in vectashield (Brunschwig Chemie, Amsterdam, The Netherlands), and visualized with an immunofluorescence microscope (Leica Mikroskopie & Systeme, Solms, Germany).

Migration assays were performed using 6.5-mm-diameter transwells with a 5-μm pore size (Corning Costar, Amsterdam, The Netherlands). In brief, cells were washed twice with HBSS medium, resuspended in 100 μl of migration medium (IMDM plus 0.5% BSA), and placed in the upper chamber of a transwell with or without CB1- or CB2-specific inverse agonists (100 nM). In the lower chamber, 600 μl of migration medium with or without ligand (2-AG, 300 nM) was placed. After 4 h of incubation at 37°C and 5% CO2, the upper chamber was removed and the numbers of migrated cells were determined using a CASY1/TTC cell counter (Schärfe System, Reutlingen, Germany).

Two polyclonal Abs directed to the peripheral cannabinoid receptor (CB2) were used in this study to investigate expression of this receptor in human immune tissues. Specificity of the Abs was confirmed using CHO cells transfected with human CB2 (5). CB2 membrane staining was demonstrated with the Ab directed to the N-terminal extracellular domain using nonpermeabilized CHO-CB2 cells (Fig. 1,A). Membrane-bound and cytoplasmic CB2 expression was demonstrated in permeabilized CB2-expressing CHO cells (Fig. 1,B). An antiserum recognizing the intracellular C-terminal part of CB2 was also highly positive in permeabilized CHO-CB2 cells (Fig. 1,C). As expected, no positivity was observed when the latter Ab was used on nonpermeabilized CHO-CB2 cells (data not shown). Remarkably, a lower signal was evident when the N-terminal specific Ab (Fig. 1, A and B) was used as compared with the analysis with the C-terminal specific anti-CB2 (Fig. 1,C), which may suggest that the latter Ab binds CB2 with a higher affinity or that the differences in staining by the CB2 Abs may be the result of differences in epitope accessibility. No positivity at all was observed using the CB2 Abs in nontransduced CHO cells (Fig. 1, D and E). Importantly, the C-terminal anti-CB2 Ab only recognizes the receptor in an inactivated nonphosphorylated state (5, 6). The N-terminal CB2 Ab, in contrast, recognizes the receptor in both an active and inactive state.

FIGURE 1.

CB2 protein expression in CHO and CHO-CB2 cells. CB2 membrane expression (A) and intracellular CB2 protein expression (B) were shown in CHO cells transfected with human CB2 (CHO-CB2) using the N-terminal anti-CB2 Ab. Intracellular CB2 protein in CHO-CB2 cells using the C-terminal anti-CB2 Ab (C) was also investigated. CB2 protein expression was analyzed in non-CB2-transduced CHO cells using the N-terminal (D) and C-terminal (E) anti-CB2 Abs, respectively. Intracellular CB2 was analyzed in permeabilized cells (see Materials and Methods). The filled areas show CB2 protein expression; the gray lines represent background staining (only secondary Ab).

FIGURE 1.

CB2 protein expression in CHO and CHO-CB2 cells. CB2 membrane expression (A) and intracellular CB2 protein expression (B) were shown in CHO cells transfected with human CB2 (CHO-CB2) using the N-terminal anti-CB2 Ab. Intracellular CB2 protein in CHO-CB2 cells using the C-terminal anti-CB2 Ab (C) was also investigated. CB2 protein expression was analyzed in non-CB2-transduced CHO cells using the N-terminal (D) and C-terminal (E) anti-CB2 Abs, respectively. Intracellular CB2 was analyzed in permeabilized cells (see Materials and Methods). The filled areas show CB2 protein expression; the gray lines represent background staining (only secondary Ab).

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Primary follicles.

