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INFLAMMATION AND IMMUNOPHARMACOLOGY
Department of Pharmacology and Toxicology and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan
Received March 24, 2003; accepted May 27, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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cannabidiol >
S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl)
methanone mesylate (WIN 55212-3) cannabinol. Cannabinoid-induced
inhibition of PMA/Io-stimulated interleukin-2 was not attenuated by the
presence of both SR144528 and SR141716A. Using pertussis toxin to address the
role of G protein-coupled receptors in this response, it was determined that
pertussis toxin treatment did not attenuate cannabinol-induced inhibition of
PMA/Io-stimulated interleukin-2. With the demonstration that
cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2 was not
mediated via CB1 or CB2, alternative targets of cannabinoids in T cells were
examined. Specifically, it was demonstrated that cannabinoids elevated
intracellular calcium concentration in resting splenocytes and that the
cannabinol-induced elevation in intracellular calcium concentration was
attenuated by treatment with both SR144528 and SR141716A. Interestingly,
pretreatment of splenocytes with agents that elevate intracellular calcium
concentration inhibited PMA/Io-stimulated interleukin-2 production, suggesting
that an elevation in intracellular calcium concentration might be involved in
the mechanism of interleukin-2 inhibition. These studies suggest that immune
modulation produced by cannabinoids involves multiple mechanisms, which might
be both cannabinoid receptor-dependent and -independent.
). With respect to acquired immunity, modulation of
interleukin-2 production is one of the more sensitive targets of cannabinoids.
It is interesting that cannabinoids either enhance or inhibit interleukin-2
secretion, depending on the experimental conditions, including age of the
animals and magnitude of cellular activation (Nakano et al.,
1992,
1993;
Jan and Kaminski, 2001). Using
an optimum stimulatory concentration of PMA/Io, cannabinol treatment inhibited
interleukin-2 protein secretion by splenic T cells
(Condie et al., 1996). This
inhibition correlated with a decrease in expression of phosphorylated
extracellular signal-regulated kinase mitogen-activated protein kinase
(Faubert and Kaminski, 2000).
Furthermore, it was determined that cannabinol inhibited transcription factor
binding to the proximal activator protein-1 and distal nuclear factor of
activated T cells motifs in the interleukin-2 promoter
(Condie et al., 1996;
Faubert and Kaminski, 2000;
Yea et al., 2000), which might
also contribute to the inhibition of interleukin-2. Thus, although the
mechanism of cannabinoid-induced inhibition of interleukin-2 in
PMA/Io-stimulated splenocyte is understood to occur via inhibition of
transcriptional activation of interleukin-2
(Yea et al., 2000), the
upstream signals contributing to inhibition of transcription have not been
completely elucidated. Likewise, the role of cannabinoid receptors in the
inhibition of interleukin-2 by cannabinoid treatment has remained elusive.
The identification of cannabinoid receptors provided a putative mechanism
for cannabinoid-mediated effects (Matsuda
et al., 1990; Munro et al.,
1993). Cannabinoid receptors, both CB1 and CB2, belong to the G
protein-coupled family of receptors and can couple negatively to adenylate
cyclase (for review, see Matsuda,
1997). In addition, both receptors have been shown to activate
extracellular signal-regulated kinase mitogen-activated protein kinase in
cannabinoid receptor-transfected cell lines. The CB1 receptor has also been
shown to couple negatively to N- and Q-type calcium channels and positively to
inwardly rectifying and A-type potassium channels (for review, see
Matsuda, 1997). CB1 expression
is highest in the CNS but has also been detected in several peripheral tissues
(including most immune system cells), whereas CB2 expression is highest in
cells of the immune system (Bouaboula et
al., 1993; Galiegue et al.,
1995; Schatz et al.,
1997). It is for that reason that many of these studies were
conducted with cannabinol, a ligand that exhibits 10-fold greater affinity for
the CB2 receptor compared with CB1 (Munro
et al., 1993).
