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Originally published online as doi:10.1189/jlb.0306224 on October 13, 2006

Published online before print October 13, 2006
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(Journal of Leukocyte Biology. 2007;81:336-343.)
© 2007 by Society for Leukocyte Biology

Transcriptional regulation of the cannabinoid receptor type 1 gene in T cells by cannabinoids

Christine Börner*, Volker Höllt*, Walter Sebald{dagger} and Jürgen Kraus*,1

* Department of Pharmacology and Toxicology, University of Magdeburg, Magdeburg, Germany; and
{dagger} Physiologische Chemie II (Biozentrum), University of Würzburg, Würzburg, Germany

1Correspondence: Department of Pharmacology and Toxicology, University of Magdeburg, Leipzigerstr. 44, 39120 Magdeburg, Germany. E-mail: juergen.kraus{at}medizin.uni-magdeburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of cannabinoids (CBs) are mediated by two types of receptors, CB1 and CB2. In this report, we investigated whether CBs regulate gene expression of their cognate receptors in T cells and studied underlying mechanisms in CD4+ Jurkat T cells. Transcription of the CB1 gene was strongly induced in response to {Delta}9-tetrahydrocannabinol (THC), whereas the CB2 gene was not regulated. The induction of CB1 gene expression is mediated by CB2 receptors only, as demonstrated by using the CB1 and CB2 agonists R(+)-methanandamide and JWH 015, respectively, and combinations of THC plus CB1- and CB2-specific antagonists. After activation of CB2 receptors, the transcription factor STAT5 is phosphorylated. STAT5 then transactivates IL-4. Induction of IL-4 mRNA as well as IL-4 protein release from the cells are necessary for the following induction of the CB1 gene. This was demonstrated by using decoy oligonucleotides against STAT5, which blocked IL-4 and CB1 mRNA induction, and by using the IL-4 receptor antagonist IL-4 [R121D,Y124D], which blocked the up-regulation of CB1 gene transcription. Transactivation of the CB1 gene in response to IL-4 is then mediated by the transcription factor STAT6, as shown by using decoy oligonucleotides against STAT6. An increase in CB1-mediated phosphorylation of MAPK in cells prestimulated with CB2-specific agonists suggests up-regulation of functional CB1 receptor proteins. In summary, up-regulation of CB1 in T lymphocytes in response to CBs themselves may facilitate or enhance the various immunomodulatory effects related to CBs.

Key Words: interleukin-4 • STAT • IFN-{gamma} • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of cannabinoids (CBs) are mediated mainly by two types of receptors, CB1 and CB2, which belong to the family of Gi/Go-protein-coupled receptors. CB1 receptors are expressed abundantly in the nervous system. CB2 receptors are expressed predominantly in non-neuronal tissues and therefore, are often called "peripheral" CB receptors [1 ]. However, albeit at lower levels, CB1 receptors are also expressed in the periphery, in particular, in cells of the immune system. In PBMC, e.g., the ratio between CB1 and CB2 receptors is ~1:3, respectively [2 ]. Furthermore, CB1 receptors are expressed in the CD4+ T cell line Jurkat [3 ]. Expression of the CB1 gene is regulated by various stimuli, behavioral conditions, and diseases. For example, up-regulation of CB1 mRNA in the brain was reported in major depression [4 ]. In contrast, brain expression of the gene was found to be down-regulated in Huntington’s disease [5 ] and after chronic alcohol abuse [6 ]. With respect to regulation of CB1 in cells of the immune system, up-regulation of the gene has been found, for example, in splenocytes and Jurkat cells after activation [3 , 7 ] and in Raji B cells and THP-1 monocytes in response to LPS and PMA, respectively [8 ]. It is interesting that regulation of CB1 gene expression by CBs themselves appears to be opposite in cells of the nervous and the immune system. Whereas down-regulation of CB1 mRNA by CBs has been observed in neuronal cells and brain areas [9 , 10 ], an up-regulation of CB1 gene expression is suggested in immune cells. Thus, it was reported that PBMC of marijuana smokers contain higher amounts of the gene’s mRNA than the cells of persons who do not smoke marijuana [2 ]. Cytokines could serve as molecular links, which mediate transcriptional regulation of CB-controlled target genes. Recently, we demonstrated that the µ-opioid receptor gene is such a CB-induced target gene in Jurkat cells and that its induction is mediated via up-regulation and release of IL-4 [11 ]. Regulation of cytokine expression in immunocytes after stimulation with CBs is well established. Most prominently, CBs up-regulate the expression of cytokines, which are typical for Th2 cells, such as IL-4 and IL-5, and down-regulate the expression of cytokines, which are typical for Th1 cells, such as IFN-{gamma}, IL-2, and IL-12 [11 12 13 14 ]. In this report, the homologous regulation of CB receptor genes by CBs themselves and the involvement of the Th cell cytokines IL-4 and IFN-{gamma} were investigated in Jurkat T cells. Whereas CB2 mRNA expression was not influenced, a drastic increase in mRNA for the CB1 gene was found after treatment with CBs, which was mediated by IL-4.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
The T cell line Jurkat E6.1 and the acute lymphoblastic leukemia cell line CEM cells were cultivated in RPMI-1640 medium (Cambrex Bio Science, Verviers, Belgium) supplemented with 10% FCS and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin, Cambrex Bio Science). The isolation of primary human T cells has been described earlier [15 ]. Twenty hours before stimulation experiments, all cells, including controls, received fresh medium with 1% FCS. {Delta}9-Tetrahydrocannabinol (THC; used at 500 nM), R(+)-methanandamide (used at 500 nM), and cycloheximide (used at 5 µg/ml) were purchased from Sigma (Taufkirchen, Germany). JWH 015 (used at 250 nM) and AM 281 and AM 630 (both used at 500 nM) were obtained from Tocris (Bristol, UK). IL-4 and IFN-{gamma} were purchased from R&D Systems (Wiesbaden, Germany) and used at 5 ng/ml [150 World Health Organization (WHO) units] and 100 ng/ml (1000 WHO units), respectively. For assessment of cell viability, 3 x 105 Jurkat cells were cultured under various conditions (see Results). Cell viability was determined after 6, 24, 48, and 72 h using a trypan blue dye (Sigma).

