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Cannabinoid Receptor Type 1- and 2-mediated Increase in Cyclic AMP Inhibits T Cell Receptor-triggered Signaling

Cannabinoid Receptor Type 1- and 2-mediated Increase in Cyclic AMP Inhibits T Cell... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 51, pp. 35450 –35460, December 18, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cannabinoid Receptor Type 1- and 2-mediated Increase in Cyclic AMP Inhibits T Cell Receptor-triggered Signaling Received for publication, April 9, 2009, and in revised form, October 16, 2009 Published, JBC Papers in Press, October 26, 2009, DOI 10.1074/jbc.M109.006338 ‡ § ‡ § ‡1 Christine Bo¨ rner , Michal Smida , Volker Ho¨ llt , Burkhart Schraven , and Ju¨ rgen Kraus ‡ § From the Departments of Pharmacology and Toxicology and Molecular and Clinical Immunology, University of Magdeburg, 39120 Magdeburg, Germany The aim of this study was to characterize inhibitory mecha- beneficial in the above mentioned diseases but on the other nisms on T cell receptor signaling mediated by the cannabinoid hand may also cause immunosuppression. Many effects of receptors CB1 and CB2. Both receptors are coupled to G pro- endogenous cannabinoids like anandamide and exogenous i/o teins, which are associated with inhibition of cyclic AMP forma- cannabinoids like 9-tetrahydrocannabinol (THC), the psy- tion. In human primary and Jurkat T lymphocytes, activation of choactive compound of Cannabis sativa, are mediated by the CB1 by R()-methanandamide, CB2 by JWH015, and both by G protein-coupled cannabinoid receptors CB1 and CB2. i/o 9-tetrahydrocannabinol induced a short decrease in cyclic While CB1 was found to be expressed predominantly in neuro- AMP lasting less than 1 h. However, this decrease was followed nal cells and only to a lesser extend in peripheral cells like by a massive (up to 10-fold) and sustained (at least up to 48 h) immune effector cells, CB2 was considered to be the “periph- increase in cyclic AMP. Mediated by the cyclic AMP-activated eral” cannabinoid receptor, being expressed in cells of the protein kinase A and C-terminal Src kinase, the cannabinoids immune system (5). However, recent reports suggest the ex- induced a stable phosphorylation of the inhibitory Tyr-505 of pression of CB2 in neuronal cells, as well (6, 7). Moreover, the leukocyte-specific protein tyrosine kinase (Lck). By thus although normally expressed in very low amounts only, recent arresting Lck in its inhibited form, the cannabinoids prevented work from our laboratory demonstrated a massive up-regula- the dephosphorylation of Lck at Tyr-505 in response to T cell tion of functional CB1 receptors in T lymphocytes in response receptor activation, which is necessary for the subsequent initi- to cannabinoids themselves, IL-4, and T cell activation (8–10). ation of T cell receptor signaling. In this way the cannabinoids In addition, a number of other receptors have been shown to inhibited the T cell receptor-triggered signaling, i.e. the activa- mediate the effects of cannabinoids, which include vanilloid tion of the -chain-associated protein kinase of 70 kDa, the receptors, peroxisome proliferator-activated receptors and the linker for activation of T cells, MAPK, the induction of interleu- formerly “orphan” designated receptors GPR55 and GPR119 kin-2, and T cell proliferation. All of the effects of the cannabi- (reviewed in Ref. 11). noids were blocked by the CB1 and CB2 antagonists AM281 and Despite of a number of reports, it remains largely unresolved AM630. These findings help to better understand the immuno- which types of cannabinoid receptors mediate IL-2 inhibition suppressive effects of cannabinoids and explain the beneficial and what are the detailed molecular mechanisms. With regard effects of these drugs in the treatment of T cell-mediated auto- to the receptors, it was demonstrated independently by differ- immune disorders like multiple sclerosis. ent groups, using either selective agonists and antagonists (12) or transgenic mice lacking CB2 (13) that CB2 is involved in mediating the effect of cannabinoids on IL-2. However, it was Cannabinoids are discussed as drugs for the treatment of also suggested that neither CB1 nor CB2 receptors mediated the effect of cannabinoids on IL-2 (14). Recently, it was re- neuroinflammatory diseases and T cell-mediated autoimmune ported that the inhibitory effect of the endogenous cannabinoid disorders such as, for example, multiple sclerosis. Their poten- tial usefulness for such disorders is emphasized by the fact that 2-arachidonyl glycerol on IL-2 production in T cells was depen- cannabinoids affect functions of both neuronal and immune dent on peroxisome proliferator-activated receptor  (15). Thus far, no reports showed involvement of CB1 in the canna- effector cells. Cannabinoids produce a variety of immuno- binoid-mediated inhibition of IL-2, which is probably due to modulatory effects (reviewed in Refs. 1 and 2). Among these effects, the cannabinoid-mediated inhibition of interleukin the fact that high amounts of CB1 are only present in T cells (IL)-2 production of activated T lymphocytes (3, 4) may be after defined stimulation of the cells (see above). With regard to the mechanisms of the cannabinoid-mediated inhibition of IL-2, it was shown that the drugs inhibit the T cell receptor * This work was supported by research grants from the government of the (TCR)-induced activities of the transcription factors NF-B state of Sachsen-Anhalt (to V. H. and B. S.), a grant from the University of Magdeburg, Faculty of Medicine (Initialprojekt) (to C. B.), and Deutsche and NFAT (4, 16, 17). This is interesting, because it is known Forschungsgemeinschaft Grant KR 1740/10-1 (to J. K.). that the expression of IL-2 in activated T cells is almost exclu- To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, University of Magdeburg, 44 Leipzigerstrasse, 39120 Magdeburg, Germany. Tel.: 49-391-6715372; Fax: 49-391-6715869; E-mail: kinase; PKA, cAMP-dependent protein kinase; TCR, T cell receptor; Zap 70, [email protected]. -chain-associated protein kinase of 70 kDa; cAMPS-Rp, (R)-adenosine, The abbreviations used are: IL, interleukin; Csk, C-terminal Src kinase; THC, cyclic 3,5-hydrogenphosphorothioate; CFSE, carboxyfluorescein succin- 9-tetrahydrocannabinol; Lck, leukocyte-specific protein tyrosine kinase; imidyl ester; FACS, fluorescence-activated cell sorter; siRNA, small interfer- LAT, linker for activation of T cells; MAPK, mitogen-activated protein ing RNA; MAEA, metanandamide. 35450 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 This is an Open Access article under the CC BY license. Cannabinoid-inhibited T Cell Signaling via cAMP sively regulated at the level of transcription by these factors. In with a preference for CB1 (MAEA; Sigma), JWH015 with a pref- addition, it was shown that activation of the mitogen-activated erence for CB2 (Tocris, Bristol, UK), and WIN 55,212-2 mesy- protein kinase (MAPK) is inhibited by cannabinoids in lympho- late, which activates CB1 and CB2 (Tocris). Antagonists were cytes (18). However, detailed molecular mechanisms linking AM281 (selective for CB1: K of CB1, 12 nM; K of CB2, 4200 nM; i i the effects of cannabinoids to the TCR-triggered signaling are Tocris) and AM630 (selective for CB2: K of CB1, 5152 nM; K of i i missing and will be the topic of this communication. CB2, 31.2 nM; Tocris). The K values were taken from Ref. 5. The The TCR-mediated activation of T cells induces a defined PKA inhibitor (R)-adenosine, cyclic 3,5-hydrogenphosphoro- signaling cascade, at the end point of which transcription of thioate (cAMPS-Rp; Tocris) was used at 100 M. IL-2 is induced (19–21). In nonactivated, resting T cells, this Quantitative Real Time Reverse Transcription-PCR—Per cascade is tonically repressed by constitutive phosphorylation sample, 10 Jurkat cells were used. The isolation of total RNA of the negative regulatory site of the leukocyte-specific protein and cDNA synthesis is described earlier (8). The PCRs were tyrosine kinase (Lck) at Tyr-505. This phosphorylation is medi- done in a total volume of 20 l on a LightCycler instrument ated by the C-terminal Src kinase (Csk) (22). Upon engagement using the LightCycler-Fast Start DNA Master SYBR Green I kit of the TCR, the inhibitory effect of Csk on Lck is abolished (both from Roche) according to the manufacturer’s sugges- allowing initiation of the TCR-induced signaling cascade (23– tions. Primers and conditions for the PCRs specific for -actin, 25). One of the earliest events in this cascade is then the phos- CB1, and CB2 are described in detail earlier (8). The PCRs for phorylation and thereby the activation of the -chain-associ- IL-2 transcripts were performed with 5-GAAGGCCACA- ated protein kinase of 70 kDa (Zap 70). Activated Zap 70 in turn GAACTGAAACATCT-3 and 5-CTGTTCAGAAATTCTA- phosphorylates the adaptor protein linker for activation of T CAATGGTTG-3 primers, with a preincubation for 8 min at cells (LAT), leading to the induction of calcium flux and activa- 95 °C and 50 cycles with5sat95 °C,5sat65 °C,and10sat tion of the MAPK cascades. In this way, the signal is transduced, 72 °C (specific T  81,75 °C). and the transcription factors AP-1, NF-B, and NFAT are acti- Proliferation Assay—The cells were washed once with phos- vated and finally transactivate the IL-2 gene. After being phate-buffered saline and loaded with 0.1 M carboxyfluores- released, IL-2 induces a variety of well defined subsequent cein succinimidyl ester (CFSE; Molecular Probes, Go¨ttingen, immune responses (26). It is known that the activity of Csk to Germany) at 37 °C for 10 min. Loading was stopped by the phosphorylate the negative regulatory site of Lck at Tyr-505 can addition of 5% fetal calf serum, and the cells were washed with be further enhanced by cAMP-dependent protein kinase A phosphate-buffered saline. CFSE-loaded cells were pretreated (PKA). Thus, PKA exhibits an inhibitory effect upon TCR sig- with cannabinoids for 2 h and then inoculated on a 24-well plate naling by stabilizing the inhibited state of Lck (27, 28). Here we coated with anti-CD3/anti-CD28. Three days later, prolifera- report on the molecular linkage between CB1 and CB2 and T tion was measured on a FACSCalibur (Becton Dickinson). cell signaling and present a mechanism by which cannabinoids Reporter Genes and Transfection—Reporter gene constructs inhibit IL-2 production of activated human T cells via these are based on pBLCAT2 (30) and contain binding sequences for receptors. AP-1 (5-AAACATATGATTCACCAGGCA-3), NF-B(5- AAAGTTGAGGGGACTTTCCCAGGCCT-3), and NFAT EXPERIMENTAL PROCEDURES (5-GCCCAAAGAGGAAAATTTGTTTCATA-3) cloned 5 T Cell Culture, Induction of CB1, T Cell Activation, and to the herpes simplex thymidine kinase promoter. Transfection Reagents—Peripheral blood mononuclear cells were isolated was performed by electroporation (210 V, 950 microfarads) from heparinized blood collected from healthy volunteers as using a Gene Pulser II (Bio-Rad). The day after transfection, described (29). Primary human T cells and the human T cell line the cells were transferred to anti-CD3/anti-CD28- or anti- Jurkat (Kab 14) were cultivated in RPMI 1640 medium (Lonza mouse-immunoglobulin-coated dishes with or without canna- Verviers SPRL, Verviers, Belgium) supplemented with 10% fetal binoids. After 72 h of transient expression, the reporter gene calf serum and antibiotics (100 units/ml penicillin and 100 was assayed via a chloramphenicol acetyl transferase-enzyme- mg/ml streptomycin; Lonza Verviers SPRL). We would like to linked immunosorbent assay purchased from Roche Applied note that no cannabinoid receptor-overexpressing cells were Science. used in our experiments. CB2 is expressed in the naive cells Western Blots—Western blots were performed as previously constitutively. The endogenous CB1 was induced by stimula- described (8, 31, 32). For antigen activation, 10 Jurkat cells/ tion of the cells with IL-4 as described (9). Briefly, the cells sample were pelleted, resuspended in anti-CD3/anti-CD28 received IL-4 (5 ng/ml; R & D Systems, Wiesbaden, Germany) monoclonal antibodies, and incubated at 37 °C for 2 min. and were incubated for 3 days. Then the medium was replaced The activation was stopped with 1 ml of ice-cold phosphate- by fresh medium without IL-4, and the cells were cultured for buffered saline, and the cells were pelleted again and lysed. additional 24 h. An aliquot of the cells was tested to assure the For protein detection, the following primary antibodies were induction of CB1 transcripts (data not shown). For these cells, used: actin C-11, Csk C-20 (both from Santa Cruz, Heidel- the terms “stimulated cells” or “CB1- and CB2-expressing cells” berg, Germany); phospho-p44/42 MAPK Tyr-202/Tyr-204, will be used. T cells were activated with 100 l of each anti-CD3 phospho-LAT Tyr-191, phospho-Zap 70 Tyr-319, and phos- monoclonal antibodies (OKT3) and anti-CD28 monoclonal pho-Lck Tyr-505 (all from Cell Signaling Technology/New antibodies (248.23.2) obtained from hybridoma supernatants England Biolabs, Frankfurt, Germany). All of the antibodies are produced in our laboratories (29). Cannabinoid agonists were from rabbits. For protein detection, the following secondary THC, which