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(Journal of Leukocyte Biology. 2001;69:841-849.)
© 2001 by Society for Leukocyte Biology

Role of mitogen-activated protein kinases in the differential regulation of interleukin-2 by cannabinol

Tong-Rong Jan and Norbert E. Kaminski

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cannabinoids can paradoxically regulate interleukin-2 (IL-2) expression either positively or negatively. This study investigated the mechanism responsible for cannabinol-mediated IL-2 modulation. In primary murine splenocytes and EL4.IL-2 T cells, the contrasting effects of cannabinol on IL-2 secretion depended on the magnitude but not the mode of T-cell activation. Suboptimal activation of T cells in the presence of cannabinol produced an enhancement of IL-2 secretion, which was paralleled by an increase in nuclear phospho-extracellular-regulated kinase (ERK) 1/2. In contrast, T cells activated with stimuli that were optimized to induce maximal IL-2 secretion elicited a marked suppression in the production of this cytokine when cultured in the presence of cannabinol. Moreover, cannabinol-mediated enhancement of IL-2 secretion by splenocytes was attenuated to various degrees by staurosporine, Ro-31-8220, and KN93. These results suggest that the enhancement of IL-2 secretion by cannabinol is associated with an increase in ERK mitogen-activated protein kinase, which is protein kinase C and calmodulin-kinase dependent.

Key Words: Cannabinol • Interleukin-2 • Mitogen-activated protein kinase • Protein kinase C • Calcium/calmodulin-dependent protein kinases • T lymphocyte.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T lymphocytes have been widely established as sensitive cellular targets for alterations by cannabinoids, as evidenced by decreased mitogen- and antigen-induced proliferation, T-cell-dependent antibody responses, and altered cytokine expression [reviewed in ref. 1 2 ]. With respect to the modulation of T-cell cytokines by cannabinoids, interleukin (IL)-2 has been extensively investigated. The interest in the modulation of IL-2 by cannabinoids is related to the critical role played by this cytokine in immune regulation. Initiation of IL-2 gene transcription is a hallmark of T-cell activation. Consequently, IL-2 functions to stimulate clonal expansion of T cells and to promote T-cell-mediated immune responses. The IL-2 gene, which in resting T cells exhibits almost no basal-level expression, is transcriptionally regulated by several trans-acting factors including activating-protein 1 (AP-1), nuclear factor (NF)-AT, CREB, OCT, and NF-{kappa}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 {Delta}9-tetrahydrocannabinol ({Delta}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 {Delta}9-THC are dependent on the mode of T-cell activation. Moreover, the enhancement by {Delta}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 {Delta}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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. CBN was provided by the National Institute on Drug Abuse. CBN was reconstituted in absolute ethanol, aliquots were taken, and these aliquots were stored at -80°C. Working solutions were prepared freshly prior to CBN addition to cell cultures. Purified hamster anti-mouse CD3{varepsilon} (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 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential effects of CBN on IL-2 production induced by optimal versus suboptimal activation stimuli
To differentially induce IL-2 protein secretion/production by murine primary splenocytes, cells were stimulated with either soluble or immobilized anti-CD3 alone or in combination with soluble anti-CD28. Stimulation of splenic T cells with immobilized CD3 (iCD3; 2 µg/mL coated overnight) alone for 48 h induced only modest IL-2 production (131 ± 2 U/mL of IL-2 activity in the culture supernatant), which could be dramatically potentiated by addition of soluble anti-CD28 (2 µg/mL; iCD3/CD28; 3062 ± 161 U/mL of IL-2). Likewise, stimulation of splenic T cells with soluble anti-CD3 alone (sCD3; 2 µg/mL) or in combination with anti-CD28 (sCD3/CD28; 2 µg/mL of each antibody) could also induce IL-2 production (101 ± 10 and 109 ± 8 U/mL of IL-2, respectively); however, the magnitude of stimulation was modest as compared to that with iCD3/CD28. These control studies demonstrated the magnitude of IL-2 induction by iCD3/CD28, a strong (optimal) activation stimulus, and by relatively weak (suboptimal) stimuli (i.e., iCD3 alone, sCD3, and sCD3/CD28) under the experimental conditions used in the present investigation. The effect of CBN on IL-2 production induced by the various stimuli, either monoclonal antibodies or PMA/Io (80 nM/1 µM), was examined in splenocytes. Cells were pretreated with CBN and/or vehicle (0.1% ethanol) for 30 min followed by activation with antibodies or PMA/Io. As illustrated in Figure 1A , the magnitude of IL-2 induced by stimuli that had been previously optimized for maximum IL-2 expression was significantly inhibited by CBN. For example, iCD3/CD28-induced IL-2 production was suppressed by CBN in a concentration-dependent manner (10–20 µM), which is similar to previous reports demonstrating the inhibition of IL-2 by CBN in PMA/Io-activated T cells. In contrast, T cells activated with a suboptimal stimulus, sCD3, sCD3/CD28, or iCD3 alone, exhibited significantly enhanced IL-2 secretion when cultured in the presence of CBN (Fig. 1B) . It is important to emphasize that CBN treatment in the absence of a T-cell activation stimulus could not induce detectable amounts of IL-2 in the culture supernatants (Fig. 1B , Fig. 2 ). Moreover, enhancement of IL-2 by CBN was concentration dependent with the enhancing effect by CBN being more pronounced in the presence of anti-CD28 (Fig. 2) . IL-2 enhancement by CBN treatment was also demonstrated in activated EL4.IL-2 cells, a murine thymoma widely used in studies of IL-2 regulation and expression [5 8 ]. It has been known that EL4.IL-2 cells can be induced to secrete IL-2 in response to phorbol ester (i.e., PMA) alone or in combination with calcium ionophore (i.e., Io). To suboptimally activate EL4.IL-2 cells, suboptimal concentrations of PMA (2–10 nM) were used that induced modest production of IL-2 as compared with a high concentration of PMA (100 nM) in the presence and/or absence of ionomycin (Fig. 3 ). Preliminary concentration response experiments demonstrated that treatment of EL4.IL-2 cells with PMA concentrations below 2 nM PMA resulted in no measurable IL-2 activity in the culture supernatant (data not shown). Based on these preliminary experiments, 2–10 nM PMA was functionally defined as a suboptimal stimulus for IL-2 production by EL4.IL-2 cells. It is interesting that pretreatment of EL4.IL-2 cells with CBN (10 or 20 µM) 30 min prior to activation by suboptimal concentrations of PMA (2 and 5 nM) resulted in a significant increase in IL-2 production (Fig. 3A) . Conversely, EL4.IL-2 cells activated with a high concentration of PMA (100 nM) or PMA/Io and cultured in the presence of CBN exhibited an inhibition of IL-2 production (Fig. 3B) . Similar to the results with splenocytes, CBN alone did not induce measurable amounts of IL-2 production by EL4.IL-2 cells (Fig. 3) . Collectively, these results confirm that CBN-mediated enhancement or inhibition of IL-2 expression was governed by the magnitude of the activation stimulus with which T cells are activated rather than the mode of activation.