Synthesis of cannabinoid receptor antagonists SR141716A and SR144528, which
antagonize actions at CB1 and CB2, respectively
(Rinaldi-Carmona et al., 1994;
Rinaldi-Carmona et al., 1998),
allowed for the determination of cannabinoid receptor function for many of the
effects of these compounds. This is particularly true for the CB1 receptor in
that many of the cannabinoid-induced CNS effects were prevented by SR141716A
treatment (for review, see Matsuda,
1997). Furthermore, use of CB1 receptor knockout mice confirmed
that the CB1 receptor mediated many of the CNS effects produced by
cannabinoids (catalepsy, hypomotility, and hypothermia)
(Ledent et al., 1999;
Steiner et al., 1999;
Zimmer et al., 1999). CB1 is
present in many immune cells at relatively low levels, and there are a few
instances in which CB1 was determined to mediate immune system effects of
cannabinoids (for review, see Berdyshev,
2000). On the other hand, CB2 is believed not to play a role in
the CNS effects of cannabinoids (Griffin
et al., 1999; Buckley et al.,
2000), yet has been shown to be important for immune system
effects, particularly for cannabinoid-induced inhibition of antigen processing
and presentation in macrophages (McCoy et
al., 1999; Buckley et al.,
2000). Therefore, the objective of these studies was to
investigate the mechanism, including the role of CB1 and CB2, of
cannabinoid-induced inhibition of interleukin-2 production in
PMA/Io-stimulated mouse splenocytes.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Animals. Pathogen-free female B6C3F1 mice, 6 weeks of age, were purchased from Charles River Laboratories, Inc. (Wilmington, MA). On arrival, mice were randomized, transferred to plastic cages containing sawdust bedding (5 animals/cage), and quarantined for 1 week. Mice were given food (Purina Certified Laboratory Chow) and water ad libitum. Mice were not used for experimentation until their body weight was 17 to 20 g. Animal holding rooms were maintained at 21-24°C and 40 to 60% relative humidity with a 12-h light/dark cycle.
Preparation of Lymphocyte Culture. Mice were sacrificed and spleens or thymi were aseptically removed. Single cell suspensions (5 x 106 cells/ml) were prepared and cells were cultured in RPMI 1640 medium supplemented with 100 units penicillin/ml, 100 units of streptomycin/ml, 50 µM 2-mercaptoethanol, and 2% bovine calf serum.
Interleukin-2 ELISA. Splenocytes were either treated with antagonist or vehicle for 30 min at 37°C, followed by cannabinoid agonist treatment for 30 min at 37°C. Alternatively, splenocytes were treated with ionomycin, A23187 [GenBank] , or thapsigargin for 30 min at 37°C. The cells were then treated with 40 nM PMA/0.5 µM ionomycin for 24 h in media containing 2% bovine calf serum in 48-well culture plates at 0.5 ml/well. Cells were harvested and supernatants were collected and assayed in triplicate for interleukin-2 using an ELISA. Briefly, 96-well plates were coated for 1 h at 37°C with a purified rat anti-mouse interleukin-2 antibody (BD PharMingen, San Diego, CA), followed by blocking with 3% bovine serum albumin in phosphate-buffered saline/Tween 20 for 30 min at 37°C. Supernatants from splenocytes were incubated for 1 h at 37°C, followed by addition of biotinylated anti-mouse interleukin-2 antibody (BD PharMingen) for 1 h at RT. Streptavidin peroxidase was then incubated for 1 h at RT. Finally, tetramethylbenzidine substrate and 6 N H2SO4 were added for color development, and samples were read with an EL-808 plate reader (Bio-Tek Instruments, Winooski, VT) at 450 nm. Samples were quantified from a standard curve prepared with interleukin-2 standard (mouse recombinant interleukin-2; BD PharMingen).
cAMP Determinations. Splenocytes were suspended in 5 ml of 2% bovine calf serum and treated with Gey's solution (5 ml/spleen). The splenocytes were swirled on ice for 5 min to lyse the red blood cells, followed by two washes in 2% bovine calf serum. Splenocytes were resuspended in RPMI 1640 medium supplemented with 1 mg/ml fatty-acid poor bovine serum albumin (Calbiochem, La Jolla, CA). Cells in 1-ml aliquots of cells were treated in triplicate with cannabinoid agonist for 30 min at 37°C. The cells were then treated with 25 µM forskolin (Sigma-Aldrich) for 15 min at 37°C. The extraction, release, and quantitation of cAMP from cells were performed using cAMP assay kits (Diagnostic Products, Los Angeles, CA). For pertussis toxin treatment, cells were either treated with pertussis toxin (100 ng/ml, Sigma-Aldrich) or not treated for 24 h before subsequent agonist treatment.
