Introduction

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Cannabinoids, which are a class of biologically active compounds originally derived from the plant, Cannabis sativa, are best known for their psychoactive and immunomodulatory properties. Although the mechanisms responsible for their broad range of physiological effects have remained controversial, the isolation and cloning of two distinct cannabinoid receptors, CB1 (Matsuda et al., 1990) and CB2 (Munro et al., 1993), have provided important insights. Both receptors are negatively coupled to adenylate cyclase via a pertussis toxin-sensitive G_i/G_o GTP-binding protein (Howlett, 1985; Howlett et al., 1985; Kaminski et al., 1994). As a result, in the presence of cannabinoids, leukocytes exhibit a marked inhibition of the cAMP signaling cascade as evidenced by a decrease in cAMP formation, protein kinase A activity, and DNA binding by cAMP response element binding protein (CREB; Herring et al., 1998). In addition, several other molecular pathways have been implicated in mediating cannabimimetic activity, including alterations in intracellular calcium regulation (Mackie and Hille, 1992; Yebra et al., 1992), alterations in mitogen-activated protein kinase (MAPK) signaling (Bouaboula et al., 1995, 1996), and the release of arachidonic acid (Burstein et al., 1994). The effect of the modulation of these distinct signaling pathways by cannabinoids on leukocyte function presently are poorly understood.

Structurally, CB1 and CB2 share approximately 44% identity, with this increasing to approximately 68% when comparing the transmembrane domains, the portion of the receptor that constitutes the putative ligand-binding pocket (Munro et al., 1993). Interestingly, despite these differences between the two receptor types, most cannabinoid receptor agonists displayed surprisingly similar binding affinities to both the CB1 and the CB2 receptor (Showalter et al., 1996). One notable exception is cannabinol (CBN), a plant-derived cannabinoid that exhibits higher binding affinity for CB2 than for CB1 (Munro et al., 1993; Felder et al., 1995). Differences are also presumed to exist in the tissue distribution of the two major forms of cannabinoid receptors, as suggested by measurements of CB1 and CB2 mRNA expression. CB1 is highly expressed within the central nervous system (CNS) and modestly expressed within the immune system (Kaminski et al., 1992; Schatz et al., 1997). Conversely, CB2 does not appear to be expressed within the CNS but is the predominant form of the cannabinoid receptor expressed by leukocytes (Kaminski et al., 1992; Munro et al., 1993). This tissue-specific distribution of CB1 and CB2 is further suggested by the fact that CBN, which has modest activity within the CNS, has equal, if not greater, binding affinity (Schatz et al., 1997) and biological activity (Schatz et al., 1997) in leukocytes than -9-tetrahydrocannabinol (⁹-THC), the primary psychoactive constitutent in C. sativa. The profile of cannabinoid receptor tissue distribution is significant because it suggests that CB2-selective ligands may have the potential of serving as immune modulators that would be devoid of CNS activity.

More recently, studies aimed at elucidating the mechanism responsible for immunosuppressive activity of cannabinoids have focused on changes in cytokine production and on the intracellular events responsible for these changes. Recently, we reported that both ⁹-THC and CBN produced a marked inhibition of interleukin (IL)-2 secretion and steady-state mRNA expression in primary mouse spleen cells and the murine T-cell line EL4 (Condie et al., 1996). The objective of the present study was to further characterize the mechanism by which cannabinoids inhibit the expression of IL-2 by activated T-cells. The IL-2 gene, which is highly regulated and exhibits virtually no basal level of expression in resting cells, is rapidly induced on T-cell activation. IL-2 transcription is controlled by a number of cis-acting elements directly upstream of the promoter and span the region from 326 to 52 bp (Novak et al., 1990). This region has been termed the minimal essential promoter/enhancer region of the IL-2 gene and is capable of full activation of IL-2 transcription (Fujita et al., 1986; Durand et al., 1987). DNA footprinting has revealed that there are specific DNA/protein interactions at the nuclear factor of activated T-cells (NF-AT) sites (293 to 263 and 130 to 140), activator protein-1 (AP-1) sites (185 to 179 and 151 to 145), B site (206 to 195), and octamer protein (Oct) sites (256 to 247 and 79 to 70; Ullman et al., 1990). Here we report that concommitant with CBN-mediated inhibition of IL-2 transcription in EL4 cells, there is an inhibition in DNA binding activity by several transcription factors that are known to play a critical role in the activation of the IL-2 gene.