Immunohistochemical staining of spleen sections using a C-terminal (inactive CB2 receptor) or N-terminal anti-CB2 receptor Ab (nondiscriminating) showed CB2 expression in the follicular B cell areas. In primary follicles, the cells were highly positive using the C-terminal specific CB2 Ab (Fig. 2, A and B). Weaker expression was observed using the N-terminal CB2 Ab (Fig. 2, C and D). These data suggest that mainly inactive CB2 receptors are present on cells in the primary follicles. Specificity was evident, because binding of the C-terminal Ab was completely blocked when the Ab was preincubated with its specific peptide (Fig. 2,E). No staining was observed when only the secondary Ab was used (Fig. 2 F). Exactly the same results were obtained when normal lymph nodes were studied (data not shown).

FIGURE 2.

CB2 expression in primary follicles of the human spleen. CB2 expression is demonstrated using the C-terminal (A, ×5; B, ×20) and the N-terminal (C, ×5; D, ×20) anti-CB2 Abs, respectively. Specificity of the C-terminal anti-CB2 Ab was confirmed by preincubation with a specific C-terminal peptide (E, ×5). Staining with only the secondary Ab (F, ×5) served as a negative control. P = primary follicle; red staining = C- or N-terminal CB2 Ab staining; blue staining = counterstaining with hematoxylin.

FIGURE 2.

CB2 expression in primary follicles of the human spleen. CB2 expression is demonstrated using the C-terminal (A, ×5; B, ×20) and the N-terminal (C, ×5; D, ×20) anti-CB2 Abs, respectively. Specificity of the C-terminal anti-CB2 Ab was confirmed by preincubation with a specific C-terminal peptide (E, ×5). Staining with only the secondary Ab (F, ×5) served as a negative control. P = primary follicle; red staining = C- or N-terminal CB2 Ab staining; blue staining = counterstaining with hematoxylin.

Close modal

Secondary follicles.

CB2 expression patterns in the secondary follicles of lymph nodes were also determined using the same Abs. The C-terminal CB2-specific Ab mainly recognized cells present in the MZ and the marginal zone (MGZ) of the secondary follicles (Fig. 3, A–C). The N-terminal specific CB2 receptor Ab only weakly recognized cells in these zones (Fig. 3,E). In contrast, using this Ab, strong staining was observed in the GCs of the secondary follicles (Fig. 3, D–F), whereas only weak staining was found in those regions using the C-terminal Ab (Fig. 3, A–C). Strong binding of the CB2 N-terminal specific Ab suggests high receptor numbers present on the cells in the GCs. These results also suggest that cells in the GC mainly express active CB2, whereas inactive CB2 is present on cells in the MZ and MGZ of the secondary follicles. Remarkably, most prominent staining with the two Abs occurred in the cytoplasm of the cells (Fig. 3, C and F, insets).

FIGURE 3.

Single immunohistochemical analysis in primary and secondary follicles of the human lymph node. C-terminal anti-CB2 Ab staining pattern is depicted in A (×1.25), B (×10), and C (×20; inset, ×40). N-terminal anti-CB2 Ab-staining pattern is shown in D (×1.25), E (×10), and F (×20; inset, ×40). Insets, Indicate CB2 staining in the cytoplasm of the cells. P = primary follicle; s = secondary follicle. Filled rectangles represent magnifications of areas of interest. Red staining = C- or N-terminal CB2 staining; blue staining = counterstaining with hematoxylin.

FIGURE 3.

Single immunohistochemical analysis in primary and secondary follicles of the human lymph node. C-terminal anti-CB2 Ab staining pattern is depicted in A (×1.25), B (×10), and C (×20; inset, ×40). N-terminal anti-CB2 Ab-staining pattern is shown in D (×1.25), E (×10), and F (×20; inset, ×40). Insets, Indicate CB2 staining in the cytoplasm of the cells. P = primary follicle; s = secondary follicle. Filled rectangles represent magnifications of areas of interest. Red staining = C- or N-terminal CB2 staining; blue staining = counterstaining with hematoxylin.

Close modal

Inactive CB2 receptors on resting B cells.

