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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3605-3615
Modulation and Functional Involvement of CB2 Peripheral Cannabinoid
Receptors During B-Cell Differentiation
By
Pierre Carayon,
Jean Marchand,
Danielle Dussossoy,
Jean-Marie Derocq,
Omar Jbilo,
Annie Bord,
Monsif Bouaboula,
Sylvaine Galiègue,
Paul Mondière,
Géraldine Pénarier,
Gérard Le Fur,
Thierry Defrance, and
Pierre Casellas
From the Immunology Department, Sanofi Recherche, Montpellier,
France; and the Immunité et vaccination, INSERM U404, Institut
Pasteur de Lyon, Lyon, France.
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ABSTRACT |
Two subtypes of G-protein-coupled cannabinoid receptors have been
identified to date: the CB1 central receptor subtype, which is mainly
expressed in the brain, and the CB2 peripheral receptor subtype, which
appears particularly abundant in the immune system. We investigated the
expression of CB2 receptors in leukocytes using anti-CB2 receptor
immunopurified polyclonal antibodies. We showed that peripheral blood
and tonsillar B cells were the leukocyte subsets expressing the highest
amount of CB2 receptor proteins. Dual-color confocal microscopy
performed on tonsillar tissues showed a marked expression of CB2
receptors in mantle zones of secondary follicles, whereas germinal
centers (GC) were weakly stained, suggesting a modulation of this
receptor during the differentiation stages from virgin B lymphocytes to
memory B cells. Indeed, we showed a clear downregulation of CB2
receptor expression during B-cell differentiation both at transcript
and protein levels. The lowest expression was observed in GC
proliferating centroblasts. Furthermore, we investigated the effect of
the cannabinoid agonist CP55,940 on the CD40-mediated proliferation of
both virgin and GC B-cell subsets. We found that CP55,940 enhanced the
proliferation of both subsets and that this enhancement was blocked by
the CB2 receptor antagonist SR 144528 but not by the CB1 receptor
antagonist SR 141716. Finally, we observed that CB2 receptors were
dramatically upregulated in both B-cell subsets during the first 24 hours of CD40-mediated activation. These data strongly support an
involvement of CB2 receptors during B-cell differentiation.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
 9-TETRAHYDROCANNABINOL
(9-THC) is the principal psychoactive component in
preparations of Cannabis sativa (marijuana, hashish).1 Its effects are mainly mediated via the CB1
central cannabinoid receptor,2 which belongs to the family
of G-protein-coupled seven-transmembrane domain
proteins.3,4 Activation of CB1 receptor, which is coupled
to a Gi protein, leads to inhibition of adenylyl cyclase5
and N-type voltage-dependent calcium channels.6 Moreover,
the CB1 receptor is functionaly coupled to mitogen-activated protein
kinase (MAPK) cascade7 and regulates the krox-24 (egr-1) gene.8 In addition to a wide range of physiological effects on the central nervous system, cannabinoid ligands have been reported to affect the immune system.9,10 At high concentrations,
they modulate proliferative responses of T
lymphocytes,11,12 cytotoxic T-cell activity,13
humoral response,14 microbiocidal activity, cytokine
production, and antigen processing by macrophages.15-17 Cannabinoid ligands also act at physiological concentrations by inhibiting the synthesis of tumor necrosis factor- (TNF-) by human large granular lymphocytes18 and the activation of
mast cells.19 All of these effects strongly suggested that
cannabinoid-induced immune modulation may be mediated at least in part
through a cannabinoid receptor-associated mechanism.
Recently, a second cannabinoid receptor has been cloned.20
This receptor, CB2, shows 44% identity with the CB1 receptor. It has
been defined as peripheral cannabinoid receptor, because it is mainly
localized in cells of the immune system. Among these cells, low levels
of CB2 receptors have been localized in B-cell areas of different rat
lymphoid tissues such as spleen, lymph nodes, and Peyer's
patches.21 The CB2 receptor, which is also linked to a Gi
protein, displays pharmacological and biochemical properties similar to
those of the CB1 receptor. It inhibits adenylyl cyclase
activities,22 activates the MAPK pathway, and induces krox-24 expression,23 but does not modulate the activity of calcium channels.24 Recently, it has been shown that, via
CB2 receptor, cannabinoid ligands cause a dose-dependent increase of
B-cell proliferation induced through cross-linking of surface Igs or
ligation of the CD40 antigen.25
With CB2 receptors being considerably more abundant in the immune
system than CB1 receptors,26,27 we used antibodies (Abs) raised against the C-terminal tail of the CB2 receptor to investigate its expression in leukocytes. With the highest level being observed in
B cells, we studied the expression of CB2 receptors in tonsillar B
cells and found that it was modulated during B-cell differentiation. Finally, we assessed the function of the CB2 receptor by demonstrating that (1) cross-linking of CD40 by monoclonal antibodies (MoAbs) increased CB2 receptor expression on both virgin and GC B cells and (2)
CB2 receptors act as coreceptors of CD40-induced proliferation of these
two B-cell subsets.