activates CB1 and CB2 (Sigma), metanandamide antibody was used: anti-rabbit IgG (GE Healthcare). For quan- DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35451 Cannabinoid-inhibited T Cell Signaling via cAMP tification of Western blot signals, densitometric analysis was performed using National Institutes of Health Image 1.63 software. Down-regulation of Csk with siRNA—The commercially available siRNA against human Csk and a nonmatching control siRNA (siRNA-A; both from Santa Cruz) were used. The siRNAs were transfected into Jurkat T cells by electroporation (210 V, 950 microfarads) using a Gene Pulser II (Bio-Rad). The cells were then cultured in RPMI 1640 for 4 days to ensure Csk protein down-regulation. Cyclic AMP Measurement—Jurkat cells were incubated with cannabinoids for 0 h (controls) up to 9 days. The cells were then lysed with 50 mM HCl for 30 min on ice. The competitive cAMP-enzyme-linked immunosorbent assay was performed according to a described procedure (33). Statistical Analysis—For statistical evaluations between two groups of samples, Student’s t tests were performed. Multiple comparisons were performed with analysis of variance, fol- lowed by Tukey’s multiple comparison post-test. RESULTS Effects of Cannabinoids on IL-2 mRNA in Activated T Cells— First, CB1- and CB2-mediated effects of cannabinoids on the anti-CD3/anti-CD28-triggered induction of IL-2 mRNA was determined in Jurkat T cells. In naive cells, which express CB2 abundantly, but only very small amounts of CB1 (Fig. 1A), THC, a mixed agonist for CB1 and CB2, caused a significant inhibi- tion of the anti-CD3/anti-CD28-triggered IL-2 mRNA produc- FIGURE 1. Inhibition of anti-CD3/anti-CD28-triggered IL-2 transcription tion, which was dependent on the time of incubation of the cells by cannabinoids. Jurkat T cells received a single dose of cannabinoids (THC, 500 nM, activates both CB1 and CB2; MAEA, 500 nM, preference for CB1; with the cannabinoid prior to the anti-CD3/anti-CD28 stimu- JWH015, 250 nM, preference for CB2) and were incubated with the drugs up to lation. Interestingly, incubation of the cells with the drug for 24 48 h. Then cells received fresh medium and were activated with anti-CD3/ anti-CD28 to induce IL-2 mRNA. The cells were lysed 4 h after the anti-CD3/ and 48 h completely inhibited the anti-CD3/anti-CD28-medi- anti-CD28 activation for quantitative reverse transcription-PCR. The amount ated induction of IL-2. A similar effect was observed when the of IL-2-specific transcripts relative to that of -actin is plotted. The cells that CB2-selective agonist JWH015 was tested. In line with the fact were not incubated with cannabinoids served as controls, for which the anti- CD3/anti-CD28-mediated induction of IL-2-specific transcripts was set to that only very small amounts of CB1 are present in naive Jurkat 100%. The data represent the means  S.E. of at least three independent cells, the CB1-selective agonist MAEA had no significant effect experiments performed in duplicate. A, experiments performed in naive Jur- on the anti-CD3/anti-CD28-induced IL-2 mRNA levels. We kat cells expressing CB2. The data sets for THC and JWH015 at 2, 4, 24, and 48 h are significantly different from controls (p 0.001). B, experiments performed have previously shown that stimulation of T cells with IL-4 in IL-4-stimulated Jurkat cells expressing CB1 and CB2. The data sets for THC, markedly induces CB1 mRNA and functional receptor proteins MAEA, and JWH015 at 2, 4, 24, and 48 h are significantly different from con- trols (p  0.001). (9) without altering the expression of CB2. In contrast to naive cells, in IL-4-stimulated Jurkat cells, which express CB1 and CB2 (Fig. 1B), the anti-CD3/anti-CD28-evoked IL-2 induction cells, not only THC and JWH015 but also MAEA inhibited the was not only inhibited by THC and JWH015 but also was inhib- anti-CD3/anti-CD28-triggered activities of NF-B and NFAT. ited significantly by MAEA, which has a strong preference for Again, AP-1 activity was not influenced. In the CB1- and CB2- CB1. Together, these data indicate that both CB1 and CB2 expressing cells, the inhibitory effect of THC on NF-B and mediate the cannabinoid-induced inhibition of anti-CD3/anti- NFAT activities was significantly stronger than the effects of CD28-triggered IL-2 production in the Jurkat T cells. the selective CB1 and CB2 agonist alone. This indicates that Effects of Cannabinoids on TCR-activated AP-1, NF-B, and both receptors mediate the inhibitory effect of the drugs. NFAT—It is known that IL-2 is almost exclusively regulated at Effects of Cannabinoids on T Cell Proliferation—Because we the level of transcription. Therefore, it was next tested whether observed a drastic reduction in IL-2 production upon cannabi- cannabinoids had an effect on TCR-induced activities of the noid treatment and because IL-2 is a well defined autocrine transcription factors AP-1, NF-B, and NFAT, which are the factor required for proper T cell proliferation, we investigated key factors for transactivation of the IL-2 gene (Fig. 2). In naive the effect of cannabinoids on the anti-CD3/anti-CD28-induced CB2-expressing Jurkat cells, THC and JWH015 significantly cell division. Therefore, IL-4-stimulated, CB1- and CB2-ex- inhibited the anti-CD3/anti-CD28-triggered activities of NF- pressing primary human T cells were loaded with CFSE, and its B and NFAT as revealed by transient transfection of reporter dilution at each cell division was measured by flow cytometry genes. In contrast, the activity of AP-1 was not significantly (Fig. 3). Untreated control cells underwent up to three rounds reduced. In IL-4-stimulated CB1- and CB2-expressing Jurkat of division. However, treatment of cells with increasing doses of 35452 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 3. Effect of cannabinoids on T cell proliferation. IL-4-stimulated primary human T cells were loaded with CFSE and then left untreated (Co)or pretreated with a single dose of cannabinoids for 2 h prior to inoculation on FIGURE 2. Modulation of anti-CD3/anti-CD28-triggered activities of AP-1, anti-CD3/anti-CD28 coated dishes. Three days later, cell proliferation was NF-B, and NFAT by cannabinoids. Naive Jurkat cells expressing CB2 (light determined by CFSE dilution measured by FACS analysis. Two independent gray), and IL-4-stimulated Jurkat cells expressing CB1 and CB2 (dark gray) experiments were performed. A, representative FACS profiles for control cells were transfected with thymidine kinase (tk)-chloramphenicol acetyl transfer- and cells treated with MAEA (10 M) and JWH015 (5 M). The bars with the ase (CAT) reporter genes with binding sites for AP-1, NF-B, and NFAT. Then numbers above the profiles indicate how many rounds of division these cells cells were incubated either on immunoglobulin- or anti-CD3/anti-CD28- underwent. Note that the gate was set only on viable cells, although no coated dishes in the absence (controls, Co) or presence of THC (500 nM), MAEA increase in cell death after cannabinoid treatment was observed. B and C, (500 nM), or JWH015 (250 nM) for 72 h. The inducibility by anti-CD3/anti-CD28 inhibition of T cell proliferation at different concentrations of cannabinoids. was determined. The means of at least two independent experiments per- The percentage of cells in each round of division for different concentrations formed in duplicate  S.E. are displayed. Secondary comparisons are indi- of MAEA (B) and JWH015 (C) is depicted. cated by brackets.*, p  0.05; **, p  0.01; ***, p  0.001. antagonist AM630 each applied separately together with THC either MAEA or JWH015 resulted in increasing numbers of reduced the inhibitory effect of THC without completely abol- cells that were unable to proliferate (division phase 0) and ishing it. Only the combination of AM281 and AM630 together decreasing numbers of cells that underwent divisions. with THC abolished the inhibitory effect of THC completely, Effect of Cannabinoids on the Proximal TCR Signaling indicating that the effect of the drugs is mediated by both CB1 Cascade—To characterize mechanisms of the CB1- and CB2- and CB2. Consequently, the CB1 agonist MAEA as well as the mediated inhibition of the anti-CD3/anti-CD28-induced IL-2 CB2 agonist JWH015 inhibited the anti-CD3/anti-CD28-in- production, we investigated the effects of cannabinoids on the duced phosphorylation of MAPK in the stimulated Jurkat cells. proximal signaling cascade downstream of the TCR. Therefore, Inhibition of TCR Signaling by Cannabinoids Involves PKA, effects on the activation of MAPK, LAT, and Zap 70 were deter- Csk, and Lck—To more precisely identify the molecular sites, at mined in Western blot experiments using phospho-specific which CB1 and CB2 communicate with T cell signaling, we next antibodies (Fig. 4). Activation of Jurkat cells with anti-CD3/ examined effects of cannabinoids on Lck, the kinase responsi- anti-CD28 induced a robust phosphorylation of the three pro- ble for initiating TCR signaling and the activation of Zap 70. In teins. In naive CB2-expressing Jurkat cells, this phosphoryla- contrast to MAPK, LAT, and Zap 70, Lck is phosphorylated at tion was significantly reduced when the cells were incubated Tyr-505 in resting T cells, by which signaling downstream of the with THC for2hor longer prior to their activation. The effect TCR is prevented and becomes dephosphorylated upon TCR lasted at least 48 h. In stimulated CB1- and CB2-expressing activation. As shown in Fig. 4, the cannabinoids inhibited the cells, the effect was similar, and moreover it was significantly anti-CD3/anti-CD28-triggered dephosphorylation of Lck at stronger compared with the effect in the naive cells, which Tyr-505. It is known that the phosphorylation of the inhibitory express CB2 only. This suggested that both CB1 and CB2 con- Tyr-505 of Lck is mediated by the kinase Csk. Therefore, we tribute to the inhibition of TCR-activated MAPK, LAT, and asked whether Csk is involved in the cannabinoid-mediated Zap 70. To prove this, agonists and antagonists with a strong inhibition of the TCR signaling using a siRNA-based approach preference for one of these receptors were used, and the phos- (Fig. 6). Transient transfection of CB1- and CB2-expressing phorylation of MAPK was monitored (Fig. 5). Thus, in CB1- Jurkat cells with siRNA directed against human Csk resulted in and CB2-expressing Jurkat and primary human T cells, the an strong85% down-regulation of the protein compared with addition of the CB1 antagonist AM281, as well as the CB2 cells transfected with a nonmatching control siRNA and DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35453 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 5. The inhibition of the anti-CD3/anti-CD28-induced phosphory- lation of MAPK by cannabinoids is mediated by CB1 and CB2. A, IL-4- stimulated Jurkat cells (above) and IL-4-stimulated primary human T cells (below) received a single dose of cannabinoids and were incubated with the drug for 4 h according to the scheme below the gels. In some samples, the cells were co-incubated with antagonists (agonists: THC, 500 nM; MAEA, 500 nM; JWH015, 250 nM; and antagonists: AM281, 500 nM; AM630, 500 nM). Then the cells received fresh medium, were activated with anti-CD3/anti-CD28 for 2 min as indicated, and were lysed. The blots were probed for phospho-MAPK and actin as shown. Representative Western blot experiments are depicted. B, quantification of Western blot signals of at least two individual experiments performed in duplicate normalized to actin. All of the samples are compared with the anti-CD3/anti-CD28 activated samples shown in group 2, which were FIGURE 4. Effects of cannabinoids on the anti-CD3/anti-CD28-induced set to 100%. Secondary comparisons are indicated by brackets. **, p  0.01; phosphorylation of Lck, Zap 70, LAT, and MAPK. A, naive Jurkat cells ***, p  0.001. expressing CB2 (above) and IL-4-stimulated Jurkat cells expressing CB1 and CB2 (below) received a single dose of THC (500 nM) and were incubated with the drug for various times according to the scheme below the gels. Then the Modulation of Intracellular cAMP in T Cells by Can- cells received fresh medium, were activated with anti-CD3/anti-CD28 for 2 nabinoids—Because PKA is activated by cAMP, we next stud- min as indicated, and were lysed. The blots were probed for phospho-specific ied the regulation of cAMP by CB1 and CB2 agonists. When proteins (P-) and actin as shown. Representative Western blot experiments are depicted. B, quantification of Western blot signals of at least two individ- THC and JWH015 were applied to naive Jurkat cells expressing ual experiments performed in duplicate normalized to actin. All of the sam- only CB2 (Fig. 8A), both drugs caused a decrease in the cAMP ples are compared with the anti-CD3/anti-CD28 activated samples shown in formation at the 15-min time point. However, this short initial group 2, which were set to 100%. Secondary comparisons are indicated by brackets.