Calcium Determination. Splenocytes were treated with Gey's solution to lyse the red blood cells. Cells were then washed twice in Ca2+-KREB buffer (129 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1 mM CaCl2, 5 mM NaHCO3, 10 mM HEPES, 2.8 mM glucose, and 0.2% bovine serum albumin). Intracellular calcium concentration was determined by measuring the fluorescence of fura-2 dye, which is dually excited at 340 and 380 nm. Fura-2/AM dye (1 µM; Molecular Probes, Eugene, OR) was added to the cells and incubated for 30 min at RT in the dark. Cells were harvested, washed three times with Ca2+-KREB buffer to remove extracellular fura-2 dye, and readjusted to 5 x 106 cells/ml in Ca2+-KREB buffer. Cells were stored at RT in the dark until used. Cells were placed in a 3-ml quartz cuvette with constant stirring. Calcium determinations were performed at RT with a Spex 1681 0.22 spectrometer with dual excitation at 340 and 380 nm and emission at 510 nm (all slit widths were 1 mm). Intracellular calcium concentration calculations were based on maximum and minimum calcium values, as assessed with use of 0.1% Triton X and 250 mM EGTA, respectively. The dissociation constant for the Fura-2-calcium complex was 1.45 x 10-7. For studies conducted in the absence of extracellular calcium, the KREB buffer was prepared as above without CaCl2 and supplemented with 0.02 mM EGTA.
Statistical Analysis. The mean ± S.E. was determined by a
parametric analysis of variance for each treatment group. When significant
differences were detected, treatment groups were compared with the appropriate
control with the Dunnett's two-tailed t test
(Dunnett, 1955).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Herring and Kaminski, 1999;
Yea et al., 2000). Thus, a
characterization of the putative involvement of the cannabinoid receptors in
cannabinoid-mediated inhibition of interleukin-2 production using splenocytes
was conducted. It is notable that splenocytes express mRNA for both
cannabinoid receptors (Schatz et al.,
1997); thus; the CB1 and CB2 receptor antagonists were used in
combination in these studies. As presented in
Fig. 1, interleukin-2 was
readily induced by PMA/Io treatment and was inhibited by cannabinol (1-20
µM) in a concentration-dependent manner. These studies were conducted with
micromolar concentrations of cannabinoid compounds because it has been
previously demonstrated that these concentrations are required in vitro due to
both the lipophilicity of the cannabinoids and the serum concentration
dependence for many of the cannabinoid effects
(Faubert and Kaminski, 2000).
Pretreatment of splenocytes with a combination of antagonists (0.5 µM/0.5
µM, 2.5 µM/2.5 µM, and 5 µM/5 µM SR144528/SR141716A) did not
significantly attenuate the cannabinol-mediated inhibition of interleukin-2
production at any cannabinol concentration tested. The antagonists alone did
not significantly affect the levels of PMA/Io-stimulated interleukin-2
production at concentrations up to 0.5 µM/0.5 µM SR144528/SR141716A.
However, it is notable that at higher concentrations, including 2.5 µM/2.5
µM and 5 µM/5 µM SR144528/SR141716A, the antagonist alone inhibited
PMA/Io-induced interleukin-2 production by at least 30% compared with the
vehicle controls. (Fig. 1,
inset). In other experiments, SR144528/SR141716A at higher concentrations (2.5
µM/2.5 µM and 5 µM/5 µM) produced an even greater decrease in
interleukin-2 production, indicating that cells cultured for extended periods
(24 h) in the presence of these antagonists were functionally affected by both
of these agents. No evidence of inverse agonist activity was observed with
either antagonist. The estimated IC50 values for cannabinol were
15.4 µM in the absence of antagonists and 13.3 µM, 18.1 µM, and 16.8
µM in the presence of 0.5 µM/0.5 µM, 2.5 µM/2.5 µM, and 5
µM/5 µM SR144258/SR141716A, respectively.