	Materials and Methods

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Reagents and Cell Culture. All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Cannabinol was provided by the National Institute on Drug Abuse. Cannabinol was reconstituted in absolute ethanol, aliquoted, and stored under nitrogen at 80°C. Working solutions were prepared fresh just before the addition to culture. The C57BL/6 mouse thymoma EL4 was obtained from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 100 U of penicillin/ml, 100 U of streptomycin/ml, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, and 5% FCS (Life Technologies). EL4 cells (5 × 10⁵ cells/ml) were pretreated with CBN (1, 5, 10, 15, and 20 µM), vehicle (0.1% ethanol), or media alone (NA) for 1 h and then stimulated with phorbol-12-myristate-13-acetate (PMA, 80 nM) plus ionomycin (Io, 1 µM) at 37°C in 5% CO₂.

Electrophoresis Mobility Shift Assay (EMSA). Nuclear proteins were prepared as previously described (Herring et al., 1998). Briefly, EL4 cells were lysed with HB buffer (10 mM HEPES, 1.5 mM MgCl₂), and the nuclei were pelleted by centrifugation at 6700g for 5 min. Nuclei were lysed using a hypertonic buffer (30 mM HEPES, 1.5 mM MgCl₂, 450 mM KCl, 0.3 mM EDTA, and 10% glycerol) that contained 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin and leupeptin, after which the samples were centrifuged at 17,500g for 15 min, and the supernatant was retained. Double-stranded deoxyoligonucleotides containing the distal NF-AT site (5'-GAGGAAAATTTG-3') of the IL-2 promoter (Jain et al., 1993), AP-1 proximal (AP-1p) site of the IL-2 promoter (5'-AGAGTCA-3'; Novak et al., 1990), the Oct (5'-ATGCAAAT-3') and the NF-B site (5'-GGGGACTTTCCC-3'; Pierce et al., 1988), and AP-1 consensus (AP-1c) site (5'-TGACTCA-3') were synthesized and end-labeled with [-³²P]dATP using Ready To-Go T₄ polynucleotide kinase (Pharmacia, Piscataway, NJ). Nuclear proteins (5 µg) were incubated with 1 µg of poly(dI/dC) in binding buffer (100 mM NaCl, 30 mM HEPES, 1.5 mM MgCl₂, 0.3 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin and leupeptin) for 10 min at room temperature and then for 20 min at room tempeature for binding after the addition of labeled probe. Protein/DNA complexes was separated from free probe using a 4% acrylamide gel in 1× TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA). DNA binding specificity was verified in all of the experiments using unlabeled competitors (50-fold molar excess ³²P-unlabeled probe). After electrophoresis, the gels were dried and autoradiographed. Bands were quantified using a densitometer visual imaging system (Bio-Rad, Hercules, CA).

IL-2 ELISA Assay. The IL-2 ELISA was performed as previously described (Condie et al., 1996). Briefly, recombinant mouse IL-2 (as standard), purified rat anti-mouse IL-2 antibody, and biotinylated anti-mouse IL-2 antibody were purchased from PharMingen (San Diego, CA). Splenocytes (1 × 10⁶ cells/ml) or EL4 cells (2 × 10⁵ cells/ml) were cultured in triplicate in 24-well cell culture plates (Corning Glass Works, Corning, NY). After 24-h stimulation with PMA (80 nM)/Io (1 µM), supernatants were collected and quantified for IL-2 activity by ELISA. The IL-2 levels were determined by comparison with a standard curve of recombinant mouse IL-2.

Plasmid Construction. Plasmids were constructed as previously described (Han et al., 1998). Briefly, a minimal promoter vector containing no enhancer, pCAT-Promoter (where CAT is chloramphenicol acetyl transferase), was purchased from Promega (Madison, WI). To construct p(NF-AT)₃-CAT, p(AP-1)₃-CAT, p(Oct)₃-CAT, and p(NF-B)₃-CAT, BglII-adhering oligonucleotides containing three copies of each consensus recognition motif, either NF-AT, AP-1, Oct, or NF-B, were synthesized and cloned into the pCAT-Promoter vector, respectively. Cloning was confirmed through a comparison of EcoRI-digested fragments from each recombinant plasmid and pCAT-Promoter vector. pIL-2-CAT (578) was kindly provided by Dr. Ellen Rothenberg. The promoters were then purified with Quiagen Plasmid Kit (Quiagen Inc., Chatsworth, CA) and quantified for transient transfection studies.