Dual immunohistochemistry using B cell-specific Abs (Table I) has been conducted in combination with the anti-CB2 receptor Abs. First, we studied the cells carrying inactive CB2 receptors. Almost all cells in the primary follicles and in the MZ and MGZ of secondary follicles coexpressed (dark purple) CD79a (pan B cell marker; blue) and the inactive CB2 (red) on their surfaces (Fig. 4,A). The same results were obtained with another pan B cell marker, CD20 (data not shown). Similarly, significant expression of inactive CB2 (red membrane/cytoplasmic staining) was evident in IgD-expressing resting follicular B cells (dark purple membrane staining) in primary follicles and MZ/MGZ of secondary follicles (Fig. 4 B). No staining was observed when the primary Abs were omitted in the immunohistochemical analysis (data not shown).

FIGURE 4.

CB2 coexpression analysis in normal human lymph nodes. Coexpression was analyzed in primary and secondary follicles using the C-terminal anti-CB2 Ab with the pan B cell marker CD79a (A, ×10) or with IgD (B, ×20). Using the N-terminal anti-CB2 Ab, coexpression of CD79a-expressing cells (C, ×10 and ×40) or IgD-expressing cells (D, ×10) was also analyzed. Detailed coexpression analysis of CD79a-positive GC cells using the C-terminal anti-CB2 Ab is shown in E (×10). Open and filled arrows indicate coexpression; red arrows indicate lack of coexpression. Red staining = C- or N-terminal CB2; blue staining = CD79a (A, C, and E) or IgD (B and D); purple staining = coexpression. The filled rectangle represents the magnification of the area of interest. P = primary follicle.

FIGURE 4.

CB2 coexpression analysis in normal human lymph nodes. Coexpression was analyzed in primary and secondary follicles using the C-terminal anti-CB2 Ab with the pan B cell marker CD79a (A, ×10) or with IgD (B, ×20). Using the N-terminal anti-CB2 Ab, coexpression of CD79a-expressing cells (C, ×10 and ×40) or IgD-expressing cells (D, ×10) was also analyzed. Detailed coexpression analysis of CD79a-positive GC cells using the C-terminal anti-CB2 Ab is shown in E (×10). Open and filled arrows indicate coexpression; red arrows indicate lack of coexpression. Red staining = C- or N-terminal CB2; blue staining = CD79a (A, C, and E) or IgD (B and D); purple staining = coexpression. The filled rectangle represents the magnification of the area of interest. P = primary follicle.

Close modal

CB2 receptors on activated B cells in the GC.

Next, we investigated the immunophenotype of the CB2 receptor-expressing cells in the GC of the secondary follicles in lymph nodes. CB2 receptor-expressing cells in the GC (as demonstrated with low staining with C-terminal CB2 Ab and strong staining with N-terminal Ab) showed strong positivity with the pan B cell marker CD79a (Fig. 4,C). These cells also expressed CD20 (data not shown), but were mostly IgD negative (Fig. 4,D). As expected, the CD79a-positive cells in the GC showed no costaining with the C-terminal CB2 Ab (Fig. 4 E). Altogether, these data suggest the abundant expression of active CB2 receptors on B cells in the GCs.

To investigate whether the cells in the GC, expressing an active CB2 receptor, indeed represent activated B lymphocytes engaged in the GC response, we analyzed coexpression of CD40 or Ki-67 with CB2. The cells in the GC showing intense staining with anti-N-terminal CB2 were highly CD40 and Ki67 positive as well (Fig. 5, A and B). No costaining was observed using anti-Ki-67 and the C-terminal specific CB2 Ab (Fig. 5 C).

FIGURE 5.

Coexpression of CD40- and Ki67-positive GC cells. Coexpression of CD40- and Ki67-positive cells in the GCs of secondary follicles of lymph nodes using the N-terminal anti-CB2 Ab (A, ×10 and ×20, respectively; B, ×20 and ×40, respectively). The C-terminal anti-CB2 Ab was used to determine coexpression of Ki-67-positive GC cells (C, ×20 and ×40, respectively). Open rectangles represent magnifications of areas of interest. Costaining is indicated by purple membrane staining of CB2 and CD40 (A) or red membrane staining (N-terminal CB2) with blue nuclear staining (Ki-67; B). Cells are either highly red (membrane/cytoplasmatic) or blue (nuclear) stained when costaining is absent (C).

FIGURE 5.