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Effect of Pertussis Toxin on Cannabinol-Induced Inhibition of
PMA/Io-Stimulated Interleukin-2 Production. Due to the inability of the
cannabinoid receptor antagonists to attenuate the cannabinol-induced
inhibition of interleukin-2 production, pertussis toxin was used to examine
the potential role of G
i/Go protein
involvement in this effect. Pertussis toxin (100 ng/ml) pretreatment for 24 h
resulted in an increase (29.0%) in forskolin-stimulated cAMP production in
splenocytes, which was statistically different from forskolin-stimulated
splenocytes in the absence of pertussis toxin (data not shown). However,
pertussis toxin pretreatment (10 and 100 ng/ml) did not attenuate the
cannabinol-induced inhibition of PMA/Io-stimulated interleukin-2 production
(Fig. 2). The estimated
IC50 values for interleukin-2 inhibition by cannabinol were 16.3
µM in the absence of pertussis toxin and 27.7 and 17.9 µM in the
presence of 10 and 100 ng/ml pertussis toxin, respectively. Pertussis toxin
did not significantly modulate interleukin-2, with the exception that there
was a modest but consistent increase in basal interleukin-2 secreted from the
cells that were treated with 100 ng/ml pertussis toxin (approximately 10-15
units/ml; Fig. 2, inset).
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Effect of Cannabinoid Receptor Antagonists on WIN-Induced Inhibition of PMA/Io-Stimulated Interleukin-2 Production. Another characteristic of a receptor-dependent mechanism is differential effects with stereoisomers. One such cannabinoid stereoisomer pair is the synthetic compounds WIN 55212-2 and WIN 55212-3. Both WIN 55212-2 ("active" isomer) and WIN 55212-3 ("inactive" isomer) inhibited PMA/Io-induced interleukin-2 production in a concentration-dependent manner; however, WIN 55212-2 demonstrated robust, and consistently greater, inhibition than observed with WIN 55212-3 (Fig. 3, A and B). The IC50 values for WIN-induced inhibition of interleukin-2 were 3.3 µM for WIN 55212-2 and 14.8 µM for WIN 55212-3. Interestingly, WIN 55212-3 inhibited interleukin-2 as robustly as cannabinol, a ligand that exhibits selectivity for the CB2 receptor. Furthermore, the antagonists (0.5 µM/0.5 µM, 2.5 µM/2.5 µM, or 5 µM/5 µM SR144528/SR141716A) did not attenuate the inhibition of interleukin-2 by either WIN isomer (Fig. 3, A and B). In the presence of SR141716A/SR144528, the IC50 values were as follows: 3.9, 5.2, and 4.1 µM for WIN 55212-2 in the presence of 0.5 µM/0.5 µM, 2.5 µM/2.5 µM, or 5 µM/5 µM SR144528/SR141716A, respectively; 14.9, 15.1, and 12.2 µM for WIN 55212-3 in the presence of 0.5 µM/0.5 µM, 2.5 µM/2.5 µM, or 5 µM/5 µM SR144528/SR141716A, respectively.
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Effect of Cannabinoid Receptor Antagonists on Cannabidiol-Induced
Inhibition of PMA/Io-Stimulated Interleukin-2 Production. In light of the
above-mentioned results, cannabidiol, another plant-derived cannabinoid was
used to assess the role of cannabinoid receptors in cannabinoid-induced
interleukin-2 inhibition. Cannabidiol was previously determined to exhibit low
affinity for the CB2 receptor in HL-60 cells
(Munro et al., 1993) and did
not inhibit forskolin-stimulated cAMP production in CB1-transfected Chinese
hamster ovary cells (Matsuda et al.,
1990). Collectively, these previous studies indicate that
cannabidiol exhibits low affinity for both CB1 and CB2. In the present study,
we show that in splenocytes, cannabidiol (1-20 µM) robustly inhibited
PMA/Io-stimulated interleukin-2 production with an IC50 value of
4.1 µM, and this inhibition was not attenuated by 0.5 µM/0.5 µM or 5
µM/5 µM SR144528/SR141716A (Fig.