Transfection and CAT Assay. Transient transfections were performed using a general DEAE-dextran method with slight modifications (Han et al., 1998). A total of 3.5 × 10⁷ EL4 cells were washed with Tris-buffered saline (TBS) and incubated in 7 ml of buffer containing 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na₂HPO₄, 0.7 mM CaCl₂, and 0.5 mM MgCl₂ plus 5 µg of each plasmid and 200 µg of DEAE-dextran/ml at 37°C for 40 min. Cells were washed with HEPES-buffered saline (140 mM NaCl, 5 mM KCl, 0.75 mM Na₂HPO₄, 6 mM dextrose, and 25 mM HEPES), resuspended in 5% FCS RPMI, and cultured separately in seven tissue culture plates at 37°C in 5% CO₂. Twenty-three hours after transfection, cells were treated with the indicated concentrations of CBN for 1 h and then stimulated with 80 nM PMA/1 µM Io for 18 h. Cells were then harvested, washed with PBS, and freeze-thawed three times in 100 µl of 0.25 mM Tris-HCl, pH 7.4, with the use of liquid N₂. The supernatants were isolated, and equal amounts of proteins were incubated in the CAT reaction mixture containing 0.1 µCi of [¹⁴C]chloramphenicol, 0.7 mM acetyl-coenzyme A, and 0.14 M Tris-HCl, pH 7.4, at 37°C for 240 min for pIL-2-CAT and p(Oct)₃-CAT, 1 h for p(NF-AT)₃-CAT and p(AP-1)₃-CAT, and 30 min for p(NF-B)₃-CAT. The degree of acetylation was assessed through the use of thin-layer chromatography, autoradiography, and liquid scintillation counting. The CAT activity was calculated as the ratio of enzyme activity. The CAT activity in the PMA/Io-treated group was arbitrarily assigned a relative value of 100%.

	Results

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Effect of Cannabinol on IL-2 Promoter Activity. To examine whether the previously reported decrease in IL-2 steady-state mRNA expression and IL-2 secretion by CBN in activated murine T-cells (Condie et al., 1996) was due to the inhibition of the IL-2 gene transcription, the 5' regulatory region (from +50 to 578 bp) of the IL-2 gene linked to the CAT reporter gene, p(IL-2)-CAT, was used. Specifically, the effect of CBN on CAT expression was measured in PMA/Io-activated EL4 cells that had been transiently transfected with p(IL-2)-CAT. As shown in Fig. 1, CBN inhibited PMA/Io-induced CAT activity in a concentration-dependent manner. The magnitude of inhibition by CBN closely correlated with previously observed inhibition in IL-2 mRNA steady-state expression and protein secretion (Condie et al., 1996). IL-2 promoter activity was approximately 104, 89, 61, and 47% of the control (PMA/Io alone) in the presence of 1, 5, 10, and 15 µM CBN, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of CBN on the transcriptional activity of the IL-2 promoter. EL4 cells (5 × 105 cells/ml) cultured in RPMI 1640 supplemented with 5% FCS were transiently transfected with a reporter plasmid, pIL-2-CAT (578 to 0). The transfected cells were pretreated with vehicle (lane 2, 0.1% ethanol) and CBN (5, 10, and 15 µM) for 1 h and then stimulated with PMA (80 nM)/(1 µM) for 18 h at 37°C. In addition, the basal level of IL-2 promoter activity was measured in unstimulated cells (lane 1). CAT activity was measured using autoradiography (A) and liquid scintilation counting (B) as described in Materials and Methods. The CAT activity in the control (vehicle lane) was arbitrarily assigned a value of 100%. All other groups were compared with the control. The results are representative of at least two independent experiments.

