Department of Pharmacology and Toxicology. Michigan State University, East Lansing, Michigan
Correspondence: Norbert E. Kaminski, Ph.D., Department of Pharmacology and Toxicology, 315 Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824. E-mail: kamins11{at}pilot.msu.edu
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Key Words: Cannabinol • Interleukin-2 • Mitogen-activated protein kinase • Protein kinase C • Calcium/calmodulin-dependent protein kinases • T lymphocyte.
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B. The corresponding responsive elements for these transcription
factors located in the 5'-proximal promoter/enhancer region of the IL-2
gene are critical for the expression of the IL-2 gene [3
4
]. In a variety of experimental cell culture systems using
primary lymphoid cells and cell lines, cannabinoids inhibited IL-2
expression by T cells activated with phorbol ester plus calcium
ionophore (PMA/Io) [5
6
]. Concordantly, PMA/Io-induced
NF-AT binding and AP-1 DNA binding were strongly inhibited by
cannabinoid treatment [7
8
]. The promoter activity of
reporter plasmids driven by the IL-2 promoter or multiple consensus
sequences for NF-AT was suppressed by cannabinoids in transiently
transfected murine thymoma EL4.IL-2 cells. A relatively transient
attenuation by cannabinol (CBN) of the promoter activity driven by
multiple consensus AP-1 motifs was also observed in the same system
[8
]. In addition, PMA/Io-mediated activation of the
extracellular regulated kinase (ERK) mitogen-activated protein (MAP)
kinases (p44mapk and p42mapk) was found to be
down-regulated by CBN in murine primary spleen cells
[7
]. As ERKs are critical for the activation of AP-1 DNA
binding, these findings suggest a potential mechanism for the
inhibition of IL-2 expression by cannabinoids through the disruption of
MAP kinase-associated signaling resulting in suppression of AP-1
activation and subsequent IL-2 expression.
It is notable that the MAP kinase cascade is one of the major cellular
signaling pathways modulated by cannabinoids. Ligand binding to
cannabinoid receptors has been reported to induce activation, instead
of the aforementioned inhibition of the ERK MAP kinases in Chinese
hamster ovary cells transfected with high levels of cannabinoid
receptors in the absence of any additional activation stimuli
[9
10
]. The positive modulation of MAP kinases by
cannabinoids is in contrast to our previous studies of IL-2 regulation
by CBN. However, it is notable as well as paradoxical that both
positive and negative regulation of IL-2 by cannabinoids has been
reported [11
12
13
]. For instance, Nakano et
al. have shown that 
9-tetrahydrocannabinol
(9-THC) inhibited mitogen-induced IL-2 production but
also enhanced anti-CD3 antibody-induced IL-2 production and
proliferation of murine spleen cells [11
]. Thus, it
appears that the contrasting effects by 9-THC are
dependent on the mode of T-cell activation. Moreover, the enhancement
by 9-THC of anti-CD3-induced IL-2 production was
reported to be influenced by the age of mice from which splenocytes
were isolated, and a possible mechanism for
9-THC-mediated enhancement of anti-CD3-induced IL-2
production has been proposed to be mediated by an increase in
cytoplasmic free calcium [14
]. There are striking
differences among the various investigations of IL-2 modulation by
cannabinoids which make comparisons between these studies difficult,
including differences in animal ages, cell preparations, cell
activation stimuli, and culture conditions. In light of these apparent
discrepancies, the objective of the present studies was to determine
whether the mode and/or the magnitude of T-cell activation is an
influencing factor for the effect of cannabinoids on IL-2 expression
and, if so, to determine the role of ERK MAP kinases. The effects of
CBN on IL-2 production induced by suboptimal versus optimal stimuli
were studied in murine primary splenocytes and EL4.IL-2 cells. CBN was
selected for this investigation because it is an immunomodulatory
plant-derived cannabinoid that exhibits low affinity for the central
cannabinoid receptor (CB1) and modest central nervous system activity.
Central nervous system-inactive or minimally active cannabinoids
possessing immunomodulatory activity represent a potentially novel
class of therapeutic agents [15
16
]. In this
investigation, CBN elicited contrasting effects on IL-2 production by T
cells depending on the magnitude of the T-cell-activation stimuli used.
Consistent with previous reports, CBN attenuated IL-2 production that
was induced by stimuli optimized for maximum IL-2 expression.