*, p  0.05; **, p  0.01; ***, p  0.001. decrease was followed by a significant and sustained elevation of cAMP levels upon longer incubation of the cells with the untransfected cells (Fig. 6A). In the cells transfected with siRNA drugs, which climbed up to 5-fold over control levels. No directed against Csk, the inhibitory effect of the CB1 and CB2 significant changes in cAMP levels in the naive Jurkat cells were agonists on TCR signaling was highly significantly blocked, as observed after incubation with MAEA. In stimulated CB1- and demonstrated by monitoring the phosphorylation of LAT. In CB2-expressing Jurkat cells, however (Fig. 8B), incubation with contrast, using cells transfected with the control siRNA, no MAEA resulted in significantly elevated cAMP levels as well. It influence on the inhibitory effect of the cannabinoids was is noteworthy that incubation of the stimulated cells with THC observed (Fig. 6, B and C). This suggested that the effects of resulted in strongly elevated cAMP levels, which reached a the cannabinoids are transduced via Csk. It is known that the maximum of almost 10-fold over controls. The above shown activity of Csk is enhanced by PKA-mediated phosphorylation effects of cannabinoids were blocked by the CB1- and CB2- of Csk. To test whether the ability of the cannabinoids to inhibit selective antagonists AM281 and AM630 (data not shown). TCR signaling is dependent on PKA, we applied the PKA antag- Experiments in stimulated CB1- and CB2-expressing primary onist cAMPS-Rp (Fig. 7). Indeed, preincubation of CB1- and human T cells (Fig. 8C) gave qualitatively identical results as CB2-expressing Jurkat cells with cAMPS-Rp significantly those obtained in stimulated Jurkat cells. However, because pri- reversed the inhibitory effects of CB1 and CB2 ligands on the mary T cells are much smaller than Jurkat cells, they contained anti-CD3/anti-CD28-induced T cell signaling, as demon- less cAMP/10 cells. A dose-response curve using the cAMP strated by monitoring the phosphorylation states of LAT and accumulation of stimulated Jurkat cells after4hof incubation (Tyr-505)-Lck. with the drugs as a read-out is shown in Fig. 8D. Note that the 35454 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 7. Influence of the PKA inhibitor cAMPS-Rp on the effect of can- nabinoids on anti-CD3/anti-CD28-induced phosphorylation of Lck and LAT. A, according to the scheme below the gels, IL-4-stimulated Jurkat cells FIGURE 6. Effect of down-regulation of Csk on the cannabinoid-inhibited were treated with cAMPS-Rp (100 M). One hour later, cannabinoids were TCR signaling. A, down-regulation of Csk by siRNA. Western blot experi- added to the cells, and the cells were incubated for 4 h with the drugs accord- ments showing the expression of Csk and actin in IL-4-stimulated Jurkat cells ing to the scheme (THC, 500 nM; MAEA, 500 nM; JWH015, 250 nM). Then the 4 days after siRNA transfection. The cells were either untransfected ()or cells received fresh medium, were activated with anti-CD3/anti-CD28 for 2 transfected with a nonmatching control siRNA (co) or siRNA directed against min as indicated in the scheme, and were lysed. Representative Western blot human Csk (Csk). B, down-regulation of Csk abolishes the inhibition of TCR experiments detecting phospho- (P-) Lck and LAT and actin are shown. signaling by cannabinoids. Jurkat cells were transfected with Csk siRNA B, quantification of Western blot signals of at least two individual experiments (above) and control siRNA (below). Four days later, the cells were treated with performed in duplicate normalized to actin. The samples are compared with cannabinoids for 4 h according to the scheme below the gels (THC, 500 nM; the anti-CD3/anti-CD28 activated samples shown in group 2, which were set MAEA, 500 nM; JWH015, 250 nM). Then the cells received fresh medium and to 100%. Secondary comparisons are indicated by brackets.*, p 0.05; **, p were activated with anti-CD3/anti-CD28 for 2 min as indicated. Representa- 0.01; ***, p  0.001. tive results monitoring phospho-LAT and actin are shown. C, quantification of Western blot signals of at least two individual experiments performed in duplicate normalized to actin. The samples are compared with the anti-CD3/ intracellular cAMP, which is mediated by G proteins and that anti-CD28-activated samples shown in group 2. Secondary comparisons are there is an additive effect mediated by the two cannabinoid indicated by brackets. Gray columns, control siRNA; white columns, Csk siRNA. ***, p  0.001. receptors. To get a further idea of cAMP regulation via CB1 and CB2, the acute effects (after 15 min of exposure) of MAEA, doses of cannabinoids chosen for the inhibition of TCR signal- JWH015, AM281, and AM630 were compared between Jurkat ing in this study relate to the middle of the linear part of the cells that were pretreated chronically (for 8 h) with either curves. Experiments with pertussis toxin demonstrated that MAEA, JWH015, AM281, or AM630 and Jurkat cells that were both the initial decrease in cAMP levels caused by the cannabi- not pretreated (see Fig. 11). Although in the nonpretreated cells noids at the 15-min time point (Fig. 8E) as well as the subse- acute exposure with MAEA and JWH015 produced inhibition quent increase in cAMP measured at the 2 h time point (Fig. 8F) of cAMP formation, we observed stimulation of cAMP forma- were dependent on G proteins. To get a first idea of how long tion after acute exposure with these agonists in cells, which the increase in cAMP provoked by the cannabinoids might last, were chronically pretreated with the same agonist. Interest- naive Jurkat cells were treated once with THC, and cAMP was ingly, although the antagonists had no acute effects in the non- measured after different times. These studies revealed that pretreated cells, acute exposure with AM281 and AM630 pro- cAMP levels remained elevated at least 7 days (Controls, 1.3  duced stimulation of cAMP formation in cells that were 0.2 pmol/10 cells; cells stimulated for 2 days, 6.6  1.0 pmol/ chronically pretreated with MAEA and JWH015, respectively. 6 6 10 cells, p  0.001; 3 days, 6.5  1.1 pmol/10 cells, p  0.001; These data indicate that chronic agonist treatment of the 4 days, 5.8  1.0 pmol/10 cells, p  0.001; 7 days, 6.1  1.6 Jurkat cells induces sensitization of CB1 and CB2, resulting 6 6 pmol/10 cells, p  0.001; and 9 days, 2.7  1.2 pmol/10 cells, in a switch from inhibition to stimulation of cAMP forma- p  0.05). Fig. 8G demonstrates that both CB1 and CB2 con- tion. These experiments were performed in naive and IL-4- tribute to the effect on cAMP in an additive manner. Thus, the stimulated Jurkat cells. Again, in line with the previous combined effect of MAEA and JWH015 is approximately dou- results, effects of CB1 and CB2 ligands were observed in bly as strong as the effects of the agonists alone. Also, the effect IL-4-stimulated cells only, whereas in the naive cells only the of THC in stimulated CB1- and CB2-expressing Jurkat cells is effects of CB2 were observed. almost doubly as strong as the effect of the drug in naive CB2- Possible Role of Endogenous Cannabinoids in the Regulation expressing cells. Similar results were obtained with WIN of T Cell Signaling—It was demonstrated that endocannabi- 55,212-2 mesylate, which is another agonist of both receptors. noids are synthesized in lymphocytes and involved in the phys- Together, the cAMP data indicated that activation of both CB1 iological regulation of immune functions (34). Therefore, we and CB2 in T cells induces strong and sustained elevation of next tested the hypothesis that endogenous cannabinoids may DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35455 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 8. Modulation of cAMP formation in T cells by cannabinoids. A–C, time-dependent modulation of cAMP in naive Jurkat cells expressing CB2 (A), IL-4-stimulated Jurkat cells expressing CB1 and CB2 (B), and IL-4-stimulated primary human T cells expressing CB1 and CB2 (C). The cells received a single dose of cannabinoids (THC, 500 nM; MAEA, 500 nM; JWH015, 250 nM) and were incubated with the drugs for up to 48 h. Then cells were harvested, and the amounts of cAMP were determined. The data represent the means  S.E. of at least three independent experiments performed in triplicate. In A, the data sets for incubation with THC and JWH015 for 15 min and for 2 h and longer are significantly different from controls (p 0.01). In B, the data sets for incubation with THC, MAEA, and JWH015 for 15 min and for 2 h and longer are significantly different from controls (p 0.01). In C, the data sets for incubation with THC, MAEA, and JWH015 for 15 min, 4 h, and 24 h are significantly different from controls (p  0.01). D, formation of cAMP relative to different doses off cannabinoids. IL-4-stimulated Jurkat cells were incubated with the drugs for 4 h. Then cAMP was determined. The data represent the means S.E. of at least two independent experiments performed in triplicate. E and F, the cannabinoid-induced cAMP modulation is mediated by G proteins. IL-4-stimulated Jurkat cells were treated i/o with pertussis toxin (PTX, 10 ng/ml) for 16 h, then cannabinoids (see above) were added, and the cells were incubated for either 15 min (E)or2h(F). Then cAMP was determined. G, contribution of CB1 and CB2 to cAMP formation by cannabinoids. Naive Jurkat cells (light gray) and IL-4-stimulated Jurkat cells (dark gray) received a single dose of cannabinoids (see above; WIN, WIN 55,212-2 mesylate, 250 nM) and were incubated with the drugs for 2 h. Then cAMP was determined. The data represent the means  S.E. of at least two independent experiments performed in triplicate. *, p  0.05; **, p  0.01; ***, p  0.001. 35456 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 9. Effect of AM 251 and AM630 on anti-CD3/anti-CD28-triggered IL-2 transcription. Naive Jurkat cells expressing CB2 (light gray) and IL-4- stimulated Jurkat cells expressing CB1 and CB2 (dark gray) were incubated with the cannabinoid antagonists AM281 (500 nM, selective for CB1) and AM630 (500 nM, selective for CB2) for 48 h or left untreated, as depicted in the scheme below. Then cells were activated with anti-CD3/anti-CD28 to induce IL-2 mRNA, according to the scheme. The cells were lysed 4 h after the anti- FIGURE 10. Effect of AM 251 and AM630 on cAMP formation. IL-4-stimu- CD3/anti-CD28 activation for quantitative reverse transcription-PCR. The lated Jurkat cells expressing CB1 and CB2 were incubated with the cannabi- amount of IL-2-specific transcripts relative to that of -actin is plotted. For noid antagonists AM281 (500 nM, selective for CB1) and AM630 (500 nM, selec- easier comparison, the levels of IL-2 mRNA after anti-CD3/anti-CD28 activa- tive for CB2) for 48 h or left untreated as depicted in the scheme below. Then tion were set to 100%. The data represent the means  S.E. of at least three cAMP was determined. The data represent the means  S.E. of at least three independent experiments performed in duplicate. The asterisks define signif- independent experiments performed in triplicate. *, p 0.05; **, p 0.01; ***, icantly different values compared with controls (group 1). †, IL-4-stimulated p  0.001. cells; #, naive cells (significantly different samples compared with the respec- tive anti-CD3/anti-CD28-activated samples (group 3)). A further comparison is indicated by a bracket. One symbol, p  0.05; two symbols, p  0.01; three DISCUSSION symbols, p  0.001. In this report, the question of how cannabinoids inhibit the TCR-mediated production of IL-2 in T lymphocytes was be involved in the regulation of the IL-2 production in activated addressed. In the key step that links the drugs to the lymphocyte Jurkat T cells. If this was the case, blocking of CB1 and CB2 signaling, activation of CB1 and CB2 causes an increase in should increase the IL-2 mRNA levels (Fig. 9). Indeed, we ob- cAMP, which activates PKA. The kinase then activates the Csk/ served significantly increased anti-CD3/anti-CD28-induced Lck pathway, leading to stabilization of the inhibited, Tyr-505- IL-2 mRNA levels in naive Jurkat cells treated with the CB2- phosphorylated state of Lck. Consequently, the sequential phosphorylation of the proximal TCR signaling components selective antagonist AM630 compared with the untreated cells Zap 70, LAT, and MAPK are inhibited. Additionally, the TCR- (Fig. 9, group 5 versus group 3), whereas the CB1-selective mediated activation of the transcription factors NF-B and antagonist AM281 had no effect (group 4 versus group 3). In NFAT, which are pivotal factors for the TCR-triggered induc- IL-4-stimulated cells, treatment with AM630 (group 5 versus tion of IL-2, is inhibited. In this way cannabinoids also inhibit group 3), as well as AM281 (group 4 versus group 3) produced processes downstream of IL-2 like T cell proliferation. elevated IL-2 mRNA levels. When both antagonists were The question as to which receptors mediate the cannabi- applied simultaneously, the effect in the CB1- and CB2-ex- noid-induced inhibition of IL-2 and signaling in activated T pressing cells was significantly stronger than that in the naive cells has been addressed repeatedly but with contradictory CB2-expressing cells (group 6), indicating that both receptors results, as already mentioned in the introduction. Our results are involved in mediating this effect. In terms of cAMP regula- are well in line with those that reported the involvement of CB2 tion, treatment of Jurkat cells with the cannabinoid receptor (12, 13). Notably, here we clearly