Time-Dependent Effects of Cannabinol on NF-AT, AP-1, and Oct Binding Activity. To further characterize the mechanism by which CBN inhibits IL-2 expression, EMSA were performed to examine the time-related effects of CBN on the DNA binding activity of those trans-activating factors known to be critical for IL-2 gene regulation. Initially, our studies focused on the binding activity of NF-AT, AP-1, and Oct. For these kinetic studies, nuclear proteins were isolated from EL4 cells that had been pretreated for 60 min with vehicle (0.1% ethanol) or 20 µM CBN and then stimulated with PMA/Io for 30, 60, 120, and 240 min, respectively. Figure 2 shows that NF-AT DNA binding activity increased in a time-dependent manner over the first 240 min after PMA/Io treatment. As previously described, two distinct NF-AT/protein complexes were induced in EL4 cells (Tsuruta et al., 1995). The upper NF-AT binding complex was first detected 60 min after PMA/Io treatment, and its binding continued to increase throughout the 240-min time period. The lower complex, which was constitutively expressed, also exhibited increased binding activity after PMA/Io treatment. The PMA/Io-induced increase in DNA binding activity for both the upper and lower NF-AT complex was markedly inhibited by 20 µM CBN at each of the time points measured. Under identical cell culture and treatment conditions, AP-1 binding activity was assessed using the AP-1p derived from the IL-2 promoter. PMA/Io treatment induced a time-dependent increase in protein binding to the AP-1p motif that steadily increased during the first 240 min. In the presence of 20 µM CBN, binding activity at the AP-1p motif was most dramatically inhibited during the first 120 min after PMA/Io treatment with the inhibitory effect waining by 240 min (Fig. 3.) Cannabinol also modestly inhibited the binding activity of Oct, which was only slightly induced by PMA/Io treatment (Fig. 4). In all of the above experiments, DNA binding specificity was verified by the use of unlabeled competitors (excess ³²P-unlabeled probe; Figs. 2-4).

View larger version (106K): [in this window] [in a new window]   Fig. 2.   The time-dependent effects of CBN on NF-AT DNA binding activity. EL4 cells (5 × 105 cells/ml) cultured in RPMI 1640 supplemented with 5% FCS were pretreated with vehicle (0.1% ethanol) and 20 µM CBN for 1 h and then stimulated with PMA (80 nM)/Io (1 µM) for various periods of time before the isolation of nuclear proteins. In addition, the basal level of NF-AT DNA binding was measured in nonactivated cells (time 0). The nuclear proteins (5 µg) were resolved by EMSA as described in Materials and Methods. The arrows indicate the NF-AT DNA/protein complexes. The competitor lane included a 50-fold molar excess of the unlabeled NF-AT oligonucleotide as a competitor using the same nuclear protein as loaded in the vehicle lane for time 120 min. The results are representative of at least two independent experiments.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   The time-dependent effects of CBN on the DNA binding activity to AP-1p in the IL-2 premoter. Experiments were performed as indicated in the legend to Fig. 2. The results are representative of at least two independent experiments.

View larger version (86K): [in this window] [in a new window]   Fig. 4.   The time-dependent effects of CBN on Oct DNA binding activity. Experiments were performed as indicated in the legend to Fig. 2. The results are representative of at least two independent experiments.

Concentration-Dependent Effects of Cannabinol on NF-AT, AP-1, and Oct Binding Activity. Based on the ability of CBN (20 µM) to inhibit NF-AT, AP-1 and Oct DNA binding in PMA/Io-activated EL4 cells, concentration-response studies were performed. EL4 cells were pretreated for 60 min with vehicle (lane 4, 0.1% ethanol) and CBN (1, 5, 10, 15, and 20 µM, respectively) and then activated with PMA/Io for 120 and 240 min. At 240 min, NF-AT exhibited a concentration-related inhibition of binding that was almost completely inhibited at 20 µM CBN (Fig. 5). Conversely, and consistent with the results shown in Fig. 3, only modest inhibition was observed on AP-1p and AP-1c binding at CBN concentrations below 10 µM at 240 min (data not shown). At 120 min, binding activity was inhibited to a greater extent at both the AP-1p (Fig. 6) and AP-1c (Fig. 7A) sites compared with that at 240 min. Moreover, at 120 min after cell activation, an inhibition of binding activity was observed only on the upper NF-AT complex (Fig. 7B), which again was concentration dependent. No significant differences were observed in binding activity to an AP-1c oligonucleotide versus the AP-1p in the presence of CBN in any of the studies. DNA binding specificity was verified using unlabeled competitors and unlabeled NF-B/Rel oligonucleotide as a cocompetitor (excess ³²P-unlabeled probe; Figs. 5 and 6). To confirm whether AP-1 proteins were present in the NF-AT upper complex as suggested by the similarity in the profile of inhibition of AP-1p, AP-1c, and NF-AT (upper band), competition binding studies were performed using a ³²P-labeled NF-AT probe and a variety of unlabeled probes, including AP-1p and AP-1c. Figure 8 shows that the AP-1c, but not the AP-1p (IL-2 promoter), competed for NF-AT binding. Interestingly, competition of NF-AT binding was also observed with cold NF-B/Rel probe and is explained by the sequence homology of the DNA binding domain between NF-AT and Rel family proteins (Jain et al., 1995). A very modest degree of competion for the upper NF-AT complex by CRE was also observed.