Conversely, CBN enhanced IL-2 expression under conditions in which T
cells were suboptimally activated. In addition, our studies suggested
that the CBN-mediated enhancement of IL-2 production is mediated
through signaling pathways involving ERK MAP kinases, protein kinase C,
and calmodulin (CaM)-dependent kinases.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(145-2C11) and anti-mouse CD28 (37.51)
monoclonal antibodies were purchased from PharMingen (San Diego, CA).
Ro-31-8220 was purchased from Calbiochem (La Jolla, CA).
Animals and cell cultures
Female B6C3F1 mice 6 weeks of age were purchased from Charles
River Laboratories (Dortage, MI). On arrival, mice were randomized,
transferred to plastic cages containing sawdust bedding (five mice per
cage), and quarantined for 1 week. Mice were given food (Purina
Certified Laboratory Chow) and water ad libitum and were not used for
experimentation until their body weight was 17–20 g each (when mice
were approximately 8–14 weeks old). Animal holding rooms were kept at
21–24°C and 40–60% relative humidity with a 12-h light/dark cycle.
Spleens were isolated aseptically and made into single-cell suspensions
as described previously [17
]. The splenocytes were
cultured in RPMI 1640 medium (GIBCO BRL, Gaithersburg, MD) supplemented
with 100 U/mL of penicillin, 100 µg/mL of streptomycin, 50 µM
2-mercaptoethanol, and 2% bovine calf serum (Hyclone,
Logan, UT). The C57BL/6 mouse T-cell lymphoma line EL4.IL-2 was
obtained from American Type Culture Collection (Manassas, VA). The
EL4.IL-2 cells were cultured in RPMI 1640 medium supplemented with 100
U/mL of penicillin, 100 µg/mL of streptomycin, 50 µM
2-mercaptoethanol, 2 mM L-glutamine, and 10% bovine calf
serum. In all cases leukocytes were cultured at 37°C in 5%
CO2.
Culture conditions for induction of IL-2
Aliquots of splenocytes (2 x 106 cells/mL)
were added to 48-well tissue culture plates (200 µL/well) and
stimulated with either soluble or immobilized anti-CD3 alone or in
combination with soluble anti-CD28. Soluble antibodies were diluted
with RPMI 1640 medium and added directly to the splenocyte cultures.
Immobilization of anti-CD3 was accomplished by precoating 48-well
culture plates with the antibody and then incubating overnight (100
µL/well) at 4°C. In preliminary concentration response experiments,
both anti-CD3 and anti-CD28 antibodies were tested over a concentration
range between 0.01 and 5 µg/mL for their ability to induce IL-2. In
all experiments splenic T-cell-secreted IL-2 was measured by
enzyme-linked immunosorbent assay (ELISA), after 48 h of culture
at 37°C in 5% CO2. Aliquots of EL4.IL-2 cells (2 x
105 cells/mL) were added into 48-well culture plates (200
µL/well) and stimulated with PMA (0.1–100 nM) or PMA/Io (80 nM/1
µM) for 24 h, and the supernatants were quantified for IL-2 by
ELISA.
ELISA for IL-2 quantification
Mouse recombinant IL-2 standard, purified rat anti-mouse IL-2,
and biotinylated anti-mouse IL-2 antibodies were purchased from
PharMingen. Splenocytes (2 x 106 cells/mL) and
EL4.IL-2 cells (2 x 105 cells/mL) were cultured in
triplicate in 48-well cell culture plates (0.2 mL/well; Corning Inc.,
Corning, NY). The supernatants were collected 48 h and 24 h
after T-cell activation for splenocytes and EL4.IL-2 cells,
respectively, and quantified for IL-2 by ELISA as described previously
[18
].