4). The IC50 values for cannabidiol-induced inhibition
in the presence of the antagonists were 2.9 and 2.4 µM for 0.5 µM/0.5
µM and 5 µM/5 µM concentrations of the SR144582/SR141716A,
respectively. It was notable that the magnitude of inhibition of interleukin-2
by cannabidiol was greater than that of cannabinol, in spite of the fact that
cannabinol possesses greater CB1 and CB2 affinity.
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Cannabinol Elevated Intracellular Calcium Concentration in Resting
Splenocytes. With the demonstration that CB1 and CB2 were not involved in
cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2, other
potential T cell targets were examined. A major target of cannabinoids in T
cells is nuclear factor of activated T cells
(Faubert and Kaminski, 2000;
Yea et al., 2000), a critical
transcription factor in the regulation of interleukin-2 that is tightly
controlled by changes in [Ca2+]i (reviewed in
(Cantrell, 1996). Therefore,
generation of the calcium signal seemed a likely point of inhibition by
cannabinoid compounds. However, these studies revealed that cannabinol
treatment elevated [Ca2+]i for at least 30
min in resting splenocytes (Fig.
5A). Although this sustained elevation by cannabinol in
[Ca2+]i was consistent with other agents that
increase [Ca2+]i, such as ionomycin and
thapsigargin, the shapes of the elevation as well as the overall magnitude of
elevation in [Ca2+]i were different for
cannabinol versus ionomycin or thapsigargin
(Fig. 5, B and C).
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Effect of Zero-Extracellular Calcium Concentration on Cannabinol-Induced
Elevation in [Ca2+]i. In several cell types,
thapsigargin is a powerful tool used to determine whether the elevation in
[Ca2+]i is due to an intracellular store
release. However, as demonstrated in Fig.
5C, thapsigargin treatment of resting splenocytes induced a
sustained elevation in [Ca2+]i, which was
likely due to the activation of calcium release-activated calcium channels to
subsequently stimulate [Ca2+]e influx
(Zweifach and Lewis, 1993).
This sustained elevation prohibited the use of thapsigargin to determine
directly whether cannabinol-induced elevation of
[Ca2+]i was due to depletion of intracellular
stores. Thus, studies were performed in the presence and absence of
extracellular calcium to determine whether the cannabinol-induced elevation in
intracellular calcium was due to influx of extracellular calcium. The calcium
response induced by cannabinol was drastically reduced when the studies were
conducted in the absence of [Ca2+]e,
indicating the elevation of [Ca2+]i by
cannabinol occurred primarily via influx of extracellular calcium although a
modest increase in [Ca2+]i by cannabinol was
still observed (Fig. 6A). Ionomycin-stimulated elevation in [Ca2+]i was
used to confirm the absence of extracellular calcium in the buffer
(Fig. 6B). As expected for
ionomycin, the first phase was primarily due to release of intracellular
stores of calcium, which was not affected by the zero-extracellular calcium
concentration conditions, and the second phase was due to influx of
extracellular calcium, which was abolished under these conditions
(Cantrell, 1996).
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Effect of Cannabinoid Receptor Antagonists on Cannabinol-Induced
Elevation in [Ca2+]i. Induction of
[Ca2+]i elevation with cannabinoid treatment
has been demonstrated in a number of other cell types in a cannabinoid
receptor-dependent manner (Sugiura et al.,
1999,
2000). Cannabinol-induced
[Ca2+]i elevation in resting splenocytes was
mediated primarily via an influx of extracellular calcium, also suggesting a
putative role for membrane-bound receptors. In light of these previous
findings, SR144528 and SR141716A were used to examine the involvement of CB1
and CB2 in cannabinol-induced elevation of
[Ca2+]i in resting splenocytes and
thymocytes. Studies were conducted in thymocytes to verify that the elevation
in [Ca2+]i also occurred in a preparation
that consisted predominantly of T cells. Cannabinol-induced elevation in
[Ca2+]i was markedly attenuated by both
cannabinoid receptor antagonists (either separately or in combination) in
resting splenocytes (Table 1).