RT-PCR
Total RNA from Jurkat E6.1 cells was extracted using the Nucleospin RNA II kit from Macherey-Nagel (Düren, Germany). One microgram total RNA was used for cDNA synthesis with Moloney murine leukemia virus RT and RNase H Minus (Promega, Mannheim, Germany), diluted to 50 µl. Two microliters cDNA was used for RT-PCR reactions. Quantitative real-time RT-PCR was performed in a total volume of 20 µl on a LightCycler instrument using the LightCycler–Fast Start DNA Master SYBR Green I kit (both from Roche, Mannheim, Germany). Conditions were as as follows: ß-actin, 5'-GGTCCACACCCGCCACCAG-3' and 5'-CAGGTCCAGACGCAGGATGG-3' primers; preincubation, 8 min at 95°C, 50 cycles, 5 s at 95°C, 5 s at 60°C, and 22 s at 72°C. CB1, 5'-CACCTTCCGCACCATCACCAC-3' and 5'-GTCTCCCGCAGTCATCTTCTCTTG-3' primers; preincubation, 8 min at 95°C, 50 cycles, 5 s at 95°C, 5 s at 68°C, and 10 s at 72°C. CB2, 5'-CATGGAGGAATGCTGGGTGAC-3' and 5'-GAGGAAGGCGATGAACAGGAG-3' primers; preincubation, 8 min at 95°C, 50 cycles, 5 s at 95°C, 5 s at 70°C, and 24 s at 72°C. IL-4, 5'-GTCTCACCTCCCAACTGCTT-3' and 5'-GTTACGGTCAACTCGGTGCA-3' primers (located on exon 1 and exon 2 to avoid amplification of the splice variant IL-4 {delta} 2); preincubation, 8 min at 95°C, 50 cycles, 5 s at 95°C, 5 s at 68°C, and 10 s at 72°C. IL-13, 5'-GCTCTCACTTGCCTTGGCGGCT-3' and 5'-TCAGCATCCTCTGGGTCTTCTCGATG-3' primers; preincubation, 8 min at 95°C, 50 cycles, 5 s at 95°C, 5 s at 70°C, and 11 s at 72°C.