Western blotting
Nuclear proteins were isolated as previously described
[19
]. Briefly, cells were lysed with a hypotonic buffer
(10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, 1.5 mM MgCl2, pH 7.5), and the nuclei were pelleted
by centrifugation at 3,000 g for 5 min. Nuclear lysis was
performed using a hypertonic buffer (30 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, 1.5 mM MgCl2, 450 mM NaCl, 0.3 mM
ethylenediaminetetraacetate, and 10% glycerol) which contained 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate, and 1 µg/mL each of aprotinin and leupeptin for 15 min
on ice. After lysis, samples were centrifuged at 17,500 g
for 15 min, and the supernatant was retained for use in the Western
blotting. Nuclear protein (25 µg) was loaded in each lane of a
minigel apparatus and resolved on an 8% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gel and transferred to
nitrocellulose by electroblotting. The blot was incubated with the
primary antibody for phospho-ERK1/ERK2, rabbit polyclonal
anti-phospho-ERK1/ERK2 (Promega, Madison, WI), or the primary antibody
for total ERK1/ERK2, goat polyclonal anti-ERK1/ERK2 (Santa Cruz
Biotechnology, Santa Cruz, CA). After washing, the blot was incubated
with an anti-rabbit horseradish peroxidase-linked immunoglobulin for
detection of phospho-ERK1/ERK2 or incubated with anti-goat horseradish
peroxidase-linked immunoglobulin for detection of total ERK1/ERK2,
followed by exposure to enhanced chemiluminescence (ECL) Western
blotting detection reagents (Amersham, Arlington Heights, IL). Bands
were quantified using a densitometer visual imaging system (Bio-Rad,
Hercules, Calif.).
Statistical analysis
The mean plus or minus standard error was determined for each
treatment group in the individual experiments. Homogeneous data were
evaluated by a parametric analysis of variance, and Dunnett’s
two-tailed t-test was used to compare treatment groups to
the vehicle control when significant differences were observed
[20
].
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Figure 1. Comparison of the effects of CBN on IL-2 secretion by splenocytes
treated with various activation stimuli. Splenocytes (2 x
106 cells/mL) were either untreated (NA) or pretreated with
CBN and/or vehicle (VH; 0.1% ethanol) for 30 min followed by
stimulation with (A) the optimal activation stimulus—immobilized
anti-CD3 plus anti-CD28 (iCD3/CD28; 2 µg/mL) or PMA/Io (PI; 80 nM/1
µM)—or (B) the suboptimal stimulus—soluble anti-CD3 (sCD3; 2
µg/mL), soluble anti-CD3 plus anti-CD28 (sCD3/CD28; 2 µg/mL of each
antibody), or immobilized anti-CD3 alone (iCD3). After 48 h of
culture, supernatants were harvested, and IL-2 was assayed by ELISA.
Data are expressed as the means ± SE of triplicate
cultures. *, P < 0.05 as compared with the VH control
group. N.D., IL-2 protein was below the level of quantification.
Results are representative of three independent experiments.
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Figure 2. Concentration-dependent enhancement by CBN of IL-2 secretion by sCD3-
or sCD3/CD28-treated splenocytes. Splenocytes (2 x
106 cells/mL) were either untreated (NA), or pretreated
with CBN (1–20 µM) and/or VH (0.1% ethanol) for 30 min followed by
treatment with sCD3 (2 µg/mL) or sCD3/CD28 (2 µg/mL of each
antibody). After 48 h of culture, supernatants were harvested, and
IL-2 was assayed by ELISA. Data are means ± SE of
triplicate cultures. *, P < 0.05 as compared with the
VH control group. N.D., IL-2 protein was below the level of
quantification. Results are representative of three independent
experiments.
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Figure 3. The effects of CBN on the IL-2 production induced by PMA or PMA plus
ionomycin in EL4.IL-2 cells. (A) EL4.IL-2 cells (2 x
105 cells/mL) were either untreated (NA), or pretreated
with CBN (0.1–20 µM) and/or VH (0.1% ethanol) for 30 min followed
by stimulation with PMA (2 and 5 nM) for 24 h at 37°C. (B)
EL4.IL-2 cells (2 x 105 cells/mL) were pretreated
with CBN (15 µM) or VH for 30 min followed by stimulation with PMA
(2, 5, 10, and 100 nM) or PMA/Io (PI; 80 nM/1 µM) for 24 h at
37°C. IL-2 in the supernatants was quantified by ELISA. Data are
means ± SE of triplicate cultures. *,
P < 0.05 as compared to the VH control group. N.D.,
IL-2 protein was below the level of quantification. Results are
representative of three independent experiments.