Coexpression of CD40- and Ki67-positive GC cells. Coexpression of CD40- and Ki67-positive cells in the GCs of secondary follicles of lymph nodes using the N-terminal anti-CB2 Ab (A, ×10 and ×20, respectively; B, ×20 and ×40, respectively). The C-terminal anti-CB2 Ab was used to determine coexpression of Ki-67-positive GC cells (C, ×20 and ×40, respectively). Open rectangles represent magnifications of areas of interest. Costaining is indicated by purple membrane staining of CB2 and CD40 (A) or red membrane staining (N-terminal CB2) with blue nuclear staining (Ki-67; B). Cells are either highly red (membrane/cytoplasmatic) or blue (nuclear) stained when costaining is absent (C).

Close modal

CB2 receptor-specific migration is enhanced by CD40-specific stimulation

Coexpression of CD40 and active CB2 receptors on B cells in the GC prompted us to investigate a functional interaction between those two molecules. CB2 has been described as a G protein-coupled receptor (GPCR) involved in migration following exposure to the endocannabinoid, 2-AG (7). We studied CB2/CD40 interaction using the Raji B cell line as a model. First, CD40 membrane expression on Raji was demonstrated by flow cytometry (Fig. 6,A). Using the N-terminal specific CB2 Ab, protein expression was observed in the cytoplasm of these cells (Fig. 6,C), as was observed by the immunohistochemical analysis (Fig. 3). CB2 membrane expression was undetectable (Fig. 6 B).

FIGURE 6.

CD40 and CB2 expression in Raji cell line. Flow cytometry of Raji cells determining CD40 expression (A). CB2 membrane expression on nonpermeabilized cells (B) and intracellular CB2 protein in permeabilized cells (C) were analyzed using the N-terminal specific anti-CB2 receptor Ab. The filled areas represent CD40 (A)- or CB2 (B)-positive cells; the gray lines (A and B) represent background staining (only secondary Ab). The green dots represent CB2 protein in the cytoplasm; blue counterstain with 4′,6′-diamidino-2-phenylindole reflects the nuclei of Raji cells.

FIGURE 6.

CD40 and CB2 expression in Raji cell line. Flow cytometry of Raji cells determining CD40 expression (A). CB2 membrane expression on nonpermeabilized cells (B) and intracellular CB2 protein in permeabilized cells (C) were analyzed using the N-terminal specific anti-CB2 receptor Ab. The filled areas represent CD40 (A)- or CB2 (B)-positive cells; the gray lines (A and B) represent background staining (only secondary Ab). The green dots represent CB2 protein in the cytoplasm; blue counterstain with 4′,6′-diamidino-2-phenylindole reflects the nuclei of Raji cells.

Close modal

Next, transwell assays were conducted to investigate migration in response to distinct stimuli (Fig. 7). Raji cells moderately migrated upon stimulation with 2-AG (Fig. 7,A). However, following stimulation with an anti-sCD40 Ab, enhanced migration was observed upon exposure to 2-AG. This migration was specific because the CB2 receptor inverse agonist (SR144528) completely abolished this effect. The CB1 receptor-specific antagonist (SR 141716) did not influence the sCD40-enhanced migration (Fig. 7,A). An iCD40-specific Ab, when added in 10-fold excess, completely abolished the sCD40-enhanced 2-AG-induced migration of Raji cells (Fig. 7,B). nCD40 Abs serving as a control did not enhance 2-AG-induced migration (Fig. 7 B). CB2 membrane expression did not detectably increase following exposure to the sCD40 Ab, as determined by flow cytometry (data not shown).

FIGURE 7.

Migration of Raji cells in response to distinct stimuli. Cells were stimulated for 24 h in the presence or absence of sCD40 mAb (K7; 100 nM), sCD40 mAb (100 nM), plus iCD40 (5D12; 1000 nM), or nCD40 (100 nM). Migration was investigated in the presence or absence of the CB2 ligand 2-AG (300 nM). Upper wells, Contained either nonstimulated or stimulated cells in the presence or absence of CB1 (SR141716; 100 nM) or CB2-specific inverse agonists (SR 144528; 100 nM). The y-axis shows percentage of migration from an input of 1 × 105 cells. A, Represents the mean migration with the SEs of the means of three individual experiments.