Treatment with either SR141716A and/or SR144528, in the absence of cannabinol,
did not significantly elevate the [Ca2+]i
(data not shown). Similar to splenocytes, cannabinol treatment elevated
[Ca2+]i in resting thymocytes. Because
thymocytes do not express CB1 mRNA (Schatz
et al., 1997), only the CB2 antagonist SR144528 was used to
address the putative role of the CB2 receptor in cannabinol-induced elevation
in [Ca2+]i in thymocytes. As presented in
Table 2, SR144528 partially
attenuated the cannabinol-induced elevation in
[Ca2+]i. Interestingly, in thymocytes only,
SR144528 treatment induced an elevation in
[Ca2+]i (approximately 40 nM) independent of
the cannabinol concentration used, suggesting that SR144528 was acting as a
partial agonist.
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Agents That Elevate [Ca2+]i Inhibited
PMA/Io-Stimulated Interleukin-2 Production. Results from the
abovementioned experiments suggest that cannabinol elevated
[Ca2+]i in resting splenocytes and thymocytes
in a cannabinoid receptor-dependent manner. A number of previous studies have
reported that a premature elevation in
[Ca2+]i results in inhibition of T cell
activation (Gallichio et al.,
1994; Nghiem et al.,
1994). Indeed, pretreatment of splenocytes with ionomycin, over a
wide concentration range, for 30 min before activation resulted in a drastic
inhibition of interleukin-2 production. This result was confirmed with two
other agents that elevate [Ca2+]i, A23187
[GenBank]
,
and thapsigargin (Fig. 7). The
concentrations used for both ionomycin and thapsigargin are in the same range
as those used in the [Ca2+]i determination
studies, providing a correlation between the magnitude of
[Ca2+]i elevation and the magnitude of
interleukin-2 inhibition.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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cannabidiol > WIN 55212-3 cannabinol. The rank order for efficacy as
assessed by the ability of the agonist to produce 100% inhibition of
PMA/Io-induced interleukin-2 at the maximum concentration tested was WIN
55212-2 > cannabidiol > WIN 55212-3 > cannabinol. In splenocytes, the
Ki for WIN 55212-2 was lower than that for cannabinol
(Schatz et al., 1997). In
HL-60 cells, rank order binding activity to the CB2 receptor has been reported
to be 
9-THC cannabinol >> cannabidiol
(Munro et al., 1993). In
addition, there was a 1000-fold difference in affinity between the WIN
stereoisomers in CB2-transfected COS cells
(Slipetz et al., 1995). It has
also been reported that cannabinol exhibited a 10-fold higher affinity for the
CB2 receptor, the predominant cannabinoid receptor expressed in the immune
system (Bouaboula et al., 1993;
Galiegue et al., 1995;
Schatz et al., 1997). Indeed,
cannabinol exhibits immunomodulating effects
(Condie et al., 1996;
Herring et al., 1998;
Faubert and Kaminski, 2000;
Yea et al., 2000), but, as
demonstrated here, so did cannabidiol and WIN 55212-3, both of which exhibit
low binding affinity for CB1 and CB2.
In contrast to the data presented here, Buckley et al.
(2000) determined that
9-THC-induced inhibition of interleukin-2 in -CD3
plus peritoneal macrophage-stimulated T cells was lost in CB2 receptor
knockout mice; however, the experimental design was very different from the
one described here. In the present study, cannabinoid-induced inhibition of
interleukin-2 from splenic T cells was measured directly. In the study by
Buckley et al. (2000),
activated peritoneal macrophages derived from either wild-type or CB2 receptor
knockout mice were treated with vehicle or 9-THC for 4 h.
Subsequently, the macrophages were cocultured with a helper T cell hybridoma,
which produced interleukin-2 in response to the macrophages plus -CD3.
In these studies, the macrophages were used as costimulators for optimal
interleukin-2 production because they presumably express the B7 molecule
required to bind the costimulatory T cell molecule, CD28 (for review, see
Harris and Ronchese, 1999).
However, the expression level of B7 on the macrophages was not measured in
either the wild-type or CB2 receptor knockout mice, nor in response to
9-THC treatment. Furthermore, the helper T cell hybridoma
that produced interleukin-2 in response to -CD3 plus peritoneal
macrophages was not assayed for cannabinoid receptor expression. These
critical issues greatly influence the interpretation of the results.
Nevertheless, the authors concluded that the loss of cannabinoid-induced
inhibition of interleukin-2 was due to the loss of CB2 in the stimulator
macrophages and not through direct effects on the helper T cell hybridoma
(Buckley et al., 2000).