Decoy oligonucleotides
The decoy oligonucleotide approach and the sequences, efficiency, and specificity of the oligonucleotides were described in detail in previous publications from our group [11 , 16 17 18 19 ]. Briefly, in the decoy approach, double-stranded oligonucleotides with specific binding sequences for transcription factors are brought into living cells to selectively disrupt the function of these factors. In the cells, transcription factors then interact with an excess of decoy oligonucleotides instead of binding to the natural regulatory motifs of genes. Jurkat E6.1 cells were incubated with decoy oligonucleotides (160 nM) for 16 h prior to stimulation of cells with THC. As demonstrated earlier, Jurkat E6.1 cells take up the decoy oligonucleotides passively. Oligonucleotides were synthesized by Metabion (Martinsried, Germany). Sequences (only the + strand is shown) were: STAT5: 5'-GATCGCATTTCGGAGAAGACG-3'; nSTAT5: 5'-GATCGCATTACGGAGTAGACG-3'; STAT6: 5'-CTAGTTCTTCTCAGAAGCATATGT-3'; nSTAT6: 5'-CTAGTTGATCTCAGATCCATATGT-3'; GATA3: 5'-CTAGAGGAAGTCTTCAGATAAAAAAGATAACAA-3'; nGATA3: 5'-CTAGAGGAAGTCTTCACTTAAAAAACTTAACAA-3'.

Western blot analysis
For STAT5 Westerns, 2 x 106 cells were seeded in RPMI-1640 medium with 1% FCS. After 18 h, cells were treated with THC or vehicle (ethanol). After stimulation for different times, cells were pelleted and lysed. Cell lysis, blotting, and antibody incubations were performed as described [11 ]. Aliquots of 20 µl were separated on a 7% polyacrylamide gel. Primary phospho-STAT5-specific [P-STAT5(Tyr694)-R] and antibodies against unphosphorylated STAT5 (C-17) proteins were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). STAT6 Westerns were performed similarly. Stimulation of cells with IL-4 was for 10 min and THC for 48 h. The IL-4 antagonist was added 1 h prior to stimulation. Primary antibodies were P-STAT6(Tyr641)-R and STAT6 (M-20), both from Santa Cruz Biotechnology. MAPK Westerns were preincubated cells with JWH 015 (250 nM) for 5 days. Stimulation with R(+)-methanandamide (500 nM) was for 5 min (phospho-p44/42-MAPK antibody, E10, Cell Signaling Technology, Frankfurt, Germany; ERK 2 antibody, C-14, Santa Cruz Biotechnology). Secondary antibodies were antirabbit and antimouse Ig from Amersham Biosciences (Braunschweig, Germany).

Statistical analysis
For statistical evaluation, Student’s t-tests were performed. Stars and daggers indicate significantly different values (*/{dagger}, P<0.05; **/{dagger}{dagger}, P<0.01; ***/{dagger}{dagger}{dagger}, P<0.001).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of CB1 gene transcripts in T lymphocytes by CBs and involvement of IL-4
Jurkat E6.1 T cells express low levels of CB1 gene transcripts (Fig. 1A ). As demonstrated by quantitative real-time RT-PCR, incubation of the cells for 48 h with THC, an agonist for CB1 and CB2 receptors, resulted in a drastic increase of mRNA for the CB1 gene (Fig. 1A and 1E) , whereas mRNA for the CB2 gene was not affected (data not shown). Dose-dependent induction of CB1 gene transcripts using THC doses up to 500 nM, which was commonly used in our experiments, is depicted in Figure 1B . Higher doses of THC were not applied, as such doses decreased the number of living cells (Fig. 1C) . Thus, cytotoxic effects of THC at the 48-h time-point, which was the usual incubation time in our experiments, were observed using 2.5 and 12.5 µM THC. In contrast, the survival rate of cells was similar for cells that recieved no THC and cells recieving 100 and 500 nM THC. As an additional control, the survival rate of Jurkat cells in growth media containing different amounts of FSC was tested, as for our experiments, cells were kept up to 72 h in medium containing 1% FCS (Fig. 1D) . Within 72 h, no significant increase in cell death was observed in cells cultivated with 1% or 10% FCS. Furthermore, cell growth was similar in both groups as well (data not shown). In contrast, growth (data not shown) and survival of cells were decreased strongly in cells cultivated without serum (Fig. 1D) . The effect of THC on CB1 gene transcription was mediated via CB2 receptors but not via CB1 receptors, as demonstrated by stimulation of Jurkat cells with R(+)-methanandamide, which preferentially binds to CB1 receptors and the CB2-specific agonist JWH 015 (Fig. 1E) . This finding was supported further by experiments using THC plus a CB1-specific antagonist (AM 281), in which an induction was observed, and THC plus a CB2-specific antagonist (AM 630), in which the induction was not observed (Fig. 1E) . Experiments with cycloheximide indicate that the CB1 gene induction by CBs involves novel protein expression (Fig. 1E) . It has been shown previously that CBs induce expression of IL-4 [11 , 13 , 14 ]. Therefore, a possible in-volvement of IL-4 in the induction of CB1 was investigated. The IL-4 receptor antagonist IL-4 [R121D,Y124D] inhibited induction of the CB1 gene by THC, indicating that release of IL-4 from the cells is essential (Fig. 1E) . When IL-4 was added to the cell medium, the effect of THC could be mimicked, resulting in a similar induction of the CB1 gene mRNA (Fig. 1E) . The Th1 cytokine IFN-{gamma}, which often has antagonistic effects compared with IL-4, had no significant effect on CB1 mRNA levels in Jurkat cells (1.0±0.2 for untreated cells vs. 2.1±0.8 for IFN-{gamma}-treated cells; Fig. 1E ). To demonstrate that the CB2-dependent effect of CBs on transcriptional induction of the CB1 gene is not restricted to the Jurkat T cell model, additional experiments were performed in CEM cells and in primary human T cells (Fig. 2 ). Although not as pronounced as in Jurkat cells, an induction of CB1 transcripts by THC and the CB2-specific agonist JWH 015 was observed in both cell types, indicating that this effect may occur in T cells generally.