View larger version (131K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent effects of CBN on NF-AT DNA binding activity. EL4 cells (5 × 105 cells/ml) culture in RPMI 1640 supplemented with 5% FCS were pretreated with vehicle (lane 4, 0.1% ethanol) and CBN (1, 5, 10, 15, and 20 µM) for 1 h and then stimulated with PMA (80 nM)/Io (1 µM) for 240 min. In addition, the basal level of NF-AT DNA binding was measured in unstimulated cells (lane 2), as was the induced level of NF-AT binding in PMA/Io-stimulated cells cultured in the absence of vehicle (lane 3). The nuclear proteins (5 µg) were resolved by EMSA as described in Materials and Methods. The arrows indicate the DNA/protein complexes. The competitor lanes included a 50-fold molar excess of the unlabeled NF-AT and NF-B oligonucleotide as competitors using the same nuclear protein isolate as in lane 3. The results are representative of at least two independent experiments.

View larger version (131K): [in this window] [in a new window]   Fig. 6.   Concentration-dependent effects of CBN on the DNA binding activity to AP-1p. Experiments were performed as indicated in the legend to Fig. 5 with the exception that nuclear proteins were isolated 120 min after PMA (80 nM)/Io (1 µM) stimulation. The competitor lanes included a 50-fold molar excess of the 32P-unlabeled AP-1p and NF-B oligonucleotide as competitors using the same nuclear protein isolate as in lane 3. The results are representative of at least two independent experiments.

View larger version (73K): [in this window] [in a new window]   Fig. 7.   Concentration-dependent effects of CBN on AP-1 consensus and NF-AT DNA binding. EL4 cells (5 × 105 cells/ml) culture in RPMI 1640 supplemented with 5% FCS were pretreated with vehicle (lane 1, 0.1% ethanol) and CBN (1, 5, and 10 µM) for 1 h. The pretreated cells were then stimulated with PMA (80 nM)/Io (1 µM), and the nuclear proteins were isolated 120 min later. DNA binding activity was assayed after incubation of the nuclear proteins with either 32P-labeled AP-1 consensus or NF-AT probe. The nuclear proteins (5 µg) were resolved by EMSA as described in Materials and Methods. The arrows indicate the DNA/protein complexes. The results are representative of at least two independent experiments.

View larger version (127K): [in this window] [in a new window]   Fig. 8.   The influence of competitor probes on NF-AT DNA binding activity. EL4 cells (5 × 105 cells/ml) culture in RPMI 1640 supplemented with 5% FCS were stimulated with PMA (80 nM)/Io (1 µM), and the nuclear proteins were isolated 240 min later. The nuclear proteins (5 µg) were resolved by EMSA as described in Materials and Methods. The competitor lanes included a 50-fold molar excess of unlabeled NF-AT, AP-1p, AP-1c, NF-B, Oct, CRE, or C/EBP oligonucleotide as competitors using the same nuclear protein as loaded in PMA/Io-stimulated group. In addition, the basal level of NF-AT DNA binding was measured in unstimulated cells. The arrows indicated the NF-AT DNA/protein complexes. The data presented are representative of two independent experiments.