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MATERIALS AND METHODS |
Production of anti-CB2 receptor Abs.
Synthetic peptide derived from the predicted aminoacid-sequence of the
carboxylic tail of the human CB2 receptor (Y-P-D-S-R-D-L-D-L-S-D-C) and
bovine serum albumin (BSA)-conjugated peptide used as
immunogen were from Neosystem (Strasbourg, France). Rabbits were
injected subcutaneously with 2 mg BSA-peptide in 250 µL water and 250 µL complete Freund's adjuvant. Animals were boosted monthly under the same conditions and blood was taken 10 days after the fifth injection. Abs directed against the C-terminal part of the human CB2
receptor were immunopurified on a Bio-Rad Affi-Gel 10 modified with the
peptide (Bio-Rad, Hercules, CA) as already
described.28 Briefly, 10 mL of immune serum was incubated
overnight at 4°C with 1 mL modified gel. After extensive washings,
anti-CB2 receptor Abs were eluted with 100 mmol/L glycine-HCl, pH 1.8, and neutralized with 1 mol/L Tris-NaOH. The pooled fractions were
supplemented with 10 mg/mL BSA, concentrated, and dialyzed on a Filtron
microsep 30 kD (Filtron, Nortborough, MA). Concentrated Abs were stored in 50% glycerol at  20°C.
Immunoblotting experiments.
Membranes of wild-type hamster ovary cells (CHO-WT) and of CHO cells
stably transfected with the CB2 receptor (CHO-CB2)27 were
prepared by homogeneizing cells with polytron in 5 mmol/L Tris, pH 7.4, containing 1 mmol/L EDTA, 20 µg/mL aprotinin, and 1 mmol/L
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). The homogenate was centrifuged for 15 minutes at
2,000g. The nuclear free supernatant was centrifuged for 1 hour
at 100,000g. Immunoblotting experiments were performed on the
membrane pellets after electrophoresis on a 4% to 20% sodium dodecyl
sulfate (SDS)-polyacrylamide gel (Novex, San Diego, CA) and transfer
onto nitrocellulose filters. Proteins were electroblotted on
nitrocellulose filters (Novex). Nonspecific binding was blocked with
10% casein in TBS buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5)
for 1 hour at room temperature. The blotted filters were washed with
0.1% Tween 20 in TBS buffer and then incubated for 3 hours at room
temperature with anti-CB2 receptor Abs (1:2,000 dilution). After
another washing, peroxidase-conjugated antirabbit Ig (1:8,000 dilution;
Sigma, St Louis, MO) was added for 45 minutes at room temperature.
After 5 extensive washes, immune complexes were detected using the ECL kit on Hyperfilm-MP (Amersham, Buckinghamshire, UK) following the
supplier's instructions.
Antibodies.
The following Abs were used for flow cytometry.
Phycoerythrin-conjugated human CD4, CD8, CD20, and CD38 MoAbs were
purchased from Becton Dickinson (San Jose, CA). Tricolor
(phycoerythrin-Cy5)-conjugated human CD3 MoAbs were from Caltag (South
San Francisco, CA). Biotinylated human CD44 MoAbs were from Leinco
Technologies (Ballwin, MO). Biotinylated antihuman IgD Abs were from
Tagoimmunologicals (Burlingame, CA). The human purified CD77 MoAbs were
from Immunotech and were stained with the biotinylated antirat IgG
(mark-1) MoAbs (Immunotech). All of the biotinylated Abs were labeled
with Tricolor-conjugated streptavidin (Caltag). Rabbit anti-CB2 Abs
were labeled with fluorescein isothiocyanate (FITC)-conjugated donkey
antirabbit IgG Abs from Jackson Immunoresearch (West Grove, PA).
The following Abs were used for confocal laser scanning microscopy:
purified human CD3, CD38 MoAbs, anti-Ki67 MoAbs, antihuman IgD MoAbs,
and antifollicular dendritic cell MoAbs (HJ2) were from Dako. These
MoAbs were all shown with FITC-conjugated donkey antimouse IgG Abs.
Rabbit anti-CB2 receptor Abs were stained with Cy3-conjugated donkey
antirabbit IgG Abs from Jackson Immunoresearch.
Cells and tissues.
Cells of the human myelocytic HL60 cell line (ATCC, Rockville,
MD) were grown in RPMI 1640 (GIBCO, Grand Island, NY)
medium supplemented with 10% heat-inactivated fetal calf serum, 0.26 mg/mL glutamine, 180 IU/mL penicillin, and 0.18 mg/mL streptamycin.