Concordant with the aforementioned studies, McCoy et al.
(1999) demonstrated that the
CB2 antagonist was involved in 9-THC-induced inhibition of
antigen processing and presentation. Again, although interleukin-2 production
from T cells was used as an endpoint, the effect by 9-THC
was exerted on the macrophages used to stimulate the T cells (not the T cells
themselves) (McCoy et al.,
1999). Thus, under certain experimental conditions, the CB2
receptor seems to be important for antigen processing and/or presentation in
macrophages (McCoy et al.,
1999; Buckley et al.,
2000). However, it is important to emphasize that neither of the
two experimental designs critically evaluated the role of CB1 and CB2 in the
direct effects that cannabinoid treatment exerts on T cells to inhibit
interleukin-2.
The observation that cannabinoids elevated
[Ca2+]i in a cannabinoid receptor-dependent
manner is consistent with several other studies (Sugiura et al.,
1999,
2000). Interestingly, in
splenocytes, SR141716A provided more effective antagonism than did SR144528,
but both cannabinoid receptor antagonists partially attenuated the
cannabinol-induced elevation in [Ca2+]i. In
contrast, in thymocytes, CB2 mediated the cannabinol-induced elevation in
[Ca2+]i because SR144528 almost completely
attenuated the response to cannabinol. Although the exact mechanism
responsible for the elevation in [Ca2+]i by
cannabinoids has not been elucidated, it is tempting to speculate that
cannabinoid receptors coupled to Gs in lymphocytes to
mediate the cannabinoid-induced elevation in
[Ca2+]i. In fact, the ability of cannabinoid
receptors to couple to Gs has been suggested, particularly
for CB1 (Glass and Felder,
1997; Maneuf and Brotchie,
1997). Another interesting observation was the gradual rise in
[Ca2+]i induced by cannabinol. Although it is
notable that the calcium determinations in this study represent the mean from
many cells at any given time point, rather than for a single cell, the gradual
rise in [Ca2+]i could be attributed to at
least one of two possibilities. One possible explanation is that cannabinol
induced only a partial depletion of intracellular stores. Alternatively, a
rapid reuptake of intracellular calcium occurred concomitantly with the
calcium influx. Additional studies will be required to resolve the specific
mechanism.
Although the cannabinoid-induced inhibition of PMA/Io-stimulated
interleukin-2 was not attenuated by either the CB1 or CB2 antagonists, the
cannabinoid receptor antagonists attenuated (both alone and in combination)
the cannabinol-induced elevation in [Ca2+]i
in resting splenocytes. This differential role of the cannabinoid receptors in
these two cannabinoid-mediated effects suggests either that the elevation in
[Ca2+]i did not contribute to the
cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2 or it is one
of several contributing factors. The present investigation suggests the latter
because cannabinoids as well as agents that elevated
[Ca2+]i (i.e., ionomycin, A23187
[GenBank]
, and
thapsigargin) inhibited PMA/Io-stimulated interleukin-2 production in
splenocytes. In concordance with these observations, T cells become
unresponsive (anergic) if they receive an inappropriate or incomplete
activation signal. Anergy is a state of unresponsiveness in T cells as
characterized by decreased production of interleukin-2, and activator
protein-1 and nuclear factor-
B DNA binding activity (for review, see
Maier and Greene, 1998). In
fact, T cells become anergic, as characterized by >90% block in the ability
to produce interleukin-2, if prematurely stimulated with the calcium ionophore
A23187
[GenBank]
(Gallichio et al.,
1994). In addition, PMA/Io-induced interleukin-2 reporter gene
activity was inhibited in response to an 8-h, 2 µM ionomycin pretreatment
(Nghiem et al., 1994). This
suggests that a premature elevation in
[Ca2+]i induces anergy, depending on the time
of activation of the calcium signal relative to cellular activation and/or the
magnitude of the overall elevation in
[Ca2+]i. Presently, it is unclear whether the
time of generation or magnitude of the calcium signal (or both) contributes to
an unresponsive state. Nevertheless, this might explain how the
[Ca2+]i elevation by cannabinoids contributes
to the inhibition of interleukin-2 production in PMA/Io-stimulated
splenocytes.