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Figure 4. The effect of CBN on the ERK MAP kinase activation induced by sCD3/CD28
or PMA/Io in splenocytes. Splenocytes (2 x 106
cells/mL) were pretreated with VH (0.1% ethanol) or CBN for 30 min and
then activated with either sCD3/CD28 (2 µg/mL of each) or PMA/Io (80
nM/1 µM). At the end of the culture period, the cells were harvested,
and nuclear proteins for each treatment group were isolated and assayed
for phospho-ERK1/ERK2 (pERK) and total ERKs by immunoblotting. (A)
Time-course analysis (15 min–4 h) of the effect of CBN (10 µM) on
sCD3/CD28-induced activation of ERK1 and ERK2. (B)
Concentration-response by CBN (1, 10, and 20 µM) measuring the
activation of ERK1 and ERK2 after a 15-min sCD3/CD28 treatment of
splenocytes. (C) The effect of CBN (10 and 20 µM) on the activation
of ERK1 and ERK2 after a 15 min PMA/Io (80 nM/1 µM) treatment of
splenocytes. Molecular-mass markers are indicated on the left; the
molecular masses for ERK1 and ERK2 are 44 and 42 kDa, respectively.
Results are representative of three independent experiments.
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Figure 5. The effect of CBN on ERK MAP kinase activation in PMA (2 nM)-treated
EL4.IL-2 cells. EL4.IL-2 cells (2 x 105 cells/mL)
were pretreated with VH (0.1% ethanol) or CBN (1, 10, and 20 µM) for
30 min and then activated with PMA (2 nM) for either 15 min or 4 h. Cells were then harvested, and nuclear proteins from each group were
isolated and assayed for phospho-ERK1/ERK2 and total ERKs by
immunoblotting. Molecular mass markers are indicated on the left; the
molecular masses for ERK1 and ERK2 are 44 and 42 kDa, respectively.
Results are representative of three independent experiments.
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Figure 6. Reversal by staurosporine and Ro-31-8220, but not by wortmannin, of
CBN-mediated enhancement of the IL-2 production induced by sCD3/CD28 in
splenocytes. Splenocytes (2 x 106 cells/mL) were
pretreated with (A) wortmannin (1–100 nM) or VH (0.001% of DMSO), (B)
staurosporine (0.1–10 nM) or VH (0.002% DMSO), or (C) Ro-31-8220
(1–100 nM) or VH (0.001%DMSO) for 15 min, or they were untreated
(Control). Cells in each group were then incubated with CBN (15 µM)
or VH for CBN (0.1% ethanol) for 30 min and then activated with
sCD3/CD28 (2 µg/mL of each) for 48 h at 37°C. IL-2 in the
supernatants was quantified by ELISA. Data are means ±
SE of triplicate cultures. *, P < 0.05 as
compared with the matched VH group treated with sCD3/CD28 and CBN.
Results are representative of three independent experiments.
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Figure 7. Reversal by KN93 alone or in combination with Ro-31-8220 of
CBN-mediated enhancement of the IL-2 production induced by sCD3/CD28 in
splenocytes. Splenocytes (2 x 106 cells/mL) were
pretreated with (A) KN93 (0.1–10 µM), (B) KN93 plus Ro-31-8220 (10
nM), (C) KN93 plus Ro-31-8220 (50 nM), or vehicle [0.0005% DMSO
(control group)] for 15 min. Cells in each group were then incubated
with CBN (15 µM) or VH for CBN (0.1% ethanol) for 30 min and then
activated with sCD3/CD28 (2 µg/mL of each) for 48 h at 37°C.
IL-2 in the supernatants was quantified by ELISA. Data are expressed as
the means ± SE of triplicate cultures. *,
P < 0.05 as compared with the matched VH group treated
with sCD3/CD28 and CBN. Results are representative of three independent
experiments.
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9-THC [5
6
]. In contrast, Nakano et al. demonstrated that the IL-2
activity induced in splenic T cells by concanavalin A was inhibited by
9-THC, whereas T cells activated by anti-CD3 exhibited
enhanced IL-2 production in the presence of 9-THC
[11
]. In light of these diverging results, the focus of
the present studies was to critically evaluate whether the mode and/or
magnitude of T-cell activation is an influencing factor in the
differential effects of cannabinoids on the IL-2 expression. Anti-CD3
and anti-CD28 antibodies or PMA/Io were used under various conditions
to differentially activate primary splenic T cells. As expected,
iCD3/CD28 was a strong activation stimulus for IL-2 induction.
Conversely, in the absence of anti-CD28, the efficacy of iCD3 alone to
activate IL-2 was weak. Most striking was the observation that sCD3 and
sCD3/CD28 were also capable of inducing IL-2 secretion, although the
magnitude of induction was significantly lower as compared with
iCD3/CD28. Using this model system, we demonstrated that CBN
elicited contrasting effects on IL-2 production by splenocytes isolated
from adult mice (8–14 weeks old) and provided further insights into
the results reported by Nakano and coworkers [11
].