Mononuclear cells were isolated from Ficoll Hypaque density
centrifugation of peripheral blood obtained from 3 consenting healthy
donors (Caucasian men 26, 38, and 44 years of age). Tonsils were
obtained after obtaining approvals from children (Caucasian females 8, 8, and 10 years of age) undergoing tonsillectomy (Clinique du Parc,
Montpellier, France). For confocal microscopic studies, tonsils were
frozen in liquid nitrogen and maintained at 80°C until
staining and analysis. For flow cytometric studies, tonsils were
immediately minced, labeled, and analyzed. B cells were purified from
tonsils with magnetic beads using the Variomacs system (Tebu, Le Perray
en Yvelines, France). In the first step, tonsil T cells and monocytes
were depleted, respectively, with CD3- and CD14-coated beads. In the
second step, B cells were incubated with biotinylated anti-IgD Abs and
streptavidin-coated beads to isolate IgD+ and
IgD B cells. The purity of both B-cell populations
was greater than 95% as assessed by fluorescence-activated cell
sorting (FACS). Isolation of GC
(CD38+CD44) B cells were thus performed
by negative selection. IgD B cells were submitted to
two rounds of depletion with different anti-CD44 MoAbs (clone NKI-P2
and clone J173 purchased from Immunotech), followed by incubation with
magnetic beads coated with antimouse IgG Abs.
Leukocyte staining for flow cytometry.
Cell surface phenotyping was performed by incubating cells with
appropriate phycoerythrin and tricolor MoAbs in phosphate-buffered saline (PBS) for 30 minutes at 4°C, following the supplier's
instructions. Cells were fixed in 1% formaldehyde overnight at
4°C, washed once, and permeabilized for 10 minutes with a solution
of 0.1% saponin in PBS containing 1% BSA. Purified anti-CB2 receptor
Abs (1:1,000 dilution) were added to 106 cells in 100 µL
of the 0.1% saponin/1% BSA solution for 30 minutes. After two washes
with 0.03% saponin in PBS containing 0.3% BSA, cells were incubated
with FITC-conjugated donkey antirabbit IgG Abs (1:100 dilution) for 30 minutes, washed once with the 0.03% saponin/0.3% BSA solution, and
washed once with PBS alone. Negative controls were performed by 1 hour
of preincubation of anti-CB2 receptor Abs with the C-terminal synthetic
peptide at 20 µg/mL. The fluorescence intensity mean of each subset
was calculated by substracting the fluorescence of the irrelevant
controls (anti-CB2 receptor Abs + peptide) from that of the relevant
labeling (anti-CB2 receptor Abs).
Tissue staining for confocal microscopy.
Serial cryostat sections (9-µm thick) of tonsils were fixed in
acetone for 5 minutes at room temperature. Sections were simultaneously incubated with mouse antihuman leukocyte antigen MoAbs and rabbit anti-CB2 receptor Abs under 100 µL of PBS containing 0.5% BSA. After
three washes in the same buffer, sections were simultaneously stained
with FITC-conjugated donkey antimouse IgG Abs and Cy3-conjugated donkey
antirabbit IgG Abs, both at 1:200 dilution in PBS containing 0.5% BSA.
After two washes in the same buffer and one wash in PBS without BSA,
sections were mounted in a solution of glycerol/PBS containing the
antibleaching reagent DABCO at 50 mg/mL (Sigma). Specificity controls
were performed by 1 hour of preincubation of anti-CB2 receptor Abs with
the C-terminal synthetic peptide at 20 µg/mL.
Dual fluorescence analysis was performed using a laser confocal
microscope (LSM410; Zeiss, Oberkochen, Germany) equipped with a Plan
NEOFLUAR water immersion lens (16×; numerical aperture [NA] = 0.50). Signals were collected separately after
excitations of FITC and Cy3 at 488 and 543 nm, respectively. FITC
emission was collected using a transmission filter centered at 530 nm
and Cy3 emission using a 590-nm long-pass filter.
RNA preparation and reverse transcription-polymerase chain reaction
(RT-PCR) analysis.
Subpopulations of B cells were obtained, purified, and labeled for cell
surface phenotyping as described above. Cell sorting of 2 × 105 cells of each B-cell subsets was performed using the
Normal-C mode of a FacstarPlus cytometer (Becton
Dickinson, Erembodegen, Belgium). Purification of each B-cell subset
was checked by reanalyzing another sorting run. This procedure led to
B-cell subpopulation purities ranging from 95% to 99.5%. The mRNA
purification and conversion to single-strand cDNA were performed using
a PolyATtract series 9600J mRNA Isolation System with cDNA Synthesis
Reagents (Promega, Charbonnières, France) according to the
manufacturer's instructions.
Briefly, 105 sorted cells were centrifuged and suspended in
20 µL of extraction buffer. Each sample was then transfered to the
well of a V-bottom 96-well plate. After hybridization with a synthetic
biotinylated oligo (dT) probe and incubation with streptavidin-coated
magnetic beads, 3 -polyadenylated RNA was captured using a 96 pins Multi-Magnet (Promega, Charbonnièrès, France). After successive washes, purified mRNA was eluted into 20 µL
of water. mRNA was converted to single-strand cDNA by adding 10 µL of
reverse transcriptase master mix (containing AMV Reverse Transcriptase)
in each well of the 96-well microplate.29
Quantitation of CB2 receptor mRNA levels was performed in the
exponential phase of amplification as previously
described.30 Independent PCR amplifications of CB2 receptor
and  2-microglobulin (used as an external control) were
run in parallel as already described.26
Reaction products were analyzed using a nonisotopic microplate assay
supplied by Sanofi Diagnostics Pasteur (Marnes-la-Coquette, France).