The differential role of the cannabinoid receptors in cannabinoid-induced
[Ca2+]i and cannabinoid-induced interleukin-2
inhibition in PMA/Io-stimulated splenocytes might be due to the requirement
for cellular activation in the studies in which interleukin-2 production was
measured. The discrepancy might be associated with the observation that
cellular activation modulates cannabinoid receptor expression levels in T
cells and B cells (Carayon et al.,
1998; Noe et al.,
2000). The ability of cellular stimulation to increase or decrease
cannabinoid receptor expression depends on cell type, cell maturity, and
activation stimulus. In T cells, CB1 receptor mRNA expression was decreased in
response to PMA/Io or -CD3 treatment, suggesting that T cell activation
inhibits expression of cannabinoid receptors
(Noe et al., 2000). This
explanation is only plausible under the assumption that the cannabinoid
effects are not solely mediated via the cannabinoid receptors. Thus, assuming
a minimal role of cannabinoid receptors in cannabinoid-induced inhibition of
PMA/Io-stimulated interleukin-2, the CB1 and/or CB2-dependent component would
not be detected in activated cells. A second possible explanation for the
discrepancy is that these two responses occurred in distinct cell populations
in the splenocyte cell preparation. However, this latter scenario seemed
unlikely based on the demonstration that SR144528 attenuated the
cannabinol-induced [Ca2+]i elevation in
thymocytes, a predominantly T cell preparation.
Another interesting observation was that despite the fact that both WIN
isomers inhibited interleukin-2 production, there were consistent differences
in efficacy especially at concentrations above 10 µM, suggesting that the
two isomers differentially affected a specific cellular target, as opposed to
a nonspecific mechanism of cell perturbation. In addition, the inability of
pertussis toxin to attenuate cannabinol-mediated inhibition of
PMA/Io-stimulated interleukin-2 suggests that the cannabinoid receptors might
couple to G proteins, other than Gi/Go.
Again, it has been suggested that CB1 might couple to Gs
(Glass and Felder, 1997;
Maneuf and Brotchie,
1997).
Together, these data support the notion that cannabinoid-induced inhibition of interleukin-2 production is not solely dependent on the presence of CB1 and CB2 in mouse splenocytes. Furthermore, the present results suggest a strong likelihood of multiple parallel mechanisms for the cannabinoid-induced inhibition of interleukin-2 production based on the observation that cannabinol-induced elevation in [Ca2+]i was attenuated by the cannabinoid receptor antagonists, whereas the cannabinol-induced inhibition of PMA/Io-stimulated interleukin-2 production was not affected by the antagonists. Collectively, these results point toward the possibility that cannabinoids modulate interleukin-2 expression via a combination of cannabinoid receptor-mediated mechanisms, and CB1/CB2-independent mechanisms.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PMA/Io, phorbol ester plus calcium ionophore; CB,
cannabinoid; SR141716A,
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-H-pyrazole-3
carboxyamidehydrochloride; SR144528,
N-[(1S)-endo-1,3,3,-trimethyl bicyclo [2,2,1]
heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide;
WIN-2 (WIN 55212-2),
R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl)
methanone mesylate; WIN-3 (WIN 55212-3),
S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl)
methanone mesylate; ELISA, enzyme-linked immunosorbent assay; PMA, phorbol
12-myristate 13-acetate; RT, room temperature; AM, acetoxymethyl ester;
[Ca2+]i, intracellular calcium concentration;
[Ca2+]e, extracellular calcium concentration;
9-THC, 9-tetrahydrocannabinol; A23187
[GenBank]
,
calcimycin.
Address correspondence to: Dr. Norbert E. Kaminski, Department of Pharmacology and Toxicology, 315 National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824. E-mail: kamins11{at}msu.edu
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80% for
all treatment groups as assessed by trypan blue exclusion. *,
p < 0.05 compared with VH group, **, p <
0.05 compared with the SR 0.5 µM/0.5 µM + VH group; 
, p
< 0.05 compared with the SR 2.5 µM/2.5 µM + VH group; #, p
< 0.05 compared with the 5 µM/5 µM + VH group. Results are
representative of four separate experiments with three replicates per
treatment group.