Specifically, the present study has shown that the contrasting effect
of CBN on IL-2 secretion was dependent on the magnitude rather than the
mode of T-cell activation. Several lines of evidence support this
premise. First, CBN significantly enhanced IL-2 production by
splenocytes activated with stimuli that alone produced only a
suboptimal induction of IL-2 (i.e., iCD3, sCD3, or sCD3/CD28). Second,
CBN significantly inhibited IL-2 production by splenocytes activated
with strong inducers of IL-2 (i.e., iCD3/CD28 or PMA/Io). Third, CBN
significantly enhanced IL-2 production by EL4.IL-2 cells that were
activated with low concentrations of PMA and markedly inhibited IL-2
production by EL4.IL-2 cells that were activated by a high
concentration of PMA with or without ionomycin. It is important to
emphasize that CBN alone was incapable of inducing a detectable amount
of IL-2 production by splenocytes or EL4.IL-2 cells. Previous studies from this laboratory sought to characterize the biochemical mechanism responsible for the decrease in IL-2 gene expression by cannabinoids under the conditions of robust T-cell activation [5 8 ]. Those studies demonstrated that EL4.IL-2 cells and splenic T cells activated by a high concentration of PMA (80 nM) plus Io (1 µM) in the presence of CBN exhibited a significant inhibition of the DNA binding activity of two nuclear factors critical to the transcriptional regulation of IL-2, NF-AT and AP-1 [6 7 8 ]. Follow-up studies in which the inhibition of AP-1 by CBN was further investigated revealed a marked and concomitant inhibition of ERK MAP kinase activation in PMA/Io-activated splenocytes [7 ]. These findings were in contrast to several reports from other laboratories employing primarily CHO cells artificially transfected with high levels of cannabinoid receptors or cell lines with nonimmune origins. In those models cannabinoid treatment induced a positive activation of ERKs which occurred in the absence of any additional activation stimuli [9 10 28 ]. Based on the important role of MAP kinases in IL-2 regulation and the strong correlation between decreased IL-2 expression and the inhibition of ERK activation in our previous studies [7 8 ], ERK regulation was evaluated under conditions in which CBN produced enhanced IL-2 expression. Remarkably, a parallel up-regulation of nuclear phospho-ERKs by CBN was observed in conjunction with enhanced IL-2 production by both splenocytes and EL4.IL-2 cells. However, CBN treatment of resting splenocytes or EL4.IL-2 cells, in the absence of activation stimuli, did not produce detectable modulation of ERK (data not shown). These findings are in contrast to those demonstrating the activation of MAP kinases by cannabinoids in transfected cell systems in which cannabinoid receptors have been greatly overexpressed [9 10 ]. The present results suggest that, in leukocytes, CBN can positively and negatively modulate ERK activation but not in the absence of activators of the MAP kinase cascade. Similar to the enhancing effects of cannabinoids on IL-2 secretion, an increase in human tonsillar B-cell proliferation after cross-linking of surface immunoglobulins in the presence of low concentrations of cannabinoids has been reported and may also be influenced by an up-regulation of ERK activity [29 ]. The present study suggests that, in leukocytes, CBN-mediated modulation of ERKs occurred through effects on upstream regulators outside the MAP kinase cascade. The other possibility is that the effect of CBN on ERKs in resting cells is direct but so modest that it is below the level of detection. Although possible, the latter scenario seems unlikely since the magnitude of CBN-mediated enhancement on nuclear ERKs under certain conditions appears to be quite profound. Collectively, these independent lines of evidence imply that the activation of ERKs was modulated indirectly by CBN and that this may represent a common signaling mechanism by which cannabinoids influence biological activity.