Effects of Cannabinol on NF-B/Rel Binding Activity. NF-B is also widely established as being an important transactivator of the IL-2 gene and therefore was investigated for modulation by CBN (Novak et al., 1990). Identical to the studies above, EL4 cells were pretreated for 60 min with vehicle (0.1% ethanol) and 20 µM CBN and then stimulated with PMA/Io for 30, 60, 120, and 240 min, respectively. Somewhat surprisingly, time course studies showed an increase in NF-B binding activity in the presence of CBN within 60 min after PMA/Io stimulation (Fig. 9). A modest enhancement of NF-B DNA binding activity was also evident at 240 min. This observation was in striking contrast to the strong inhibition CBN produced on NF-B binding in PMA/Io-activated thymocytes (Herring and Kaminski, 1999) and splenocytes (unpublished observation). Follow-up concentration-response studies with CBN showed no effect on NF-B binding activity in EL4 cells at 120 min (Fig. 10). Likewise, shortening the CBN preincubation time (i.e., to 45, 30, or 15 min), in the event that the effects on NF-B may be highly transient in EL4 cells, did not alter the NF-B binding activity from what was observed after a 60-min pretreatment with CBN (data not shown).

View larger version (110K): [in this window] [in a new window]   Fig. 9.   The time-dependent effects of CBN on NF-B DNA binding activity. Experiments were performed as indicated in the legend to Fig. 2. The results are representative of at least two independent experiments.

View larger version (98K): [in this window] [in a new window]   Fig. 10.   Concentration-dependent effects of CBN on NF-B DNA binding activity. Experiments were performed as indicated in the legend Fig. 2. The competitor lanes included a 50-fold molar excess of the unlabeled NF-B oligonucleotide as a competitor using the same protein as loaded in lane 3. The results are representative of at least two independent experiments.

Effects of Cannabinol on Promoter Activities of NF-AT, AP-1, Oct, and NF-B/Rel. To evaluate the impact of CBN-mediated modulation of transcription factor DNA binding activity on gene transactivation, the effect of CBN was evaluated using the following reporter gene constructs p(NF-AT)₃-CAT, p(AP-1)₃-CAT, p(NF-B)₃-CAT, and p(Oct)₃-CAT in cells cultured in 5% FCS (Fig. 11). Concordant with the NF-AT EMSA results, CBN inhibited NF-AT promoter activity in a dose-dependent manner. Oct promoter activity was also modestly inhibited by CBN, which coincided with the Oct EMSA results. There was no effect on NF-B promoter activity by CBN, and these results also corresponded with the NF-B EMSA results obtained from EL4 cells cultured in 5% FCS. Interestingly, AP-1 promoter activity was not inhibited by CBN. Again, the AP-1 reporter gene results are consistent with the transient inhibition of AP-1 DNA binding observed in the EMSA between 120 and 240 min after PMA/Io stimulation in the presence of CBN shown in Fig. 3.

View larger version (67K): [in this window] [in a new window]   Fig. 11.   The effects of CBN on p(NF-AT)3-CAT (A), p(AP-1)3-CAT (B), p(Oct)3-CAT (C), and p(NF-B)3-CAT (D) promoter activity transiently transfected into EL4 cells (5 × 105 cells/ml). The transfected cells were cultured in RPMI 1640 media supplemented 5% FCS, pretreated with vehicle (0.1% ethanol) or CBN (1, 5, 10, and 15 µM) for 1 h, and then stimulated with PMA (80 nM)/Io (1 µM) for 18 h at 37°C. CAT activity was measured as described in Materials and Methods. In addition, the basal level of promoter activity was measured in unstimulated cells (no PMA/Io). PMA/Io-induced level of CAT activity in the absence of vehicle or CBN and were arbitrarily assigned a value of 100% to which all respective treatment groups were compared. The results are representative of at least two independent experiments.