demonstrated for the first time antagonists resulted in decreased intracellular cAMP con- that CB1 is involved as well, by comparing the effects of various centrations (Fig. 10). In line with other experiments, the agonists in cells expressing either CB2 only (naive T cells) or combination of AM281 and AM630 produced a significantly both CB1 and CB2 (stimulated T cells). Thus, the induction of stronger effect than treatment of the cells with only one sub- functional CB1 can be seen by the lack of effects of CB1 agonists stance, again indicating the involvement of both CB1 and in naive cells and the presence of effects in the stimulated cells. CB2. We next tested whether applying the cannabinoid Moreover, additional evidence for the induction of CB1 in stim- receptor antagonists would alter the basal and anti-CD3/ ulated cells comes from the observation that mixed agonists anti-CD28-activated phosphorylation status of Zap 70, LAT, such as THC and WIN55,212-2 mesylate have significantly and MAPK. However, significant changes were not detect- stronger effects in stimulated cells compared with naive cells able (data not shown). The reason for this might be that (see Figs. 1, 2, 4, 5, and 8–11). These experiments thus clearly relatively small effects, which can be well detected in terms support an earlier study from our group, demonstrating the of cAMP and IL-2 mRNA, cannot be detected easily with the functionality of IL-4-induced CB1 in T cells (9). As to the Western blot method because of the narrow linear range in amount of cannabinoid receptors in T cells, it was shown that which signals can be quantified and because of quick satura- the ratio of CB1 to CB2 is 1:1.5 in peripheral human blood tion of exposed films. (34). In IL-4-stimulated Jurkat cells, this ratio is 1:20. Never- DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35457 Cannabinoid-inhibited T Cell Signaling via cAMP reports showing that THC behaves as a partial agonist/antago- nist for CB1 and CB2 in some cell systems (see e.g. Refs. 36 and 37). Nevertheless, in our hands THC produced similar effects in terms of cAMP formation as WIN55,212-2 mesylate, which is known to activate CB1 and CB2, suggesting that THC acts in an agonistic manner at both receptors in T cells. Notably, CB1 and CB2, which produced the remarkable and long lasting increase in cAMP, belong to the class of G -cou- i/o pled receptors, which are classically associated with a decrease in cAMP levels. Indeed, we observed decreased cAMP levels in the T cells in response to cannabinoids, but only within a short initial time in a range of minutes. This is in good accordance with the bulk of literature for CB1 and CB2 (reviewed in Ref. 5). Most interestingly, at later time points, activation of CB1 and CB2 induced a massive increase in cAMP in Jurkat and primary human T cells that was also mediated by G proteins. In general, G -coupled receptors may not only produce an inhibition of adenylyl cyclase but also a “superactivation” of the enzyme that results in increased cAMP levels (38, 39). Although known for decades, the mechanisms of this phenomenon remain unre- solved. Currently, one model aiming to explain it proposes a switch between G and G protein coupling (40, 41). However, i s we showed that both the initial decrease in cAMP as well as the subsequent increase in cAMP are mediated by pertussis toxin- sensitive G proteins. An additional mechanism that contrib- utes to the modulation of cAMP is the involvement of proteins termed regulators of G protein signaling proteins, which reduce G protein activity (42). Interestingly, we observed that chronic agonist treatment of Jurkat cells induced sensitization of CB1 and CB2, which resulted in a switch from inhibition to stimu- lation of cAMP formation after acute agonist exposure. This FIGURE 11. Chronic cannabinoid receptor agonists induce receptor sen- might be explained by various alterations of cellular functions sitization. Naive Jurkat cells expressing CB2 (A) and IL-4-stimulated Jurkat induced by the receptors, such as induction of adenylyl cyclases, cells expressing CB1 and CB2 (B) were preincubated for 8 h with cannabinoid receptor agonists (MAEA, 500 nM; JWH015, 250 nM), antagonists (AM281, 500 activation of these enzymes by phosphorylation, alterations in nM; AM630, 500 nM), or vehicle at 37 °C. Then cells were washed three times the intracellular calcium concentration, or stimulation of ade- for 5 min with phosphate-buffered saline (0 °C) and then incubated for nylyl cyclases by G protein --subunits, which are known to another 15 min in a 37 °C water bath with cannabinoid receptor agonists, antagonists, or vehicle, as indicated. Then cells were lysed, and cAMP was occur after chronic treatment of G protein-coupled receptors determined. All of the samples are compared with controls (100%) that were and may result in stimulated cAMP formation (see e.g. Refs. 38, identically preincubated for 8 h, washed, and incubated for 15 min with vehi- 39, and 43). Furthermore, it is known that chronic agonist expo- cle. The asterisks define samples that are significantly different from these controls. Secondary comparisons are indicated by brackets. The data repre- sure often results in post-translational modifications of recep- sent the means  S.E. of at least three individual experiments performed in tors, such as multiple phosphorylation events, which may duplicate. *, p  0.05; **, p  0.01; ***, p  0.001. induce conformational changes in the receptors, thereby alter- theless, our results suggest that activation of either receptor ing the affinities and the potencies of agonists and also antago- leads to quantitatively similar effects with regard to inhibition nists. It is discussed that in this way a single ligand of a given of T cell signaling and IL-2 production and induction of cAMP receptor may display a whole spectrum of activity ranging from formation. This might be explained by the fact that the cells agonism to antagonism or inverse agonism (44, 45). This might contain spare CB2 receptors that are not needed for a full effec- explain why AM281 and AM630 stimulated cAMP formation tor response. Moreover, because activation of both receptors in cells that have been pretreated chronically with MAEA simultaneously resulted in almost double-fold effects com- and JWH015, whereas AM281 and AM630 behaved as classic pared with the effects observed after activation of CB1 or CB2 antagonists in nonpretreated cells. Unraveling the detailed alone, our data suggest that the receptors act in an additive mechanisms of cAMP regulation by cannabinoids in T cells will mode. It should be mentioned that several G protein-coupled be a challenging aim for future investigations. It is noteworthy receptors may form heterodimers, which also might be true for that cAMP levels remained elevated for 7 days, which, to our CB1 and CB2 and also can explain additive effects. Although knowledge, has never been reported. This is remarkable in view heterodimerization of CB1 with other receptors has been of results by Massi et al. (46), who found persistent inhibition of reported (reviewed in Ref. 35), to our knowledge, heterodimer- IL-2 production in mice even 7 days after cannabinoid treat- ization of CB1 with CB2 has not been reported thus far but ment. As we will discuss below, there is reason to believe that cannot be excluded here. It should also be noted that there are the increase in cAMP is the key step in the inhibition of T cell 35458 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP signaling by cannabinoids in T cells. Interestingly and similar to cells lies upstream of the TCR. Thus, it can easily be explained our results in immune cells, it was reported for various brain that by interference with cAMP and Lck, cannabinoids can regions that chronic THC increased cAMP levels and PKA modulate TCR-mediated effects (e.g. induced by anti-CD3) but activity (47). It was discussed that this may be one of the mech- not the effects generated by activation of pathways downstream anisms leading to drug tolerance. It was also reported for CB1- of the TCR (e.g. induced by phorbol 12-myristate 13-acetate/ overexpressing HEK293 cells (48, 49) and for CB1 in the brain ionomycin). Therefore, caution is advised when conclusions (50) that these receptors desensitize rapidly, which also may are made that are based solely on experiments, in which T cells contribute to phenomena like tolerance. Although not investi- are not fully activated. For example, it was stated in an earlier gated here, our results and those reported by Massi et al. show- report that CB1 and CB2 were not involved in the inhibition of ing the sustained effects of cannabinoids in T cells suggest that IL-2 production, based on experiments in which cells were only desensitization may play only a minor role during chronic treated with phorbol 12-myristate 13-acetate/ionomycin (15). exposure of T cells to the drugs and/or that mechanisms of However, our experiments, in which T cells were activated in a desensitization may be different in neuronal and immune cells. more physiological way at the site of the TCR, clearly show the Our results suggested that the downstream effects of cAMP involvement of CB1 and CB2. were mediated by PKA. It should be noted that the PKA inhib- Interestingly, treatment of Jurkat T cells with AM281 and itor that we used, cAMPS-Rp, inhibits PKA by preventing the AM630 resulted in decreased cAMP levels and enhanced levels holoenzyme from dissociation, whereas other PKA inhibitors of TCR-induced IL-2. Assuming that the two substances antag- interfere with the phosphorylation process of PKA. This might onize effects mediated by CB1 and CB2, respectively, which has be of importance if the released regulatory subunits have spe- been reported for various effects (reviewed in Ref. 5), our results cial tasks on their own, which cannot be excluded here. In addi- indicate that endogenous cannabinoids are involved in the tion to our cAMP and PKA data, the experiments demonstrat- TCR-triggered IL-2 production. Although their role in the ing the involvement of Csk by down-regulation of the protein physiological regulation of IL-2 production in activated T cells with siRNA, and last but not least the results showing that the remains to be established, disregulation in endogenous canna- Tyr-505-phosphorylated (i.e. inhibited) form of Lck is stabi- binoid production is currently discussed as an important step in lized by cannabinoids favor our hypothesis that inhibition of T the pathophysiology of T cell-mediated autoimmune disorders cell signaling by cannabinoids is transduced via the cAMP/ (55). It should be noted, however, that the effects of AM281 and PKA/Csk/Lck pathway. This is in accordance with other re- AM630 could be explained alternatively, if these compounds ports showing inhibition of T cell signaling by this pathway in acted as inverse agonists and the cannabinoid receptors had other systems (for a review see Ref. 20). some constitutive activity in T cells. In fact, inverse agonistic Remarkably, despite of the marked inhibition of TCR-trig- activity has been reported for both substances, and it is well gered signaling, the cannabinoids only caused inhibition of the known that at least CB1 is constitutively active in other cell activities of the transcription factors NF-B and NFAT but systems (56–58). However, at the moment, the picture in T not AP-1. This is in accordance with earlier observations (4, 16, cells is unclear, and final conclusions cannot be made here. To 17) and may be explained by the fact that cannabinoids can sum up, our results help to explain immunosuppressive effect activate AP-1 via other pathways (51), which thus would coun- of cannabinoid drugs, which may be important for the pharma- teract the inhibitory input of the drugs on AP-1 activity via the cological evaluation of these drugs, e.g. with respect to their use TCR signaling cascade. in neuroinflammatory diseases and T cell-mediated autoim- One of the downstream effects of IL-2 is the induction of T mune disorders like multiple sclerosis. In addition, our results cell proliferation. Therefore, by inhibiting the expression of may indicate a regulatory role for endogenous cannabinoids in IL-2, cannabinoids also inhibit T cell proliferation (52–54). We the production of IL-2 in T cells, which could be, for example, a demonstrated that both CB1- and CB2-selective agonists inhib- prevention of excessive T cell responses. ited T cell proliferation, although higher doses were needed for Acknowledgments—We thank Helga Tischmeyer for excellent techni- this effect than for the inhibition of the T cell signaling cascade cal assistance. The plates and reagents for the cAMP-enzyme-linked and IL-2 inhibition. Our results are very good in line with an immunosorbent assay were a kind gift from Hermann Ammer (Insti- earlier report demonstrating marked effects of THC on prolif- tute of Pharmacology, Toxicology, and Pharmacy, University of eration of primary human T cells only at high (2.5 and 5 M) Munich). In addition, we are very thankful to Hermann Ammer for doses of the drug (54). 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Cannabinoid Receptor Type 1- and 2-mediated Increase in Cyclic AMP Inhibits T Cell Receptor-triggered Signaling

Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen
Journal of Biological Chemistry – Dec 1, 2009
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10.1074/jbc.m109.006338
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 51, pp. 35450 –35460, December 18, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cannabinoid Receptor Type 1- and 2-mediated Increase in Cyclic AMP Inhibits T Cell Receptor-triggered Signaling Received for publication, April 9, 2009, and in revised form, October 16, 2009 Published, JBC Papers in Press, October 26, 2009, DOI 10.1074/jbc.M109.006338 ‡ § ‡ § ‡1 Christine Bo¨ rner , Michal Smida , Volker Ho¨ llt , Burkhart Schraven , and Ju¨ rgen Kraus ‡ § From the Departments of Pharmacology and Toxicology and Molecular and Clinical Immunology, University of Magdeburg, 39120 Magdeburg, Germany The aim of this study was to characterize inhibitory mecha- beneficial in the above mentioned diseases but on the other nisms on T cell receptor signaling mediated by the cannabinoid hand may also cause immunosuppression. Many effects of receptors CB1 and CB2. Both receptors are coupled to G pro- endogenous cannabinoids like anandamide and exogenous i/o teins, which are associated with inhibition of cyclic AMP forma- cannabinoids like 9-tetrahydrocannabinol (THC), the psy- tion. In human primary and Jurkat T lymphocytes, activation of choactive compound of Cannabis sativa, are mediated by the CB1 by R()-methanandamide, CB2 by JWH015, and both by G protein-coupled cannabinoid receptors CB1 and CB2. i/o 9-tetrahydrocannabinol induced a short decrease in cyclic While CB1 was found to be expressed predominantly in neuro- AMP lasting less than 1 h. However, this decrease was followed nal cells and only to a lesser extend in peripheral cells like by a massive (up to 10-fold) and sustained (at least up to 48 h) immune effector cells, CB2 was considered to be the “periph- increase in cyclic AMP. Mediated by the cyclic AMP-activated eral” cannabinoid receptor, being expressed in cells of the protein kinase A and C-terminal Src kinase, the cannabinoids immune system (5). However, recent reports suggest the ex- induced a stable phosphorylation of the inhibitory Tyr-505 of pression of CB2 in neuronal cells, as well (6, 7). Moreover, the leukocyte-specific protein tyrosine kinase (Lck). By thus although normally expressed in very low amounts only, recent arresting Lck in its inhibited form, the cannabinoids prevented work from our laboratory demonstrated a massive up-regula- the dephosphorylation of Lck at Tyr-505 in response to T cell tion of functional CB1 receptors in T lymphocytes in response receptor activation, which is necessary for the subsequent initi- to cannabinoids themselves, IL-4, and T cell activation (8–10). ation of T cell receptor signaling. In this way the cannabinoids In addition, a number of other receptors have been shown to inhibited the T cell receptor-triggered signaling, i.e. the activa- mediate the effects of cannabinoids, which include vanilloid tion of the -chain-associated protein kinase of 70 kDa, the receptors, peroxisome proliferator-activated receptors and the linker for activation of T cells, MAPK, the induction of interleu- formerly “orphan” designated receptors GPR55 and GPR119 kin-2, and T cell proliferation. All of the effects of the cannabi- (reviewed in Ref. 11). noids were blocked by the CB1 and CB2 antagonists AM281 and Despite of a number of reports, it remains largely unresolved AM630. These findings help to better understand the immuno- which types of cannabinoid receptors mediate IL-2 inhibition suppressive effects of cannabinoids and explain the beneficial and what are the detailed molecular mechanisms. With regard effects of these drugs in the treatment of T cell-mediated auto- to the receptors, it was demonstrated independently by differ- immune disorders like multiple sclerosis. ent groups, using either selective agonists and antagonists (12) or transgenic mice lacking CB2 (13) that CB2 is involved in mediating the effect of cannabinoids on IL-2. However, it was Cannabinoids are discussed as drugs for the treatment of also suggested that neither CB1 nor CB2 receptors mediated the effect of cannabinoids on IL-2 (14). Recently, it was re- neuroinflammatory diseases and T cell-mediated autoimmune ported that the inhibitory effect of the endogenous cannabinoid disorders such as, for example, multiple sclerosis. Their poten- tial usefulness for such disorders is emphasized by the fact that 2-arachidonyl glycerol on IL-2 production in T cells was depen- cannabinoids affect functions of both neuronal and immune dent on peroxisome proliferator-activated receptor  (15). Thus far, no reports showed involvement of CB1 in the canna- effector cells. Cannabinoids produce a variety of immuno- binoid-mediated inhibition of IL-2, which is probably due to modulatory effects (reviewed in Refs. 1 and 2). Among these effects, the cannabinoid-mediated inhibition of interleukin the fact that high amounts of CB1 are only present in T cells (IL)-2 production of activated T lymphocytes (3, 4) may be after defined stimulation of the cells (see above). With regard to the mechanisms of the cannabinoid-mediated inhibition of IL-2, it was shown that the drugs inhibit the T cell receptor * This work was supported by research grants from the government of the (TCR)-induced activities of the transcription factors NF-B state of Sachsen-Anhalt (to V. H. and B. S.), a grant from the University of Magdeburg, Faculty of Medicine (Initialprojekt) (to C. B.), and Deutsche and NFAT (4, 16, 17). This is interesting, because it is known Forschungsgemeinschaft Grant KR 1740/10-1 (to J. K.). that the expression of IL-2 in activated T cells is almost exclu- To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, University of Magdeburg, 44 Leipzigerstrasse, 39120 Magdeburg, Germany. Tel.: 49-391-6715372; Fax: 49-391-6715869; E-mail: kinase; PKA, cAMP-dependent protein kinase; TCR, T cell receptor; Zap 70, [email protected]. -chain-associated protein kinase of 70 kDa; cAMPS-Rp, (R)-adenosine, The abbreviations used are: IL, interleukin; Csk, C-terminal Src kinase; THC, cyclic 3,5-hydrogenphosphorothioate; CFSE, carboxyfluorescein succin- 9-tetrahydrocannabinol; Lck, leukocyte-specific protein tyrosine kinase; imidyl ester; FACS, fluorescence-activated cell sorter; siRNA, small interfer- LAT, linker for activation of T cells; MAPK, mitogen-activated protein ing RNA; MAEA, metanandamide. 35450 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 This is an Open Access article under the CC BY license. Cannabinoid-inhibited T Cell Signaling via cAMP sively regulated at the level of transcription by these factors. In with a preference for CB1 (MAEA; Sigma), JWH015 with a pref- addition, it was shown that activation of the mitogen-activated erence for CB2 (Tocris, Bristol, UK), and WIN 55,212-2 mesy- protein kinase (MAPK) is inhibited by cannabinoids in lympho- late, which activates CB1 and CB2 (Tocris). Antagonists were cytes (18). However, detailed molecular mechanisms linking AM281 (selective for CB1: K of CB1, 12 nM; K of CB2, 4200 nM; i i the effects of cannabinoids to the TCR-triggered signaling are Tocris) and AM630 (selective for CB2: K of CB1, 5152 nM; K of i i missing and will be the topic of this communication. CB2, 31.2 nM; Tocris). The K values were taken from Ref. 5. The The TCR-mediated activation of T cells induces a defined PKA inhibitor (R)-adenosine, cyclic 3,5-hydrogenphosphoro- signaling cascade, at the end point of which transcription of thioate (cAMPS-Rp; Tocris) was used at 100 M. IL-2 is induced (19–21). In nonactivated, resting T cells, this Quantitative Real Time Reverse Transcription-PCR—Per cascade is tonically repressed by constitutive phosphorylation sample, 10 Jurkat cells were used. The isolation of total RNA of the negative regulatory site of the leukocyte-specific protein and cDNA synthesis is described earlier (8). The PCRs were tyrosine kinase (Lck) at Tyr-505. This phosphorylation is medi- done in a total volume of 20 l on a LightCycler instrument ated by the C-terminal Src kinase (Csk) (22). Upon engagement using the LightCycler-Fast Start DNA Master SYBR Green I kit of the TCR, the inhibitory effect of Csk on Lck is abolished (both from Roche) according to the manufacturer’s sugges- allowing initiation of the TCR-induced signaling cascade (23– tions. Primers and conditions for the PCRs specific for -actin, 25). One of the earliest events in this cascade is then the phos- CB1, and CB2 are described in detail earlier (8). The PCRs for phorylation and thereby the activation of the -chain-associ- IL-2 transcripts were performed with 5-GAAGGCCACA- ated protein kinase of 70 kDa (Zap 70). Activated Zap 70 in turn GAACTGAAACATCT-3 and 5-CTGTTCAGAAATTCTA- phosphorylates the adaptor protein linker for activation of T CAATGGTTG-3 primers, with a preincubation for 8 min at cells (LAT), leading to the induction of calcium flux and activa- 95 °C and 50 cycles with5sat95 °C,5sat65 °C,and10sat tion of the MAPK cascades. In this way, the signal is transduced, 72 °C (specific T  81,75 °C). and the transcription factors AP-1, NF-B, and NFAT are acti- Proliferation Assay—The cells were washed once with phos- vated and finally transactivate the IL-2 gene. After being phate-buffered saline and loaded with 0.1 M carboxyfluores- released, IL-2 induces a variety of well defined subsequent cein succinimidyl ester (CFSE; Molecular Probes, Go¨ttingen, immune responses (26). It is known that the activity of Csk to Germany) at 37 °C for 10 min. Loading was stopped by the phosphorylate the negative regulatory site of Lck at Tyr-505 can addition of 5% fetal calf serum, and the cells were washed with be further enhanced by cAMP-dependent protein kinase A phosphate-buffered saline. CFSE-loaded cells were pretreated (PKA). Thus, PKA exhibits an inhibitory effect upon TCR sig- with cannabinoids for 2 h and then inoculated on a 24-well plate naling by stabilizing the inhibited state of Lck (27, 28). Here we coated with anti-CD3/anti-CD28. Three days later, prolifera- report on the molecular linkage between CB1 and CB2 and T tion was measured on a FACSCalibur (Becton Dickinson). cell signaling and present a mechanism by which cannabinoids Reporter Genes and Transfection—Reporter gene constructs inhibit IL-2 production of activated human T cells via these are based on pBLCAT2 (30) and contain binding sequences for receptors. AP-1 (5-AAACATATGATTCACCAGGCA-3), NF-B(5- AAAGTTGAGGGGACTTTCCCAGGCCT-3), and NFAT EXPERIMENTAL PROCEDURES (5-GCCCAAAGAGGAAAATTTGTTTCATA-3) cloned 5 T Cell Culture, Induction of CB1, T Cell Activation, and to the herpes simplex thymidine kinase promoter. Transfection Reagents—Peripheral blood mononuclear cells were isolated was performed by electroporation (210 V, 950 microfarads) from heparinized blood collected from healthy volunteers as using a Gene Pulser II (Bio-Rad). The day after transfection, described (29). Primary human T cells and the human T cell line the cells were transferred to anti-CD3/anti-CD28- or anti- Jurkat (Kab 14) were cultivated in RPMI 1640 medium (Lonza mouse-immunoglobulin-coated dishes with or without canna- Verviers SPRL, Verviers, Belgium) supplemented with 10% fetal binoids. After 72 h of transient expression, the reporter gene calf serum and antibiotics (100 units/ml penicillin and 100 was assayed via a chloramphenicol acetyl transferase-enzyme- mg/ml streptomycin; Lonza Verviers SPRL). We would like to linked immunosorbent assay purchased from Roche Applied note that no cannabinoid receptor-overexpressing cells were Science. used in our experiments. CB2 is expressed in the naive cells Western Blots—Western blots were performed as previously constitutively. The endogenous CB1 was induced by stimula- described (8, 31, 32). For antigen activation, 10 Jurkat cells/ tion of the cells with IL-4 as described (9). Briefly, the cells sample were pelleted, resuspended in anti-CD3/anti-CD28 received IL-4 (5 ng/ml; R & D Systems, Wiesbaden, Germany) monoclonal antibodies, and incubated at 37 °C for 2 min. and were incubated for 3 days. Then the medium was replaced The activation was stopped with 1 ml of ice-cold phosphate- by fresh medium without IL-4, and the cells were cultured for buffered saline, and the cells were pelleted again and lysed. additional 24 h. An aliquot of the cells was tested to assure the For protein detection, the following primary antibodies were induction of CB1 transcripts (data not shown). For these cells, used: actin C-11, Csk C-20 (both from Santa Cruz, Heidel- the terms “stimulated cells” or “CB1- and CB2-expressing cells” berg, Germany); phospho-p44/42 MAPK Tyr-202/Tyr-204, will be used. T cells were activated with 100 l of each anti-CD3 phospho-LAT Tyr-191, phospho-Zap 70 Tyr-319, and phos- monoclonal antibodies (OKT3) and anti-CD28 monoclonal pho-Lck Tyr-505 (all from Cell Signaling Technology/New antibodies (248.23.2) obtained from hybridoma supernatants England Biolabs, Frankfurt, Germany). All of the antibodies are produced in our laboratories (29). Cannabinoid agonists were from rabbits. For protein detection, the following secondary THC, which activates CB1 and CB2 (Sigma), metanandamide antibody was used: anti-rabbit IgG (GE Healthcare). For quan- DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35451 Cannabinoid-inhibited T Cell Signaling via cAMP tification of Western blot signals, densitometric analysis was performed using National Institutes of Health Image 1.63 software. Down-regulation of Csk with siRNA—The commercially available siRNA against human Csk and a nonmatching control siRNA (siRNA-A; both from Santa Cruz) were used. The siRNAs were transfected into Jurkat T cells by electroporation (210 V, 950 microfarads) using a Gene Pulser II (Bio-Rad). The cells were then cultured in RPMI 1640 for 4 days to ensure Csk protein down-regulation. Cyclic AMP Measurement—Jurkat cells were incubated with cannabinoids for 0 h (controls) up to 9 days. The cells were then lysed with 50 mM HCl for 30 min on ice. The competitive cAMP-enzyme-linked immunosorbent assay was performed according to a described procedure (33). Statistical Analysis—For statistical evaluations between two groups of samples, Student’s t tests were performed. Multiple comparisons were performed with analysis of variance, fol- lowed by Tukey’s multiple comparison post-test. RESULTS Effects of Cannabinoids on IL-2 mRNA in Activated T Cells— First, CB1- and CB2-mediated effects of cannabinoids on the anti-CD3/anti-CD28-triggered induction of IL-2 mRNA was determined in Jurkat T cells. In naive cells, which express CB2 abundantly, but only very small amounts of CB1 (Fig. 1A), THC, a mixed agonist for CB1 and CB2, caused a significant inhibi- tion of the anti-CD3/anti-CD28-triggered IL-2 mRNA produc- FIGURE 1. Inhibition of anti-CD3/anti-CD28-triggered IL-2 transcription tion, which was dependent on the time of incubation of the cells by cannabinoids. Jurkat T cells received a single dose of cannabinoids (THC, 500 nM, activates both CB1 and CB2; MAEA, 500 nM, preference for CB1; with the cannabinoid prior to the anti-CD3/anti-CD28 stimu- JWH015, 250 nM, preference for CB2) and were incubated with the drugs up to lation. Interestingly, incubation of the cells with the drug for 24 48 h. Then cells received fresh medium and were activated with anti-CD3/ anti-CD28 to induce IL-2 mRNA. The cells were lysed 4 h after the anti-CD3/ and 48 h completely inhibited the anti-CD3/anti-CD28-medi- anti-CD28 activation for quantitative reverse transcription-PCR. The amount ated induction of IL-2. A similar effect was observed when the of IL-2-specific transcripts relative to that of -actin is plotted. The cells that CB2-selective agonist JWH015 was tested. In line with the fact were not incubated with cannabinoids served as controls, for which the anti- CD3/anti-CD28-mediated induction of IL-2-specific transcripts was set to that only very small amounts of CB1 are present in naive Jurkat 100%. The data represent the means  S.E. of at least three independent cells, the CB1-selective agonist MAEA had no significant effect experiments performed in duplicate. A, experiments performed in naive Jur- on the anti-CD3/anti-CD28-induced IL-2 mRNA levels. We kat cells expressing CB2. The data sets for THC and JWH015 at 2, 4, 24, and 48 h are significantly different from controls (p 0.001). B, experiments performed have previously shown that stimulation of T cells with IL-4 in IL-4-stimulated Jurkat cells expressing CB1 and CB2. The data sets for THC, markedly induces CB1 mRNA and functional receptor proteins MAEA, and JWH015 at 2, 4, 24, and 48 h are significantly different from con- trols (p  0.001). (9) without altering the expression of CB2. In contrast to naive cells, in IL-4-stimulated Jurkat cells, which express CB1 and CB2 (Fig. 1B), the anti-CD3/anti-CD28-evoked IL-2 induction cells, not only THC and JWH015 but also MAEA inhibited the was not only inhibited by THC and JWH015 but also was inhib- anti-CD3/anti-CD28-triggered activities of NF-B and NFAT. ited significantly by MAEA, which has a strong preference for Again, AP-1 activity was not influenced. In the CB1- and CB2- CB1. Together, these data indicate that both CB1 and CB2 expressing cells, the inhibitory effect of THC on NF-B and mediate the cannabinoid-induced inhibition of anti-CD3/anti- NFAT activities was significantly stronger than the effects of CD28-triggered IL-2 production in the Jurkat T cells. the selective CB1 and CB2 agonist alone. This indicates that Effects of Cannabinoids on TCR-activated AP-1, NF-B, and both receptors mediate the inhibitory effect of the drugs. NFAT—It is known that IL-2 is almost exclusively regulated at Effects of Cannabinoids on T Cell Proliferation—Because we the level of transcription. Therefore, it was next tested whether observed a drastic reduction in IL-2 production upon cannabi- cannabinoids had an effect on TCR-induced activities of the noid treatment and because IL-2 is a well defined autocrine transcription factors AP-1, NF-B, and NFAT, which are the factor required for proper T cell proliferation, we investigated key factors for transactivation of the IL-2 gene (Fig. 2). In naive the effect of cannabinoids on the anti-CD3/anti-CD28-induced CB2-expressing Jurkat cells, THC and JWH015 significantly cell division. Therefore, IL-4-stimulated, CB1- and CB2-ex- inhibited the anti-CD3/anti-CD28-triggered activities of NF- pressing primary human T cells were loaded with CFSE, and its B and NFAT as revealed by transient transfection of reporter dilution at each cell division was measured by flow cytometry genes. In contrast, the activity of AP-1 was not significantly (Fig. 3). Untreated control cells underwent up to three rounds reduced. In IL-4-stimulated CB1- and CB2-expressing Jurkat of division. However, treatment of cells with increasing doses of 35452 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 3. Effect of cannabinoids on T cell proliferation. IL-4-stimulated primary human T cells were loaded with CFSE and then left untreated (Co)or pretreated with a single dose of cannabinoids for 2 h prior to inoculation on FIGURE 2. Modulation of anti-CD3/anti-CD28-triggered activities of AP-1, anti-CD3/anti-CD28 coated dishes. Three days later, cell proliferation was NF-B, and NFAT by cannabinoids. Naive Jurkat cells expressing CB2 (light determined by CFSE dilution measured by FACS analysis. Two independent gray), and IL-4-stimulated Jurkat cells expressing CB1 and CB2 (dark gray) experiments were performed. A, representative FACS profiles for control cells were transfected with thymidine kinase (tk)-chloramphenicol acetyl transfer- and cells treated with MAEA (10 M) and JWH015 (5 M). The bars with the ase (CAT) reporter genes with binding sites for AP-1, NF-B, and NFAT. Then numbers above the profiles indicate how many rounds of division these cells cells were incubated either on immunoglobulin- or anti-CD3/anti-CD28- underwent. Note that the gate was set only on viable cells, although no coated dishes in the absence (controls, Co) or presence of THC (500 nM), MAEA increase in cell death after cannabinoid treatment was observed. B and C, (500 nM), or JWH015 (250 nM) for 72 h. The inducibility by anti-CD3/anti-CD28 inhibition of T cell proliferation at different concentrations of cannabinoids. was determined. The means of at least two independent experiments per- The percentage of cells in each round of division for different concentrations formed in duplicate  S.E. are displayed. Secondary comparisons are indi- of MAEA (B) and JWH015 (C) is depicted. cated by brackets.*, p  0.05; **, p  0.01; ***, p  0.001. antagonist AM630 each applied separately together with THC either MAEA or JWH015 resulted in increasing numbers of reduced the inhibitory effect of THC without completely abol- cells that were unable to proliferate (division phase 0) and ishing it. Only the combination of AM281 and AM630 together decreasing numbers of cells that underwent divisions. with THC abolished the inhibitory effect of THC completely, Effect of Cannabinoids on the Proximal TCR Signaling indicating that the effect of the drugs is mediated by both CB1 Cascade—To characterize mechanisms of the CB1- and CB2- and CB2. Consequently, the CB1 agonist MAEA as well as the mediated inhibition of the anti-CD3/anti-CD28-induced IL-2 CB2 agonist JWH015 inhibited the anti-CD3/anti-CD28-in- production, we investigated the effects of cannabinoids on the duced phosphorylation of MAPK in the stimulated Jurkat cells. proximal signaling cascade downstream of the TCR. Therefore, Inhibition of TCR Signaling by Cannabinoids Involves PKA, effects on the activation of MAPK, LAT, and Zap 70 were deter- Csk, and Lck—To more precisely identify the molecular sites, at mined in Western blot experiments using phospho-specific which CB1 and CB2 communicate with T cell signaling, we next antibodies (Fig. 4). Activation of Jurkat cells with anti-CD3/ examined effects of cannabinoids on Lck, the kinase responsi- anti-CD28 induced a robust phosphorylation of the three pro- ble for initiating TCR signaling and the activation of Zap 70. In teins. In naive CB2-expressing Jurkat cells, this phosphoryla- contrast to MAPK, LAT, and Zap 70, Lck is phosphorylated at tion was significantly reduced when the cells were incubated Tyr-505 in resting T cells, by which signaling downstream of the with THC for2hor longer prior to their activation. The effect TCR is prevented and becomes dephosphorylated upon TCR lasted at least 48 h. In stimulated CB1- and CB2-expressing activation. As shown in Fig. 4, the cannabinoids inhibited the cells, the effect was similar, and moreover it was significantly anti-CD3/anti-CD28-triggered dephosphorylation of Lck at stronger compared with the effect in the naive cells, which Tyr-505. It is known that the phosphorylation of the inhibitory express CB2 only. This suggested that both CB1 and CB2 con- Tyr-505 of Lck is mediated by the kinase Csk. Therefore, we tribute to the inhibition of TCR-activated MAPK, LAT, and asked whether Csk is involved in the cannabinoid-mediated Zap 70. To prove this, agonists and antagonists with a strong inhibition of the TCR signaling using a siRNA-based approach preference for one of these receptors were used, and the phos- (Fig. 6). Transient transfection of CB1- and CB2-expressing phorylation of MAPK was monitored (Fig. 5). Thus, in CB1- Jurkat cells with siRNA directed against human Csk resulted in and CB2-expressing Jurkat and primary human T cells, the an strong85% down-regulation of the protein compared with addition of the CB1 antagonist AM281, as well as the CB2 cells transfected with a nonmatching control siRNA and DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35453 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 5. The inhibition of the anti-CD3/anti-CD28-induced phosphory- lation of MAPK by cannabinoids is mediated by CB1 and CB2. A, IL-4- stimulated Jurkat cells (above) and IL-4-stimulated primary human T cells (below) received a single dose of cannabinoids and were incubated with the drug for 4 h according to the scheme below the gels. In some samples, the cells were co-incubated with antagonists (agonists: THC, 500 nM; MAEA, 500 nM; JWH015, 250 nM; and antagonists: AM281, 500 nM; AM630, 500 nM). Then the cells received fresh medium, were activated with anti-CD3/anti-CD28 for 2 min as indicated, and were lysed. The blots were probed for phospho-MAPK and actin as shown. Representative Western blot experiments are depicted. B, quantification of Western blot signals of at least two individual experiments performed in duplicate normalized to actin. All of the samples are compared with the anti-CD3/anti-CD28 activated samples shown in group 2, which were FIGURE 4. Effects of cannabinoids on the anti-CD3/anti-CD28-induced set to 100%. Secondary comparisons are indicated by brackets. **, p  0.01; phosphorylation of Lck, Zap 70, LAT, and MAPK. A, naive Jurkat cells ***, p  0.001. expressing CB2 (above) and IL-4-stimulated Jurkat cells expressing CB1 and CB2 (below) received a single dose of THC (500 nM) and were incubated with the drug for various times according to the scheme below the gels. Then the Modulation of Intracellular cAMP in T Cells by Can- cells received fresh medium, were activated with anti-CD3/anti-CD28 for 2 nabinoids—Because PKA is activated by cAMP, we next stud- min as indicated, and were lysed. The blots were probed for phospho-specific ied the regulation of cAMP by CB1 and CB2 agonists. When proteins (P-) and actin as shown. Representative Western blot experiments are depicted. B, quantification of Western blot signals of at least two individ- THC and JWH015 were applied to naive Jurkat cells expressing ual experiments performed in duplicate normalized to actin. All of the sam- only CB2 (Fig. 8A), both drugs caused a decrease in the cAMP ples are compared with the anti-CD3/anti-CD28 activated samples shown in formation at the 15-min time point. However, this short initial group 2, which were set to 100%. Secondary comparisons are indicated by brackets.