PCR products and amplicons were captured by specific immobilized
nucleotide probes complementary to 2-microglobulin and
CB2-receptor sequences located between primers. Quantitation was
performed by hybridization with a biotinylated labeled probe and
incubation with an avidin-peroxidase conjugate. The probes used were as
follows: CB2 receptor capture probe,
5-gccaacctcacatccagcctcattcgggc-3; CB2 receptor detection
probe, 5-biotin-tgggaaccaacagatgagga-3; 2-microglobulin capture probe,
5-caattctctctccattcttcagtaagtcaac-3; and
2-microglobulin detection probe,
5-biotin-agaaagaccagtccttgctg-3. Sandwich hybridization
assays were performed as recently described,31 with slight
modifications. Briefly, 96-well microplates (Maxisorb Nunc, Rosk,
Denmark) were coated with 200 µL of the capture oligonucleotide solutions (0.5 µg/mL in PBS buffer). After overnight incubation at
room temperature, plates were washed twice, dried for 20 minutes at
55°C, and then sealed for long-term storage. Wells were
prehybridized for 30 minutes at 37°C with 200 µL of hybridization
buffer; after supernatants were discarded, 200 µL of
biotin-oligonucleotide probes (50 ng/mL in hybridization buffer)
containing 7 µL of heat-denaturated (10 minutes at 95°C) PCR
products were distributed in each well and incubated for 60 minutes at
37°C with gentle shaking. Microplates were washed six times with
200 µL of washing buffer and a second incubation with 200 µL of
extravidin-peroxidase conjugate (Sigma; 1:5,000 dilution in PBS
containing 0.3% BSA) was performed for 30 minutes at 37°C under
shaking. Plates were washed six times and the immobilized hybrid
complex was detected by addition of 200 µL of ortho-phenylene diamine
chromogenic substrate solution (OPD) for 15 minutes at 37°C. Fifty
microliters of 4N H2SO4 was added to block the
enzymatic reaction. Buffer solutions and reagents were distributed in
microplates using a Biomek 1000 automated laboratory workstation
(Beckman, Paris, France) equipped with a spectrophotometer that
measures optical densities at 492 nm.
Plasmid construction and generation of a stable cell line.
A 1.08-kb HindIII BamHI fragment encompassing the CB2
receptor coding sequence was ligated into the mammalian expression
vector pcDNA3 (In Vitrogen, San Diego, CA), placing transcription of cDNA under the strong immediate early promoter of human
cytomegalovirus. Transfection of HL60 cell line with recombinant
plasmid was performed by electroporation. Briefly, 2 × 107 cells were washed in PBS, resuspended in 1 mL PBS
containing 20 µg vector, and incubated on ice for 10 minutes.
Electroporation was performed in a 0.4-cm in diameter cuvette by using
a Bio-Rad Gene Pulser at 320 mV and 250 µF. After 10 minutes of
incubation at room temperature, electroporated cells were grown in the
medium described above. The day after, cells were seeded at 5 × 105/mL in the presence of geneticin (600 µg/mL medium) in
24-well microplates. Cells were screened 3 to 4 weeks later for
expression of CB2 receptors by flow cytometry. Positive cells were
cloned in 96-well microplates with a FacstarPlus cytometer to get a
stable transfected HL60 cell line.
Staining of wild-type (HL60-WT) and CB2 receptor-transfected (HL60-CB2)
cells was performed by incubating cells with anti-CB2 receptor Abs
(1:1,000 dilution) with or without C-terminal synthetic peptide at 20 µg/mL, as described above for leukocytes. FITC-conjugated antirabbit
IgG Abs (1:100 dilution) was used for flow cytometry, while the same
reagent linked to Cy3 fluorochrome was used for confocal microscopy.
CD40-mediated proliferation of B-cell subsets.
CD32+-L cells suspended at 2 × 106/mL
were treated for 1 hour with 75 µg/mL mitomycin C in RPMI 1640 supplemented with 0.5% heat-inactivated calf serum, 2 mmol/L
L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 5 mmol/L HEPES buffer. These cells were washed four times and plated at 5 × 103 cells per well in round-bottomed 96-well
microtiter trays. CD40 MoAbs (MAB89) were added at 100 ng/mL and
incubated for 2 hours at 37°C. Purified B-cell subsets were seeded
at 105 cells per well simultaneously with CP55,940 ligand
(generously provided by Pfizer, Groton, CT) at the
indicated concentrations. When the cannabinoid compounds SR 141716
(CB1 receptor antagonist) and SR 144528 (CB2 receptor antagonist)
from Sanofi Recherche were used, they were preincubated for 30 minutes
with B cells before the addition of CP 55,940. DNA synthesis was
determined 72 hours later by pulsing the cells with 1 µCi/well
[3H] thymidine for the last 16 hours of the culture
period. Each point was the mean of four replicates. The data shown are
representative of two separate experiments performed with two different
donors.