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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.

 
Up-regulation by CBN of the MAP kinase activation induced by suboptimal activation stimuli
The ERK MAP kinases, which have been implicated as an intracellular target responsible for contributing to certain biological actions produced by cannabinoids, also play a critical role in the regulation of IL-2 gene expression. In light of these previous findings, the effect of CBN on ERK1 and ERK2 activation was examined in the context of IL-2 modulation. Western blot analysis using antibodies specific for phospho-ERK1/ERK2 and total ERKs revealed an up-regulation by CBN of both phosphorylated ERKs in the nucleus of splenocytes activated by sCD3/CD28 (suboptimal stimulus) but not total ERKs, which served as an internal loading control for the Western blotting (Fig. 4 ). The increase in phosphorylated nuclear ERKs in CBN-treated cells was concentration dependent and transient. Peak enhancement of ERK phosphorylation was observed 15 min after sCD3/CD28 stimulation (Fig. 4A and 4B) . It is important that CBN up-regulation of ERK1/ERK2 activation was observed only in splenocytes treated with a suboptimal activation stimulus and not in resting cells (data not shown) or those cells activated by a robust activation stimulus. For example, CBN (10 and 20 µM) did not exhibit marked effects on PMA/Io (80 nM/1 µM)-induced phospho-ERK1/ERK2 at 15 min postactivation (Fig. 4C) . Similar experiments were conducted in EL4.IL-2 cells. As shown in Figure 5 , the CBN-mediated enhancement of ERK1/ERK2 activation was also demonstrable in PMA (2 nM)-activated EL4.IL-2 cells. It is interesting that enhancement of ERK1/ERK2 activation was more pronounced at 4 h postactivation than at 15 min in the PMA-activated EL4.IL-2 cells.