	Discussion

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We have previously reported that two plant-derived cannabinoids, ⁹-THC and CBN, produced a marked inhibited of IL-2 mRNA steady-state expression and IL-2 secretion in primary splenocytes and EL4 cells (Condie et al., 1996). The objective of the present study was to further explore the mechanism responsible for the modulation of IL-2 using the CB2-expressing/CB1-deficient T-cell line, EL4 (Schatz et al., 1997). Here, we demonstrate that the inhibition of IL-2 expression by CBN in EL4 cells is mediated through direct inhibition of IL-2 gene transcription. This conclusion is supported by transient transfection studies in which CBN inhibited PMA/Io-induced expression of a CAT reporter plasmid (p(IL-2)-CAT) under the control of the minimal essential region (1 to 578) of the IL-2 promoter. Extensively characterized, the regulatory region of the murine IL-2 gene contains all sites known to be essential for IL-2 expression and includes two NF-AT sites, of which the NF-AT distal motif from IL-2 promoter site is flanked by an AP-1-like site through which AP-1 complexes help to stabilize NF-AT binding; two additional AP-1-like sites (AP-1p and AP-1d), an NF-B site, and two Oct binding sites (Novak et al., 1990). Characterization of protein/DNA binding and transcriptional regulation at specific response elements after cell activation by PMA/Io treatment revealed that CBN exerted a significant inhibition of NF-AT and AP-1 DNA binding, which we believe contributes to the inhibition of IL-2 gene transcription by activated T-cells. Conversely, modest effects on either protein/DNA binding or transcriptional activity were observed at B or Oct sites.

EMSA and transient transfection assays were used to focus on individual regulatory elements within the IL-2 promoter to provide insights into which signaling cascades may be modulated by CBN. With this approach, the most significant effects by CBN were observed on NF-AT. In the absence of CBN, NF-AT DNA binding was strongly induced in EL4 cells by PMA/Io beginning at approximately 60 min and continued to increase over 240 min as evidenced by the formation of two distinct binding complexes visualized by EMSA. In activated T-cells, cytoplasmic NF-AT is dephosphorylated by the Ca²⁺-dependent phosphatase calcineurin to facilitate nuclear translocation and association with the AP-1 proteins fra-1 and JunB to form the upper of the two NF-AT DNA binding complexes (Clipstone and Crabtree, 1992; Boise et al., 1993; McCaffrey et al., 1993). Conversely, the lower NF-AT binding complex consists solely of NF-AT. Two distinct effects were observed on the profile of NF-AT DNA binding, which was directly correlated to the concentration of CBN under which EL4 cells were activated. It is important to emphasize that none of the concentration of CBN used in any of the studies discussed produced direct cytotoxicity. At CBN concentrations above 15 µM, DNA binding activity by both the upper and lower NF-AT complex was markedly inhibited and the inhibition was sustained over a 240-min period. Conversely, at CBN concentrations below 15 µM, only the upper NF-AT complex appeared to be inhibited. Although presently unclear, it is likely that the inhibition of NF-AT binding activity at the higher concentration of CBN is mediated through a disregulation in intracellular calcium. This premise is suggest by the fact that calcium is one of the primary regulators of the NF-AT signaling pathway and that both of the major NF-AT complexes were robustly inhibited.

With respect to the effects on NF-AT binding activity at concentrations below 15 µM, CBN appeared to selectively exert a greater inhibition on the upper NF-AT complex, which was most apparent at 120 min after cell activation (Fig. 7). As discussed earlier, the upper NF-AT complex contains AP-1 components fra-1 and JunB. Interestingly, DNA binding activity to the AP-1p site in the IL-2 promoter also showed greater sensitivity to the inhibitory effects by CBN at 120 than at 240 min, as evidenced by the lack of any inhibition by CBN on AP-1p binding at CBN concentrations below 20 µM. Conversely, at 2 h after PMA/Io activation, AP-1p binding was inhibited even in the presence of 10 µM CBN. A similar profile of activity was also observed with DNA binding to an AP-1c motif (data not shown). The transient inhibition by CBN on AP-1 binding activity was further suggested by the fact that there was no significant inhibition by CBN of the p(AP-1)₃-CAT expression plasmid when measured 18 h after PMA/Io activation of EL4 cells.