Modulation of CB2 receptor expression in B-cell subsets.
B-cell subsets were activated by CD40 MoAbs as described above, except
that CD32+-L cells and B cells were seeded at 5 × 104 and 106 cells per well, respectively, in
24-well microtiter trays. CB2 receptor expression was checked at 24 and
48 hours using the flow cytometric and RT-PCR analyses.
Statistical analysis.
Data were analyzed using the Dunnett's analysis of variance test. A
cut-off value of P  .05 was used to indicate statistical significance. Each experiment was repeated at least twice.
| |
RESULTS |
Production and characterization of anti-CB2 receptor polyclonal Abs.
Several rabbits were immunized with 1 of the 18 BSA-conjugated peptides
corresponding to different intracellular and extracellular parts of the
CB2 receptor. Among these, the peptide corresponding to the
intracellular 11-aminoacid sequence of the C-terminal part was the only
one that led to specific anti-CB2 receptor Abs. After five injections,
Abs from immune serum were immunopurified on a gel modified with the
synthetic peptide. The specificity of purified Abs was evaluated in
immunoblotting experiments performed with membrane of CHO cells stably
transfected with the CB2 receptor (CHO-CB2). Two bands were shown when
75 µg of protein from CHO-CB2 membranes were electrophoresed, whereas
no band was observed when the same amount of protein from the wild-type
cell line (CHO-WT) was analyzed (Fig 1).
The major band corresponded to a molecular weight of approximately 46 kD, consistent with the deduced amino acid sequence of the human CB2
receptor cDNA. The minor band corresponded to a molecular weight of
approximately 45 kD, which could represent a degraded receptor or
another form of the receptor differently glycosylated.
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| Fig 1.
Reactivity of CHO membranes to anti-CB2 receptor Abs
assayed in Western blot. CHO-WT and CHO-CB2 membranes were resolved by
SDS-PAGE, transferred to nitrocellulose, and shown with anti-CB2
receptor Abs raised against the C-terminal CB2 receptor peptide. Lane
1, molecular weight markers; lane 2, CHO-CB2 membranes; lane 3, CHO-WT
membranes.
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The specificity of immunopurified anti-CB2 receptor Abs to recognize
CB2 receptors in their native forms was achieved by studying their
binding on HL60 cells transfected with the human CB2 receptor cDNA
(HL60-CB2). Flow cytometric analysis showed that a positive staining
was obtained in HL60-CB2 cell line but not in the wild-type cell line
(HL60-WT), as shown in Fig 2A. Moreover,
inhibition of the labeling was observed when anti-CB2 receptor Abs were
preincubated with the C-terminal synthetic peptide confirming the
specificity of anti-CB2 receptor Abs. We next examined the subcellular
distribution of CB2 receptors by confocal microscopy. Figure 2B shows a
localization of CB2 receptors mainly associated with the plasma
membrane of HL60-CB2 cells.
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| Fig 2.
(A) Flow cytometric analysis of the labeling of HL60
cells transfected with CB2 receptor cDNA (HL60-CB2) by anti-CB2
receptor Abs. After formaldehyde fixation and saponin permeabilization,
HL60-CB2 (top histogram) and wild-type HL60 cells (bottom histogram)
were labeled with anti-CB2 receptor Abs preincubated (dotted line) or
not (solid line) with the C-terminal peptide of the CB2 receptor. (B)
Confocal microscopic analysis of the localization of CB2 receptors in
HL60-CB2 cells. The left side corresponds to HL60-CB2 stained with
anti-CB2 receptor Abs and the right side corresponds to HL60-CB2
stained with anti-CB2 receptor Abs preincubated with the C-terminal
peptide as negative control.
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Expression of CB2 receptors in mononuclear cells isolated from
peripheral blood and tonsils.
The expression of CB2 receptors was first assayed by flow cytometry in
peripheral blood mononuclear cells isolated from three different human
donors. As shown in Fig 3A, the levels of
CB2 receptor expression in these cells was relatively low as compared with HL60-CB2 cells. The quantitation of CB2 receptors in leukocytes showed that B lymphocytes (CD20+) expressed the highest
level of CB2 receptors, followed by NK cells (CD56+; Fig
3B). Among T-cell subsets, T8 (CD3+CD8+)
lymphocytes displayed a higher level of CB2 receptors than did T4 cells
(CD3+CD4+).
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| Fig 3.
Expression of CB2 receptors in peripheral blood
mononuclear cells. (A) Mononuclear leukocytes were isolated and labeled
for flow cytometric analysis as reported in Materials and Methods. Each
staining profile for CB2 receptor expression (solid line) was overlayed
with the negative control (dotted line) performed by preincubating
anti-CB2 receptor Abs with the C-terminal synthetic peptide.