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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.

 
Involvement of PKC and calcium/CaM-dependent protein kinases (CaM kinases), but not PI-3 kinase, in the CBN-mediated enhancement of IL-2
Since both phosphatidylinositol (PI) 3-kinase and PKC have been widely established as upstream activators of MAP kinases in T cells [reviewed in ref. 21 22 ], experiments were designed to investigate whether either of these two kinases is involved in CBN-mediated enhancement of IL-2 production by sCD3/CD28-activated splenocytes. The specific PI 3-kinase inhibitor, wortmannin, and PKC inhibitor, staurosporine, were used for these studies. Because the 50% inhibitory concentration (IC50) for PI 3-kinase inhibition by wortmannin in neutrophils has been shown to be less than 10 nM, concentrations between 1 and 100 nM were utilized for these studies [23 ]. As illustrated in Figure 6A , wortmannin (10–100 nM) alone increased the magnitude of IL-2 secretion by sCD3/CD28-activated splenic T cells (Fig. 6A , black columns). These results suggested a negative role for PI 3-kinase in the induction of IL-2 by sCD3/CD28. Nevertheless, CBN was still capable of enhancing IL-2 secretion in the presence of wortmannin, indicating that PI 3-kinase was not involved in the CBN-mediated enhancement of IL-2. In contrast, when splenocytes were pretreated with staurosporine (0.1–10 nM) prior to CBN treatment, the CBN-mediated enhancement of IL-2 was remarkably attenuated by 5 and 10 nM staurosporine (Fig. 6B) . Because the IC50 for PKC inhibition by staurosporine was determined to be approximately 9 nM [24 ], a concentration range between 0.1 and 10 nM was used to ensure its selectivity for PKC. Notably, staurosporine at these low concentrations did not interfere with the sCD3/CD28-induced IL-2 production (Fig. 6B , black columns), indicating that the ability of splenocytes to produce IL-2 in response to sCD3/CD28 is not affected by the presence of staurosporine. In addition to staurosporine, the role of PKC in CBN-mediated enhancement of IL-2 secretion was also examined by using a more specific PKC inhibitor, Ro-31-8220 (Ro), which is a structural analog of staurosporine. Ro possesses an IC50 for PKC between 5 and 27 nM [25 ]. Similar to staurosporine, Ro partially attenuated CBN-mediated enhancement of IL-2 secretion (Fig. 6C) , further supporting the involvement of PKC. However, the extent of the attenuation by Ro (50–100 nM) was rather modest compared to that of staurosporine. It is notable that staurosporine has also been reported to inhibit other calcium-dependent protein kinases, such as CaM kinases [26 ]. Thus, the role of CaM kinases in CBN-mediated enhancement of IL-2 secretion was further investigated using KN93, a competitive CaM kinase inhibitor possessing an IC50 of 13 µM [27 ]. As illustrated in Figure 7A , KN93 (5–10 µM) robustly attenuated CBN-mediated enhancement of IL-2. It is interesting that 1 µM KN93 in combination with 10 nM Ro significantly attenuated CBN-mediated enhancement of IL-2, even though either 1 µM KN93 or 10 nM Ro alone was ineffective (Fig. 7A and 7B) . Moreover, the magnitude of reversal induced by KN93 was further increased in the presence of 50 nM Ro (Fig. 7A and 7C) . These results suggest that both PKC and CaM kinases are likely involved in the mechanism by which CBN enhances IL-2 secretion by sCD3/CD28-activated splenocytes.



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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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Plant-derived cannabinoids have been reported to both positively and negatively modulate IL-2 expression by T cells [5 6 11 13 ]. Although the mechanism responsible for cannabinoid-mediated IL-2 modulation is unknown, several factors have been implicated including the method of T-cell activation and the age of animals used in the studies [11 ]. Previously, our laboratory has reported that PMA/Io-induced IL-2 expression by T cells was markedly inhibited by CBN and {Delta}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 {Delta}9-THC, whereas T cells activated by anti-CD3 exhibited enhanced IL-2 production in the presence of {Delta}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, {Delta}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, {Delta}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.


   ACKNOWLEDGEMENTS
 
This work was supported by NIDA grant DA07908.

Received April 30, 2000; revised December 10, 2000; accepted December 12, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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