FIGURE 7.

Migration of Raji cells in response to distinct stimuli. Cells were stimulated for 24 h in the presence or absence of sCD40 mAb (K7; 100 nM), sCD40 mAb (100 nM), plus iCD40 (5D12; 1000 nM), or nCD40 (100 nM). Migration was investigated in the presence or absence of the CB2 ligand 2-AG (300 nM). Upper wells, Contained either nonstimulated or stimulated cells in the presence or absence of CB1 (SR141716; 100 nM) or CB2-specific inverse agonists (SR 144528; 100 nM). The y-axis shows percentage of migration from an input of 1 × 105 cells. A, Represents the mean migration with the SEs of the means of three individual experiments.

Close modal

In this study, we investigated the distribution CB2 receptors as well as their activation status in human immune tissues using two distinct anti-CB2 Abs. Specificity of a C-terminal and a N-terminal specific CB2 Ab was demonstrated in CHO cells transfected with human CB2. We demonstrated that CB2 protein was present on the membrane as well as in the cytoplasm of these cells (Fig. 1). Applying these Abs in single immunohistochemical staining, we observed distinct expression patterns in B cell areas in human lymphoid organs. In conjunction with previous studies (5), we found CB2 receptors in the MZs using the anti-C-terminal CB2 Ab. We also observed CB2 positivity in primary follicles and in the MGZs of secondary follicles, using this antiserum. As the C-terminal specific Ab only recognizes nonphosphorylated CB2, apparently inactive CB2 receptors are present on B lymphocytes in these particular areas. It will be of interest to investigate CB2 phosphorylation in B lymphocytes, especially in relation to the Ag-specific activation and proliferation of these cells.

Only low CB2 positivity was observed in the MGZ when the N-terminal specific Ab was used. A possible explanation for the differences observed between the two Abs may be that the C-terminal specific Ab binds CB2 with a higher affinity than the N-terminal specific CB2 antiserum (Fig. 1). Alternatively, it is also possible that the differences in staining by the CB2 Abs may be the result of differences in epitope accessibility.

In this study, using the N-terminal specific Ab, we have demonstrated that various CB2-expressing B cell subsets exist. Some of these B cell populations are only weakly stained with the C-terminal anti-CB2 Ab. Therefore, the CB2 receptors on these cells are most probably phosphorylated and therefore in an active state (6). Because of the possibility of low affinity of the N-terminal specific Ab, a strong positivity most likely indicates high numbers of CB2 receptors on these cells. These cells reside in an anatomical location where B memory cells are formed and affinity maturation occurs, i.e., the GC of secondary follicles, and they express CD79a, but are mainly IgD negative (Fig. 4, C and D). B cells in primary follicles or in the MZ/MGZ of secondary follicles may express low levels of CD40; in contrast, B cells in the GCs of secondary follicles highly express CD40 as well as Ki-67, a proliferation marker (Fig. 5, A and B). Both markers are characteristic for actively cycling B cells in the GC. All in all, it appears that functional CB2 receptors are present on immunologically active B lymphocytes.