The four staining profiles were obtained after positionning a region of
interest on cells expressing CD3+ CD4+ (T4
cells), CD8+ CD3+ (T8 cells),
CD56+ (NK cells), or CD20+ (B cells). The
histograms shown are all from one donor and are representative of
three different donors. (B) Mean ± SD of CB2 receptor
fluorescence intensities in peripheral blood leukocyte subsets from
three different donors analyzed by flow cytometry as described in (A).
For each leukocyte subset, the fluorescence intensity reported on the
abcissa was calculated in arbitrary units by subtracting the irrelevant
control (anti-CB2 receptor Abs + specific peptide) from the anti-CB2
receptor Ab labeling.
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The preferential expression of CB2 receptors in B cells led us to
investigate the in situ distribution of this molecule in the B-cell
zones of secondary lymphoid organs. For this purpose, dual-immunofluorescence studies were performed on tonsil tissue sections by combining anti-CB2 receptor Abs together with several MoAbs
identifying the different compartments of the B-cell follicles. In this
tissue, the absence of CB2 receptor staining in interfollicular T-cell
areas (CD3+) was observed as well as a marked homogeneous
labeling of the mantle zones containing the resting IgD+ B
cells (Fig 4, lines I and II,
respectively). By contrast, germinal center (GC) areas displayed a
heterogeneous labeling. In some cases, they appeared labeled with the
same intensity as the mantle zone (Fig 4, lines II and IV), and in
other cases they displayed a slight decreased CB2 receptor staining
(Fig 4, lines I and V). Variations of intensity of CB2 receptor
labeling observed in some GC may be associated with the presence of a
particular cell subset in these areas. Indeed, when GC were full of
follicular dendritic cells (FDC), as shown after labeling with anti-FDC
HJ2 MoAbs, GC were entirely stained by anti-CB2 receptor Abs (Fig 4,
line IV), indicating that FDC expressed the CB2 receptor. By contrast, we found that weak expression of CB2 receptors was frequently associated with the presence of Ki67+ proliferating cells
that are localized in the dark zones of GC in human tonsils (Fig 4,
line V). These observations, which were reproducibly repeated in three
different tonsils, showed that CB2 receptors appeared preferentially
expressed in both follicular mantles and GC of B-cell zones. In GC, FDC
expressed CB2 receptors, whereas Ki67+ cells displayed
substantial lower levels of these receptors.
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| Fig 4.
In situ localization of CB2 receptors on tonsil
tissue sections. Frozen tissue sections were simultaneously labeled
with MoAbs characterizing different anatomical compartments in tonsils
and with anti-CB2 receptor Abs. The MoAb labeling are displayed in the
first column and are green-colored: CD3 (I); anti-IgD (II); CD38 (III);
anti-follicular dendritic cells (IV); anti-Ki67 (V). The anti-CB2
receptor Ab labelings are displayed in the second column and are
red-colored. The last line shows an irrelevant control performed with
the specific peptide as described in Fig 2. The third column shows the
merge colors. These data are representative of three different
tonsils.
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Regulation of the expression of CB2 receptors during B-cell
differentiation.
The low CB2 receptor expression associated with proliferating B cells
suggested a subtle regulation of this receptor during B-cell
differentiation. To confirm the above-noted histological observations,
expression of CB2 receptors was studied in purified B cells from three
different human tonsils, using anti-CB2 receptor Abs and flow
cytometry.
The expression of CB2 receptors was found to be modulated during B-cell
differentiation (Fig 5). In
CD38+ GC B cells, a decrease of CB2 receptor expression was
observed and confirmed when B lymphocytes acquired CD77, corresponding to their differentiation in centroblasts. Finally, CB2 receptor expression was restored when B cells reached their terminal stages of
differentiation to become memory B cells
(IgDCD44+CD38).
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| Fig 5.
Analysis of the regulation of expression of CB2 receptors
during B-cell differentiation. Human tonsillar B-cell subsets were
analyzed by flow cytometry ( ) and by RT-PCR ( ) to quantitate CB2
receptor expression at the protein and transcript levels, respectively.
For flow cytometric study, phenotypes of virgin B cells
(IgD+ CD38), GC B cells
(IgD CD38+), centroblasts
(IgD CD77+), and memory B cells
(IgD CD38 CD44+) were
performed on tonsillar B cells as indicated in Materials and Methods.
Results are the mean ± SD from three different donors and were
calculated as described in Fig 3. For RT-PCR, the level of mRNA in each
subset was quantitated from 2 × 105 virgin B cells, GC B
cells, centroblasts, or memory B cells sorted by FACS. CB2-receptor
mRNA contents were normalized with that of
2-microglobulin and are expressed in arbitrary units.
This experiment was repeated from two different donors with similar
results.
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These results were confirmed when the modulation of CB2 receptors was
studied at the level of mRNA content during B-cell differentiation. A
quantitative RT-PCR-based method was performed on 2 × 105 highly purified B cells belonging to different B-cell
subsets. Two analyses performed on B-cell subsets from two different
donors showed that centroblasts (CD77+) displayed a
fourfold loss in their CB2 receptor-mRNA content compared with virgin B
cells (IgD+; Fig 5).
Involvement of CB2 receptors in the proliferation of virgin and GC B
cells induced by CD40 MoAbs.