The presence of AP-1 family members in the upper NF-AT complex was investigated and confirmed by competition experiment using a ³²P-unlabeled AP-1c DNA probe (Fig. 8). Interestingly, NF-AT did not compete for binding to the AP-1p sequence, thus suggesting that fra-1/JunB dimers do not bind to the AP-1p site. Although the specific proteins that bind to the AP-1p site to transactivate IL-2 have not been identified, CREB has been demonstrated as a constituent of that protein binding complex (Chen and Rothenberg, 1993). The above findings also suggest that the inhibition of NF-AT binding at lower CBN concentrations is due to effects on AP-1-related proteins and could potentially be explained by at least one of two mechanisms: 1) direct transcriptional regulation of c-fos through CRE elements located proximally to the TATA box start site, which have been shown to confer cAMP responsiveness for this gene (Berkowitz et al., 1989), or 2) the inhibition of MAPK activity, specifically, via the inhibition of Ras. It is notable that the MAPK pathway, modulated by Ras, plays a critical role in the regulation of NF-AT indirectly through effects on AP-1. In fact, Ras can fully substitute for PMA, and together with active calcinuerin, Ras provided a full signal for NF-AT reporter gene expression (Cantrell, 1996; Genot et al., 1996). In addition, Ho et al. (1997) recently reported that pretreatment of EL4 cells with the cAMP agonist forskolin for 240 min before activation resulted in an increase in NF-AT and AP-1 report gene activity. Finally, it has been reported that the treatment of CB1- or CB2-transfected Chinese hamster ovary cells with a nanomolar concentration of CP-55940, a synthetic cannabinoid, induced a rapid increase in MAPK activity that was maximal within the first 10 min after cell treatment and then declined rapidly. These results may reflect a highly transient increase in MAPK activity on very short exposures to cannabinoids with longer cannabinoid treatment, potentially inducing either desensitization of this pathway or the up-regulation of mediators that exert a negative influence on MAPK activity.

The effects of CBN on NF-B binding activity in PMA/Io-activated EL4 cells in the present study were surprising based on our past findings that both CBN and ⁹-THC markedly inhibited NF-B DNA binding under a wide variety of conditions in a number of different leukocyte preparations. In primary leukocytes (i.e., splenocytes and thymocytes), both CBN and ⁹-THC inhibited forskolin-induced (Herring et al., 1998) and PMA/Io-induced NF-B binding by CBN in thymocytes (Herring and Kaminski, 1999). Likewise, ⁹-THC markedly inhibited forskolin- as well as lipopolysaccharide-induced NF-B binding in the macrophage cell line RAW 264.7 (Jeon et al., 1996). Here, NF-B binding was modestly increased by CBN in PMA/Io-activated EL4 cells within 30 min after activation. At time points 60 min after activation, either no effect or a modest enhancement by CBN on NF-B binding was observed. These findings are similar to those reported by Daaka et al. (1997); they observed that ⁹-THC treatment induced an increase in NF-B binding activity and an increase in the B family member RelA in the NK-like cell line NKB61A2. In addition, the increase in NF-B binding by ⁹-THC paralleled an increase in IL-2 receptor -chain gene transcription and surface expression in NKB61A2 cells (Daaka et al., 1997). Although it is unclear what effect CBN may have on IL-2 receptor expression, the present results suggest that unlike primary leukocytes, NF-B does not appear to be an important trans-activating factor in the regulation of IL-2 gene expression in EL4 cells. It is also notable that CBN produced no effect on p(NF-B)₃-CAT activity, which may be reflective of transient effects as suspected for AP-1. Only very modest effects, at high CBN concentrations, were observed on Oct DNA binding or promoter activity.

Collectively, these data suggest that the inhibition of IL-2 secretion by CBN in activated EL4 cells is mediated at the level of IL-2 gene transcription by inhibition of NF-AT and AP-1 DNA binding activity. The inhibition by CBN of NF-AT DNA binding within the first several hours immediately after cell activation was most profound on the upper complex, which contains AP-1 proteins fra-1 and JunB and was closely correlated with a decrease in AP-1p and AP-1c binding activity. The mechanism responsible for altered NF-AT and AP-1 regulation by CBN may be partially associated with the robust inhibition of the cAMP signaling cascade by cannabinoids, which ultimately results in a marked inhibition of CREB phosphorylation and binding to CRE sites. However, cannabinoids have more recently been found to also influence additional signal transduction pathways, which may further contribute to the effects cannabinoids exert on leukocyte function. Therefore, the specific upstream signaling events disrupted by CBN that lead to altered regulation of NF-AT and AP-1 remain to be elucidated. Nevertheless, the present finding that under the present experimental conditions both NF-AT and AP-1 are negatively regulated by CBN is significant because both families of transcription factors play a critical role in the regulation of a wide range of cytokines, and this observation may help to explain the diverse effects produced by cannabinoids on immune function.