Dual immunohistochemistry using pan-B cell markers confirmed previous observations that in general B lymphocytes are the leukocyte subsets expressing CB2 mRNA and protein (3, 4, 5) (Fig. 4). Furthermore, modulation of this receptor during B cell differentiation has been demonstrated (5) using the C-terminal anti-CB2 Ab. In accordance with this fact, we found CD79a, IgD-positive resting B cells in the MZs highly expressing inactive CB2 (Fig. 4, A and B). The coexpression of CB2 and CD40 on activated B lymphocytes prompted us to investigate the functional relation between those two membrane proteins. CD40 belongs to the TNFR family and is involved in B cell activation, survival, proliferation, differentiation, and Ig isotype switch (8). Previous studies in mice have shown that upon CD40 stimulation, mCB2 mRNA may be up-regulated (9). An increase in human CB2 mRNA following CD40 stimulation has been demonstrated in human tonsillar B cells as well (5). We demonstrate, using a B lymphoid cell line as a model, that migration of B cells in response to the CB2-specific ligand 2-AG was significantly enhanced following CD40 stimulation. The mechanism of activation appears to be a different one, because we neither found any increase of the CB2 protein levels, nor did the CB2 receptor activation status change following CD40 stimulation (data not shown). The peripheral cannabinoid receptor belongs to the family of seven-transmembrane GPCRs (3, 4). GPCRs are crucial to many cellular functions, such as proliferation, maturation, survival, apoptosis, or migration (10, 11, 12); Carayon et al. (5) showed moderate proliferation of virgin and GC tonsillar B cells upon stimulation with the synthetic cannabinoid agonist CP55,940 only when the cells were stimulated with CD40 mAbs. In our functional study with Raji B lymphoma cells, we observed moderate migration upon stimulation with the CB2 ligand 2-AG. Interestingly, this migration was greatly enhanced when cells were stimulated for 24 h with a stimulating CD40 mAb (Fig. 7). Furthermore, this enhanced migration was CB2 and CD40 receptor specific, because the effects could be specifically blocked by the CB2 inverse agonist (SR144528) and the CD40 antagonist (5D12). A similar observation has been reported for CXCR4 and CD40 on GC cells. CD40 pretreatment of these cells led to an increased migration upon stimulation with the CXCR4 ligand stromal-derived factor 1 (13). Apparently, exposure to CD40-activating agents is crucial for the induction of a functional response of certain GPCRs such as CB2 or CXCR4.

Studies of the potential involvement of cannabinoid receptors and stimulation by cannabinoids in the immune system have been reported previously (14, 15, 16, 17, 18, 19). In fact, immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor (20). Recently, it was also demonstrated that human Ag-presenting dendritic cells produce high levels of endocannabinoid ligand 2-AG (21) and that 2-AG is probably the physiological ligand for the cannabinoid CB2 receptor (22). These findings together with our observations that: 1) active CB2 receptors are present on immunologically active cells, and 2) CB2 response may be increased by the costimulatory molecule CD40 provide evidence that CB2 receptors may be involved in B cell activation.

The observation that CB2 receptors are present on B lymphocytes and show migratory function in a malignant B cell line raises the question as to whether this receptor is also involved in the malignant transformation of B cells, resulting in lymphomas, or whether this receptor contributes to a specific function of malignant B cells. CB2-dependent migration in vitro in a B lymphoma cell line could give a direction toward homing of malignant B cells in vivo. In the staging of B lymphomas, bone marrow infiltration is one of the important parameters. Therefore, analysis of the CB2 receptor status could play an important role as a prognostic and/or diagnostic marker, possibly predicting spreading from the initial localization of the tumor. In high-throughput non-Hodgkin lymphoma (NHL) patient screens using tissue microarrays, currently in progress to identify potential novel diagnostic and/or prognostic markers in NHL, CB2 is the most interesting one. Furthermore, a correlation between the active CB2 receptor state in normal immune tissues and NHLs can also be made to see whether in a malignant setting the functional relation between CB2 and CD40 receptors is still evident.

We thank P. Casellas (Sanofi-Synthelabo Recherche, Montpellier, France) for donation of the CB1 and CB2 receptor-specific inverse agonists SR 141716 and SR 144528, and Dr. L. Boon (PanGenetics BV, Amsterdam, The Netherlands) for donation of the sCD40 (clone 7), iCD40 (5D12), and nCD40 mAbs. We also thank Karola van Rooyen for preparation of the figures.

1

This work was supported by the Dutch Cancer Society and the Erasmus Medical Centre (revolving fund).

3

Abbreviations used in this paper: MZ, mantle zone; 2-AG, 2-arachidonoylglycerol; CHO, Chinese hamster ovary; GC, germinal center; GPCR, G protein-coupled receptor; iCD40, inhibitory CD40; MGZ, marginal zone; nCD40, nonstimulatory/noninhibitory CD40; NHL, non-Hodgkin lymphoma; RT, room temperature; sCD40, stimulatory CD40.

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