To study the function of CB2 receptors during B-cell differentiation,
tonsillar B cells were separated into two subsets corresponding to
virgin (IgD+) and GC (CD38+) B cells. We first
examined the effects of the CP55,940 cannabinoid agonist on the
proliferation of both subsets triggered with optimal concentrations of
CD40 MoAbs in the presence of CD32+-L cells. CP55,940
induced a dose-dependent increase of proliferation on both subsets
(Fig 6). The thymidine uptake mediated by
CD40 MoAbs was increased by 27% for the virgin B cells and 20% for the GC B cells in the presence of 10 nmol/L of the cannabinoid ligand.
These effects were optimal at 72 hours, although they can be detected
as soon as 24 hours. They were not inhibited by the CB1 receptor
antagonist SR 14171622 and were totally affected by the
CB2 receptor antagonist SR 144528 (Fig
7). Furthermore, we observed that CP55,940 was not able to induce by
itself any proliferation of B-cell subsets in the absence of CD40 MoAbs
(data not shown).
View larger version (17K):
[in this window]
[in a new window]
| Fig 6.
Effect of CP55,940 on the proliferation of B-cell
subsets. Virgin (A) and GC (B) B-cell subsets were induced to
proliferate in the presence of different concentrations of CP55,940 for
72 hours after ligation of CD40 antigen using CD32+ L
cells. Data shown are representative of two different experiments
performed from two different donors. *P .05.
|
|
View larger version (19K):
[in this window]
[in a new window]
| Fig 7.
CB2 receptors accounted for the CP 55,940-increased
B-cell proliferation. The CB1 receptor antagonist SR 141716 ()
and the CB2 receptor antagonist SR 144528 ( ) were preincubated at
indicated concentrations for 30 minutes with purified tonsillar B cells
before the addition of 10 nmol/L CP 55,940. B cells were induced to
proliferate for 72 hours after ligation of CD40 antigen. Data are
expressed taking as 100% the difference between [3H]
thymidine uptakes (which corresponded to 15,200 cpm) into B cells
activated with CD40 MoAbs with and without 10 nmol/L CP 55,940. *P .05.
|
|
We then examined the regulation of CB2 receptors in virgin and GC B
cells triggered by CD40 ligation. Transcripts of CB2 receptors were
dramatically upregulated in both B-cell subsets. CB2 receptor mRNA
content was maximum around 24 hours and returned to its basal level
within 48 hours (Fig 8A). This upregulation
of CB2 receptors was confirmed at the protein level using anti-CB2
receptor Abs and flow cytometry. CB2 receptors increased during the
first 24 hours and was maintained at 48 hours in virgin and GC B cells (Fig 8B). This might indicate that CD40 ligation induces the
transcription of CB2 receptors.
View larger version (15K):
[in this window]
[in a new window]
| Fig 8.
Regulation of CB2 receptor expression during
CD40-mediated activation. Virgin and GC B-cell subsets were triggered
by CD40 ligation in the presence of CD32+-L cells.
Expression of CB2 receptors were quantitated at 24 and 48 hours by
RT-PCR (A) and flow cytometry (B) as described in Figs 3 and 5. Data
are representative of two different experiments performed from two
different donors. *P .05.
|
|
| |
DISCUSSION |
Both 9-THC and the chemical analog CP55,940 exert their
psychoactive effects through the brain cannabinoid
receptor.1,32 They activate different signaling
pathways5-8 that are all inhibited by the
potent and selective SR 141716 antagonist.22 Both
cannabinoid compounds also recognize another receptor, CB2, which is
mainly localized in cells of the immune system.20 These two
receptors belong to the seven-transmembrane
G-protein-coupled receptor family. Several receptors of this
family, such as the chemokine receptors, display major functions in the
immune system through their involvement in the traffic and activation
of leukocytes33,34 as well as their implication in the
infection of immune cells by the human immunodeficiency
virus.35-37 It was thus relevant to postulate that the
large range of pharmacological effects of cannabinoid compounds
reported to date on the immune system9-19,25 could be
mediated through the CB2 receptor.
CB2 cannabinoid receptors are rather considered as orphan receptors,
because only derivatives of arachidonic acid are able to bind these
receptors as putative endogenous ligands with low affinities.38,39 Signaling induced by cannabinoid receptors has extensively been studied in transfected or gene
reporter-transformed cell lines.23,24,40,41 To contribute
to the understanding of the function of CB2 receptors in the immune
system, we decided to accurately study the expression of CB2 receptors
in lymphocytes. We raised polyclonal Abs highly specific for the
C-terminal part of this receptor. Using a semiquantitative flow
cytometric assay, we found that, in human peripheral blood, the rank
order of CB2 receptor expressions was B cells > NK cells > T8 cells > T4 cells, confirming our previously reported
results of determination of CB2 receptor mRNA expression level in these
cells.26,27 Moreover, the evaluation of the number of
secondary Abs bound per cell made it possible to estimate the CB2
receptor quantity to 2,000 receptors per B cell (data not shown).