*, p  0.05; **, p  0.01; ***, p  0.001. decrease was followed by a significant and sustained elevation of cAMP levels upon longer incubation of the cells with the untransfected cells (Fig. 6A). In the cells transfected with siRNA drugs, which climbed up to 5-fold over control levels. No directed against Csk, the inhibitory effect of the CB1 and CB2 significant changes in cAMP levels in the naive Jurkat cells were agonists on TCR signaling was highly significantly blocked, as observed after incubation with MAEA. In stimulated CB1- and demonstrated by monitoring the phosphorylation of LAT. In CB2-expressing Jurkat cells, however (Fig. 8B), incubation with contrast, using cells transfected with the control siRNA, no MAEA resulted in significantly elevated cAMP levels as well. It influence on the inhibitory effect of the cannabinoids was is noteworthy that incubation of the stimulated cells with THC observed (Fig. 6, B and C). This suggested that the effects of resulted in strongly elevated cAMP levels, which reached a the cannabinoids are transduced via Csk. It is known that the maximum of almost 10-fold over controls. The above shown activity of Csk is enhanced by PKA-mediated phosphorylation effects of cannabinoids were blocked by the CB1- and CB2- of Csk. To test whether the ability of the cannabinoids to inhibit selective antagonists AM281 and AM630 (data not shown). TCR signaling is dependent on PKA, we applied the PKA antag- Experiments in stimulated CB1- and CB2-expressing primary onist cAMPS-Rp (Fig. 7). Indeed, preincubation of CB1- and human T cells (Fig. 8C) gave qualitatively identical results as CB2-expressing Jurkat cells with cAMPS-Rp significantly those obtained in stimulated Jurkat cells. However, because pri- reversed the inhibitory effects of CB1 and CB2 ligands on the mary T cells are much smaller than Jurkat cells, they contained anti-CD3/anti-CD28-induced T cell signaling, as demon- less cAMP/10 cells. A dose-response curve using the cAMP strated by monitoring the phosphorylation states of LAT and accumulation of stimulated Jurkat cells after4hof incubation (Tyr-505)-Lck. with the drugs as a read-out is shown in Fig. 8D. Note that the 35454 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 7. Influence of the PKA inhibitor cAMPS-Rp on the effect of can- nabinoids on anti-CD3/anti-CD28-induced phosphorylation of Lck and LAT. A, according to the scheme below the gels, IL-4-stimulated Jurkat cells FIGURE 6. Effect of down-regulation of Csk on the cannabinoid-inhibited were treated with cAMPS-Rp (100 M). One hour later, cannabinoids were TCR signaling. A, down-regulation of Csk by siRNA. Western blot experi- added to the cells, and the cells were incubated for 4 h with the drugs accord- ments showing the expression of Csk and actin in IL-4-stimulated Jurkat cells ing to the scheme (THC, 500 nM; MAEA, 500 nM; JWH015, 250 nM). Then the 4 days after siRNA transfection. The cells were either untransfected ()or cells received fresh medium, were activated with anti-CD3/anti-CD28 for 2 transfected with a nonmatching control siRNA (co) or siRNA directed against min as indicated in the scheme, and were lysed. Representative Western blot human Csk (Csk). B, down-regulation of Csk abolishes the inhibition of TCR experiments detecting phospho- (P-) Lck and LAT and actin are shown. signaling by cannabinoids. Jurkat cells were transfected with Csk siRNA B, quantification of Western blot signals of at least two individual experiments (above) and control siRNA (below). Four days later, the cells were treated with performed in duplicate normalized to actin. The samples are compared with cannabinoids for 4 h according to the scheme below the gels (THC, 500 nM; the anti-CD3/anti-CD28 activated samples shown in group 2, which were set MAEA, 500 nM; JWH015, 250 nM). Then the cells received fresh medium and to 100%. Secondary comparisons are indicated by brackets.*, p 0.05; **, p were activated with anti-CD3/anti-CD28 for 2 min as indicated. Representa- 0.01; ***, p  0.001. tive results monitoring phospho-LAT and actin are shown. C, quantification of Western blot signals of at least two individual experiments performed in duplicate normalized to actin. The samples are compared with the anti-CD3/ intracellular cAMP, which is mediated by G proteins and that anti-CD28-activated samples shown in group 2. Secondary comparisons are there is an additive effect mediated by the two cannabinoid indicated by brackets. Gray columns, control siRNA; white columns, Csk siRNA. ***, p  0.001. receptors. To get a further idea of cAMP regulation via CB1 and CB2, the acute effects (after 15 min of exposure) of MAEA, doses of cannabinoids chosen for the inhibition of TCR signal- JWH015, AM281, and AM630 were compared between Jurkat ing in this study relate to the middle of the linear part of the cells that were pretreated chronically (for 8 h) with either curves. Experiments with pertussis toxin demonstrated that MAEA, JWH015, AM281, or AM630 and Jurkat cells that were both the initial decrease in cAMP levels caused by the cannabi- not pretreated (see Fig. 11). Although in the nonpretreated cells noids at the 15-min time point (Fig. 8E) as well as the subse- acute exposure with MAEA and JWH015 produced inhibition quent increase in cAMP measured at the 2 h time point (Fig. 8F) of cAMP formation, we observed stimulation of cAMP forma- were dependent on G proteins. To get a first idea of how long tion after acute exposure with these agonists in cells, which the increase in cAMP provoked by the cannabinoids might last, were chronically pretreated with the same agonist. Interest- naive Jurkat cells were treated once with THC, and cAMP was ingly, although the antagonists had no acute effects in the non- measured after different times. These studies revealed that pretreated cells, acute exposure with AM281 and AM630 pro- cAMP levels remained elevated at least 7 days (Controls, 1.3  duced stimulation of cAMP formation in cells that were 0.2 pmol/10 cells; cells stimulated for 2 days, 6.6  1.0 pmol/ chronically pretreated with MAEA and JWH015, respectively. 6 6 10 cells, p  0.001; 3 days, 6.5  1.1 pmol/10 cells, p  0.001; These data indicate that chronic agonist treatment of the 4 days, 5.8  1.0 pmol/10 cells, p  0.001; 7 days, 6.1  1.6 Jurkat cells induces sensitization of CB1 and CB2, resulting 6 6 pmol/10 cells, p  0.001; and 9 days, 2.7  1.2 pmol/10 cells, in a switch from inhibition to stimulation of cAMP forma- p  0.05). Fig. 8G demonstrates that both CB1 and CB2 con- tion. These experiments were performed in naive and IL-4- tribute to the effect on cAMP in an additive manner. Thus, the stimulated Jurkat cells. Again, in line with the previous combined effect of MAEA and JWH015 is approximately dou- results, effects of CB1 and CB2 ligands were observed in bly as strong as the effects of the agonists alone. Also, the effect IL-4-stimulated cells only, whereas in the naive cells only the of THC in stimulated CB1- and CB2-expressing Jurkat cells is effects of CB2 were observed. almost doubly as strong as the effect of the drug in naive CB2- Possible Role of Endogenous Cannabinoids in the Regulation expressing cells. Similar results were obtained with WIN of T Cell Signaling—It was demonstrated that endocannabi- 55,212-2 mesylate, which is another agonist of both receptors. noids are synthesized in lymphocytes and involved in the phys- Together, the cAMP data indicated that activation of both CB1 iological regulation of immune functions (34). Therefore, we and CB2 in T cells induces strong and sustained elevation of next tested the hypothesis that endogenous cannabinoids may DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35455 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 8. Modulation of cAMP formation in T cells by cannabinoids. A–C, time-dependent modulation of cAMP in naive Jurkat cells expressing CB2 (A), IL-4-stimulated Jurkat cells expressing CB1 and CB2 (B), and IL-4-stimulated primary human T cells expressing CB1 and CB2 (C). The cells received a single dose of cannabinoids (THC, 500 nM; MAEA, 500 nM; JWH015, 250 nM) and were incubated with the drugs for up to 48 h. Then cells were harvested, and the amounts of cAMP were determined. The data represent the means  S.E. of at least three independent experiments performed in triplicate. In A, the data sets for incubation with THC and JWH015 for 15 min and for 2 h and longer are significantly different from controls (p 0.01). In B, the data sets for incubation with THC, MAEA, and JWH015 for 15 min and for 2 h and longer are significantly different from controls (p 0.01). In C, the data sets for incubation with THC, MAEA, and JWH015 for 15 min, 4 h, and 24 h are significantly different from controls (p  0.01). D, formation of cAMP relative to different doses off cannabinoids. IL-4-stimulated Jurkat cells were incubated with the drugs for 4 h. Then cAMP was determined. The data represent the means S.E. of at least two independent experiments performed in triplicate. E and F, the cannabinoid-induced cAMP modulation is mediated by G proteins. IL-4-stimulated Jurkat cells were treated i/o with pertussis toxin (PTX, 10 ng/ml) for 16 h, then cannabinoids (see above) were added, and the cells were incubated for either 15 min (E)or2h(F). Then cAMP was determined. G, contribution of CB1 and CB2 to cAMP formation by cannabinoids. Naive Jurkat cells (light gray) and IL-4-stimulated Jurkat cells (dark gray) received a single dose of cannabinoids (see above; WIN, WIN 55,212-2 mesylate, 250 nM) and were incubated with the drugs for 2 h. Then cAMP was determined. The data represent the means  S.E. of at least two independent experiments performed in triplicate. *, p  0.05; **, p  0.01; ***, p  0.001. 35456 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP FIGURE 9. Effect of AM 251 and AM630 on anti-CD3/anti-CD28-triggered IL-2 transcription. Naive Jurkat cells expressing CB2 (light gray) and IL-4- stimulated Jurkat cells expressing CB1 and CB2 (dark gray) were incubated with the cannabinoid antagonists AM281 (500 nM, selective for CB1) and AM630 (500 nM, selective for CB2) for 48 h or left untreated, as depicted in the scheme below. Then cells were activated with anti-CD3/anti-CD28 to induce IL-2 mRNA, according to the scheme. The cells were lysed 4 h after the anti- FIGURE 10. Effect of AM 251 and AM630 on cAMP formation. IL-4-stimu- CD3/anti-CD28 activation for quantitative reverse transcription-PCR. The lated Jurkat cells expressing CB1 and CB2 were incubated with the cannabi- amount of IL-2-specific transcripts relative to that of -actin is plotted. For noid antagonists AM281 (500 nM, selective for CB1) and AM630 (500 nM, selec- easier comparison, the levels of IL-2 mRNA after anti-CD3/anti-CD28 activa- tive for CB2) for 48 h or left untreated as depicted in the scheme below. Then tion were set to 100%. The data represent the means  S.E. of at least three cAMP was determined. The data represent the means  S.E. of at least three independent experiments performed in duplicate. The asterisks define signif- independent experiments performed in triplicate. *, p 0.05; **, p 0.01; ***, icantly different values compared with controls (group 1). †, IL-4-stimulated p  0.001. cells; #, naive cells (significantly different samples compared with the respec- tive anti-CD3/anti-CD28-activated samples (group 3)). A further comparison is indicated by a bracket. One symbol, p  0.05; two symbols, p  0.01; three DISCUSSION symbols, p  0.001. In this report, the question of how cannabinoids inhibit the TCR-mediated production of IL-2 in T lymphocytes was be involved in the regulation of the IL-2 production in activated addressed. In the key step that links the drugs to the lymphocyte Jurkat T cells. If this was the case, blocking of CB1 and CB2 signaling, activation of CB1 and CB2 causes an increase in should increase the IL-2 mRNA levels (Fig. 9). Indeed, we ob- cAMP, which activates PKA. The kinase then activates the Csk/ served significantly increased anti-CD3/anti-CD28-induced Lck pathway, leading to stabilization of the inhibited, Tyr-505- IL-2 mRNA levels in naive Jurkat cells treated with the CB2- phosphorylated state of Lck. Consequently, the sequential phosphorylation of the proximal TCR signaling components selective antagonist AM630 compared with the untreated cells Zap 70, LAT, and MAPK are inhibited. Additionally, the TCR- (Fig. 9, group 5 versus group 3), whereas the CB1-selective mediated activation of the transcription factors NF-B and antagonist AM281 had no effect (group 4 versus group 3). In NFAT, which are pivotal factors for the TCR-triggered induc- IL-4-stimulated cells, treatment with AM630 (group 5 versus tion of IL-2, is inhibited. In this way cannabinoids also inhibit group 3), as well as AM281 (group 4 versus group 3) produced processes downstream of IL-2 like T cell proliferation. elevated IL-2 mRNA levels. When both antagonists were The question as to which receptors mediate the cannabi- applied simultaneously, the effect in the CB1- and CB2-ex- noid-induced inhibition of IL-2 and signaling in activated T pressing cells was significantly stronger than that in the naive cells has been addressed repeatedly but with contradictory CB2-expressing cells (group 6), indicating that both receptors results, as already mentioned in the introduction. Our results are involved in mediating this effect. In terms of cAMP regula- are well in line with those that reported the involvement of CB2 tion, treatment of Jurkat cells with the cannabinoid receptor (12, 13). Notably, here we clearly demonstrated for the