It has been widely established that the induction of the MAP kinase signaling cascade, as assessed through the phosphorylation and activation of ERKs, can be up-regulated through direct activators of PKC such as phorbol esters or by mitogens that activate the small GTP-binding protein p21ras [22 ]. Recently, agonists for GTP-binding protein (G-protein)-coupled receptors have also been implicated in the indirect activation of ERK MAP kinases via the activation of PI 3-kinase [21 ]. In fact PI-3 kinase has been identified as a critical mediator bridging signaling between G proteins and the MAP kinases [21 ]. In light of this, the role of PI 3-kinase and PKC in the CBN-mediated enhancement of IL-2 was examined. These studies showed that pretreatment with the PI-3 kinase inhibitor wortmannin alone enhanced IL-2 production by sCD3/CD28-activated splenic T cells. However, wortmannin pretreatment did not attenuate the CBN-mediated enhancement of sCD3CD28-induced IL-2 production. We interpreted these results as suggesting that PI-3 kinase is not involved in the CBN-mediated enhancement of IL-2. A second series of studies focused on the role of PKC in the CBN-mediated enhancement of IL-2. It is interesting that staurosporine, a broad calcium-dependent protein kinase inhibitor with some selectivity for PKC at 5–10 nM concentrations produced no effect on sCD3/CD28-induced IL-2 but significantly attenuated the CBN-mediated enhancement of IL-2 production. The PKC inhibitor Ro-31-8220, a staurosporine-derived analog with greater selectivity for PKC, only partially attenuated the CBN-mediated IL-2 enhancement. These data suggested that PKC and possibly other calcium-dependent protein kinases are likely involved in CBN-mediated enhancement of IL-2 secretion.
In light of the above findings implicating the involvement of other
calcium-dependent protein kinases, experiments were performed to
investigate the role of CaM kinases. The CaM kinase inhibitor, KN93,
was found to effectively attenuate CBN-mediated enhancement of IL-2. In
addition, the present data also suggest a synergistic role between CaM
kinases and PKC in CBN-mediated enhancement of IL-2 as evidenced by the
observation that the magnitude of reversal by KN93 was potentiated by
Ro-31-8220. It is notable that, in addition to PKC and PI-3 kinase,
elevated intracellular calcium can also activate the ERK MAP kinase
cascade in certain cell types [30
31
32
]. Moreover, CaM
kinases have been demonstrated as being the signaling molecules
responsible for the activation of ERKs by elevated intracellular
calcium [33
34
]. In light of the well-established role
of PKC and calcium-associated signaling as positive regulators of ERKs,
our results suggest that the CBN-induced enhancement of IL-2 described
in this investigation is mediated, at least in part, through an
up-regulation of ERK MAP kinase-associated signaling. Consistent with
this premise, cannabinoids have been identified as being capable of
mobilizing intracellular calcium [35
]. Most pertinent to
the present studies, 9-THC was previously reported to
enhance the rise in intracellular calcium in splenocytes activated with
soluble anti-CD3 [14
]. In addition, PKC was also
implicated by others as being positively modulated by cannabinoids and
involved in cannabinoid-mediated induction of the growth-related gene
Krox-24 [10
] and in isolated preparations
from rat forebrain. These results were subsequently confirmed in vitro
when it was demonstrated that the plant-derived cannabinoids, CBN,
9-THC, and cannabidiol all enhanced PKC activity
[36
]. Collectively, these studies suggest that PKC- and
calcium-associated signaling pathways can be positively regulated by
cannabinoids and that there are cellular targets involved in
CBN-induced enhancement of the IL-2 production.
In summary, this study demonstrated that CBN could elicit both positive and negative influences on IL-2 production by T cells. Whether enhancement or inhibition of IL-2 was induced by CBN was primarily dictated by the magnitude of T-cell activation. Inhibition of IL-2 by CBN was readily achieved when T cells were activated by stimuli that had been optimized for maximum IL-2 production. Conversely, CBN markedly enhanced IL-2 production under those conditions in which T cells had been stimulated with a suboptimal activator of IL-2. Both the enhancing and inhibitory effects of CBN on IL-2 appeared to be closely correlated to up-regulation and down-regulation of the ERK MAP kinases, respectively. Through the use of specific kinase inhibitors, our studies ruled out a role for PI-3 kinase but implicated the involvement of PKC and CaM kinases in the CBN-mediated enhancement of IL-2. Together, these results indicate that modulation of ERK MAP kinase-associated signaling is involved, at least in part, in the differential effects by CBN on IL-2 regulation in T cells.
Received April 30, 2000; revised December 10, 2000; accepted December 12, 2000.
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B elements by cannabinol, a minimally CNS-active cannabinoid Biochem. Pharmacol. 55,1013-1023[Medline]B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor Int. Immunol. 7,151-1619-tetrahydrocannabinol-induced stimulation of glucose metabolism in primary astrocytes Mol. Pharmacol. 54,834-843This article has been cited by other articles:
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