The preferential expression of CB2 receptors in the B-cell lineage led
us to study their regulation during B-cell differentiation. We thus
examined CB2 receptors in tonsil tissue sections by dual-color confocal
microscopy and found that CB2 receptors were restricted to B-cell areas
in accordance with a previous autoradiographic study of the binding of
[3H]-CP55,940 to rat immune tissues.21 In
secondary follicles, labeling by anti-CB2 receptor Abs was clearly
observed in the follicular mantle, whereas in GC, heterogeneous
staining was observed. In GC, CB2 receptor less staining was found to
be associated with the presence of proliferating cells
(Ki67+), whereas expression of CB2 receptors was found to
be associated with the presence of FDC.
Identification of different B-cell subsets along the B-cell
differentiation pathway in tonsils has recently been described using
flow cytometric technique.42 We used this technique to characterize virgin B cells, GC B cells, centroblasts, and memory B
cells. During B-cell differentiation process, a dramatic downregulation of CB2 receptor labeling was observed when B cells left the virgin B-cell stage to become centroblasts. CB2 receptor expression was restored at the end of differentiation when memory B cells appeared. The polyclonal anti-CB2 receptor Abs target the intracytoplasmic CB2
receptor C-terminal tail. Therefore, the decrease in CB2 receptor staining in centroblasts may be explained either by a decrease in CB2
receptor transcription or a receptor modification at the Ab recognition
site after intracellular signaling. To exclude the
latter, CB2 receptor transcripts were quantitated in highly purified
B-cell subsets. A decrease in CB2 receptor mRNA level was also observed
in centroblasts, confirming the downregulation of CB2 receptors at the
protein level during B-cell differentiation.
The original distribution of CB2 receptors among cells of the immune
system and their fine modulation during B-cell differentiation suggested that these receptors may exert their function on immune cells
depending on their lineages, their stages of differentiation, and their
partitioning at different locations within secondary lymphoid organs.
Our previous observations that cannabinoid ligands enhanced human
B-cell proliferation mediated by cross-linking of surface Igs, whereas
no effect was noticeable on human T-cell proliferation mediated by
phytohemagglutinin (PHA), argued in favor of a B-cell
lineage-specific expression of CB2 receptors.25 Moreover,
cannabinoid agonists effects were totally inhibited by pertusis toxin,
demonstrating that cannabinoid receptors are coupled to a Gi protein in
B cells. To examine the pattern of expression in the mature B-cell
compartment, we compared the proliferative response of virgin and GC B
cells to the cannabinoid agonist, CP55,940, under CD40-MoAb challenge.
Low concentrations of CP55,940 enhanced the proliferation of both
subsets in the presence of CD40 MoAbs. This enhancement was mediated by
CB2 receptors, because the selective CB2 receptor antagonist SR 144528A
inhibited CP 55,940 effects in a dose-dependent manner, whereas the CB1
receptor antagonist SR 141716A was without any effect. Moreover, the
putative endogenous CB1 cannabinoid ligand anandamide
(arachidonylethanolamide)38 was not able to enhance the
proliferation of B-cell subsets induced by CD40 MoAbs (data not shown),
confirming its lack of activity on the peripheral cannabinoid receptor
transfected in CHO cell line.43 Furthermore, stimulation of
CB2 receptors by CP55,940 in the absence of CD40 MoAbs was not
sufficient to induce a proliferation of B cells, indicating that CB2
receptors may act as coreceptors in the CD40-transduction pathway. The
CB2 receptor-mediated enhancement of the proliferation of B cells at
various stages of differentiation suggested a regulation of its
expression after exposition to CD40 MoAbs. Indeed, a strong
upregulation of CB2 receptors was observed in virgin and GC B cells
stimulated with CD40 MoAbs. We have recently shown that CP55,940
induces the activation of p42/p44 MAPK and the expression of the
growth-related gene krox-24 in a CB2 receptor-transfected cell
line.23 The activation of MAPK after ligation of the B-cell receptor is also associated to the induction of krox-24 through the
activation of p21ras pathway.44 Ligation of
CD40 by MoAbs leads to the activation of another signaling pathway that
is the stress-activated protein kinase pathway (SAPK).45
The fact that CB2 receptors may act as coreceptors in both signaling
pathways, MAPK via surface IgM and SAPK via CD40 antigen, suggests that
CB2 receptor signals may converge at a Gi protein
regulating the activity of p21ras, which is an effector
shared by the two important pathways of B-cell
differentiation.46
| |
ACKNOWLEDGMENT |
The authors thank Pierre Gros for a critical review of the manuscript
and Catherine Carayon for her secretarial assistance.
| |
FOOTNOTES |
Submitted March 19, 1998;
accepted July 8, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Pierre Carayon, PhD,
Immunology Department, Sanofi Recherche, 371, rue du Professeur
Joseph-Blayac, 34184 Montpellier Cedex 04, France; e-mail:
catherine.carayon{at}tls1.elfsanofi.fr.
| |
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Abstract
Introduction