first time antagonists resulted in decreased intracellular cAMP con- that CB1 is involved as well, by comparing the effects of various centrations (Fig. 10). In line with other experiments, the agonists in cells expressing either CB2 only (naive T cells) or combination of AM281 and AM630 produced a significantly both CB1 and CB2 (stimulated T cells). Thus, the induction of stronger effect than treatment of the cells with only one sub- functional CB1 can be seen by the lack of effects of CB1 agonists stance, again indicating the involvement of both CB1 and in naive cells and the presence of effects in the stimulated cells. CB2. We next tested whether applying the cannabinoid Moreover, additional evidence for the induction of CB1 in stim- receptor antagonists would alter the basal and anti-CD3/ ulated cells comes from the observation that mixed agonists anti-CD28-activated phosphorylation status of Zap 70, LAT, such as THC and WIN55,212-2 mesylate have significantly and MAPK. However, significant changes were not detect- stronger effects in stimulated cells compared with naive cells able (data not shown). The reason for this might be that (see Figs. 1, 2, 4, 5, and 8–11). These experiments thus clearly relatively small effects, which can be well detected in terms support an earlier study from our group, demonstrating the of cAMP and IL-2 mRNA, cannot be detected easily with the functionality of IL-4-induced CB1 in T cells (9). As to the Western blot method because of the narrow linear range in amount of cannabinoid receptors in T cells, it was shown that which signals can be quantified and because of quick satura- the ratio of CB1 to CB2 is 1:1.5 in peripheral human blood tion of exposed films. (34). In IL-4-stimulated Jurkat cells, this ratio is 1:20. Never- DECEMBER 18, 2009• VOLUME 284 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 35457 Cannabinoid-inhibited T Cell Signaling via cAMP reports showing that THC behaves as a partial agonist/antago- nist for CB1 and CB2 in some cell systems (see e.g. Refs. 36 and 37). Nevertheless, in our hands THC produced similar effects in terms of cAMP formation as WIN55,212-2 mesylate, which is known to activate CB1 and CB2, suggesting that THC acts in an agonistic manner at both receptors in T cells. Notably, CB1 and CB2, which produced the remarkable and long lasting increase in cAMP, belong to the class of G -cou- i/o pled receptors, which are classically associated with a decrease in cAMP levels. Indeed, we observed decreased cAMP levels in the T cells in response to cannabinoids, but only within a short initial time in a range of minutes. This is in good accordance with the bulk of literature for CB1 and CB2 (reviewed in Ref. 5). Most interestingly, at later time points, activation of CB1 and CB2 induced a massive increase in cAMP in Jurkat and primary human T cells that was also mediated by G proteins. In general, G -coupled receptors may not only produce an inhibition of adenylyl cyclase but also a “superactivation” of the enzyme that results in increased cAMP levels (38, 39). Although known for decades, the mechanisms of this phenomenon remain unre- solved. Currently, one model aiming to explain it proposes a switch between G and G protein coupling (40, 41). However, i s we showed that both the initial decrease in cAMP as well as the subsequent increase in cAMP are mediated by pertussis toxin- sensitive G proteins. An additional mechanism that contrib- utes to the modulation of cAMP is the involvement of proteins termed regulators of G protein signaling proteins, which reduce G protein activity (42). Interestingly, we observed that chronic agonist treatment of Jurkat cells induced sensitization of CB1 and CB2, which resulted in a switch from inhibition to stimu- lation of cAMP formation after acute agonist exposure. This FIGURE 11. Chronic cannabinoid receptor agonists induce receptor sen- might be explained by various alterations of cellular functions sitization. Naive Jurkat cells expressing CB2 (A) and IL-4-stimulated Jurkat induced by the receptors, such as induction of adenylyl cyclases, cells expressing CB1 and CB2 (B) were preincubated for 8 h with cannabinoid receptor agonists (MAEA, 500 nM; JWH015, 250 nM), antagonists (AM281, 500 activation of these enzymes by phosphorylation, alterations in nM; AM630, 500 nM), or vehicle at 37 °C. Then cells were washed three times the intracellular calcium concentration, or stimulation of ade- for 5 min with phosphate-buffered saline (0 °C) and then incubated for nylyl cyclases by G protein --subunits, which are known to another 15 min in a 37 °C water bath with cannabinoid receptor agonists, antagonists, or vehicle, as indicated. Then cells were lysed, and cAMP was occur after chronic treatment of G protein-coupled receptors determined. All of the samples are compared with controls (100%) that were and may result in stimulated cAMP formation (see e.g. Refs. 38, identically preincubated for 8 h, washed, and incubated for 15 min with vehi- 39, and 43). Furthermore, it is known that chronic agonist expo- cle. The asterisks define samples that are significantly different from these controls. Secondary comparisons are indicated by brackets. The data repre- sure often results in post-translational modifications of recep- sent the means  S.E. of at least three individual experiments performed in tors, such as multiple phosphorylation events, which may duplicate. *, p  0.05; **, p  0.01; ***, p  0.001. induce conformational changes in the receptors, thereby alter- theless, our results suggest that activation of either receptor ing the affinities and the potencies of agonists and also antago- leads to quantitatively similar effects with regard to inhibition nists. It is discussed that in this way a single ligand of a given of T cell signaling and IL-2 production and induction of cAMP receptor may display a whole spectrum of activity ranging from formation. This might be explained by the fact that the cells agonism to antagonism or inverse agonism (44, 45). This might contain spare CB2 receptors that are not needed for a full effec- explain why AM281 and AM630 stimulated cAMP formation tor response. Moreover, because activation of both receptors in cells that have been pretreated chronically with MAEA simultaneously resulted in almost double-fold effects com- and JWH015, whereas AM281 and AM630 behaved as classic pared with the effects observed after activation of CB1 or CB2 antagonists in nonpretreated cells. Unraveling the detailed alone, our data suggest that the receptors act in an additive mechanisms of cAMP regulation by cannabinoids in T cells will mode. It should be mentioned that several G protein-coupled be a challenging aim for future investigations. It is noteworthy receptors may form heterodimers, which also might be true for that cAMP levels remained elevated for 7 days, which, to our CB1 and CB2 and also can explain additive effects. Although knowledge, has never been reported. This is remarkable in view heterodimerization of CB1 with other receptors has been of results by Massi et al. (46), who found persistent inhibition of reported (reviewed in Ref. 35), to our knowledge, heterodimer- IL-2 production in mice even 7 days after cannabinoid treat- ization of CB1 with CB2 has not been reported thus far but ment. As we will discuss below, there is reason to believe that cannot be excluded here. It should also be noted that there are the increase in cAMP is the key step in the inhibition of T cell 35458 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 •DECEMBER 18, 2009 Cannabinoid-inhibited T Cell Signaling via cAMP signaling by cannabinoids in T cells. Interestingly and similar to cells lies upstream of the TCR. Thus, it can easily be explained our results in immune cells, it was reported for various brain that by interference with cAMP and Lck, cannabinoids can regions that chronic THC increased cAMP levels and PKA modulate TCR-mediated effects (e.g. induced by anti-CD3) but activity (47). It was discussed that this may be one of the mech- not the effects generated by activation of pathways downstream anisms leading to drug tolerance. It was also reported for CB1- of the TCR (e.g. induced by phorbol 12-myristate 13-acetate/ overexpressing HEK293 cells (48, 49) and for CB1 in the brain ionomycin). Therefore, caution is advised when conclusions (50) that these receptors desensitize rapidly, which also may are made that are based solely on experiments, in which T cells contribute to phenomena like tolerance. Although not investi- are not fully activated. For example, it was stated in an earlier gated here, our results and those reported by Massi et al. show- report that CB1 and CB2 were not involved in the inhibition of ing the sustained effects of cannabinoids in T cells suggest that IL-2 production, based on experiments in which cells were only desensitization may play only a minor role during chronic treated with phorbol 12-myristate 13-acetate/ionomycin (15). exposure of T cells to the drugs and/or that mechanisms of However, our experiments, in which T cells were activated in a desensitization may be different in neuronal and immune cells. more physiological way at the site of the TCR, clearly show the Our results suggested that the downstream effects of cAMP involvement of CB1 and CB2. were mediated by PKA. It should be noted that the PKA inhib- Interestingly, treatment of Jurkat T cells with AM281 and itor that we used, cAMPS-Rp, inhibits PKA by preventing the AM630 resulted in decreased cAMP levels and enhanced levels holoenzyme from dissociation, whereas other PKA inhibitors of TCR-induced IL-2. Assuming that the two substances antag- interfere with the phosphorylation process of PKA. This might onize effects mediated by CB1 and CB2, respectively, which has be of importance if the released regulatory subunits have spe- been reported for various effects (reviewed in Ref. 5), our results cial tasks on their own, which cannot be excluded here. In addi- indicate that endogenous cannabinoids are involved in the tion to our cAMP and PKA data, the experiments demonstrat- TCR-triggered IL-2 production. Although their role in the ing the involvement of Csk by down-regulation of the protein physiological regulation of IL-2 production in activated T cells with siRNA, and last but not least the results showing that the remains to be established, disregulation in endogenous canna- Tyr-505-phosphorylated (i.e. inhibited) form of Lck is stabi- binoid production is currently discussed as an important step in lized by cannabinoids favor our hypothesis that inhibition of T the pathophysiology of T cell-mediated autoimmune disorders cell signaling by cannabinoids is transduced via the cAMP/ (55). It should be noted, however, that the effects of AM281 and PKA/Csk/Lck pathway. This is in accordance with other re- AM630 could be explained alternatively, if these compounds ports showing inhibition of T cell signaling by this pathway in acted as inverse agonists and the cannabinoid receptors had other systems (for a review see Ref. 20). some constitutive activity in T cells. In fact, inverse agonistic Remarkably, despite of the marked inhibition of TCR-trig- activity has been reported for both substances, and it is well gered signaling, the cannabinoids only caused inhibition of the known that at least CB1 is constitutively active in other cell activities of the transcription factors NF-B and NFAT but systems (56–58). However, at the moment, the picture in T not AP-1. This is in accordance with earlier observations (4, 16, cells is unclear, and final conclusions cannot be made here. To 17) and may be explained by the fact that cannabinoids can sum up, our results help to explain immunosuppressive effect activate AP-1 via other pathways (51), which thus would coun- of cannabinoid drugs, which may be important for the pharma- teract the inhibitory input of the drugs on AP-1 activity via the cological evaluation of these drugs, e.g. with respect to their use TCR signaling cascade. in neuroinflammatory diseases and T cell-mediated autoim- One of the downstream effects of IL-2 is the induction of T mune disorders like multiple sclerosis. In addition, our results cell proliferation. Therefore, by inhibiting the expression of may indicate a regulatory role for endogenous cannabinoids in IL-2, cannabinoids also inhibit T cell proliferation (52–54). We the production of IL-2 in T cells, which could be, for example, a demonstrated that both CB1- and CB2-selective agonists inhib- prevention of excessive T cell responses. ited T cell proliferation, although higher doses were needed for Acknowledgments—We thank Helga Tischmeyer for excellent techni- this effect than for the inhibition of the T cell signaling cascade cal assistance. The plates and reagents for the cAMP-enzyme-linked and IL-2 inhibition. Our results are very good in line with an immunosorbent assay were a kind gift from Hermann Ammer (Insti- earlier report demonstrating marked effects of THC on prolif- tute of Pharmacology, Toxicology, and Pharmacy, University of eration of primary human T cells only at high (2.5 and 5 M) Munich). In addition, we are very thankful to Hermann Ammer for doses of the drug (54). 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