Figure 1
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  		Figure 1. Induction of CB1 mRNA in Jurkat E6.1 cells. (A) CB1 induction in response to THC. Cells were stimulated with THC (500 nM, 48 h), and CB1-specific transcripts were detected by quantitative real-time RT-PCR. US, Unstimulated cells. A representative example is shown. The inset shows amplification of the ß-actin housekeeping gene of the same cDNAs. Crossing points are as follows: (CB1/ß-actin) US1, 35.69/27.58; US2, 36.03/27.65; US3, 37.14/28.13; THC1, 28.23/28.45; THC2, 28.3/27.8; THC3, 28.08/28.4. (B) Dose response for CB1 mRNA induction. Cells were stimulated with different concentrations of THC for 48 h, and CB1 transcripts were monitored by quantitative real-time RT-PCR. Results of at least two independent experiments performed in triplicate relative to ß-actin plus SEM are shown. (C) Survival of Jurkat E6.1 cells incubated in medium containing 1% FCS treated with different concentrations of THC as indicated. (D) Survival of Jurkat E6.1 cells incubated in medium containing different concentrations of FCS. (E) CB1 mRNA induction is mediated by CB2 receptors, cycloheximide-sensitive and mediated by IL-4. Jurkat E6.1 cells were stimulated with the CB1/CB2 mixed agonist THC (500 nM), the CB1 agonist R(+)-methanandamide (MAEA; 500 nM), THC plus the CB1-specific antagonist AM 281 (500 nM), the CB2-specific agonist JWH 015 (250 nM), THC plus the CB2-specific antagonist AM 630 (500 nM), THC together with cycloheximide (CX; 5 µg/ml), THC together with the IL-4 antagonist IL-4 [R121D,Y124D] (300 nM), IL-4 (5 ng/ml), and IFN-{gamma} (100 ng/ml) for 48 h. Then, CB1 transcripts were monitored by quantitative real-time RT-PCR. Results of at least two independent experiments performed in triplicate relative to ß-actin plus SEM are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

Figure 2
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  		Figure 2. Induction of CB1 mRNA by THC and JWH 015 in the acute lymphoblastic leukemic cell line CEM and primary human T cells, which were stimulated with THC (500 nM) or the CB2-specific agonist JWH 015 (250 nM), and CB1 transcripts were monitored by quantitative real-time RT-PCR. Results of at least two independent experiments performed in triplicate relative to ß-actin plus SEM are shown. *, P < 0.05; ***, P < 0.001.

 
CB-mediated induction of IL-4
Next, experiments were performed to demonstrate the induction of IL-4 in response to THC. Thus, THC stimulation of Jurkat E6.1 T cells resulted in a 4.7-fold increase in IL-4 mRNA after 48 h (Fig. 3 ). Again, an induction was only observed when cells were stimulated with the CB2 receptor-specific agonist JWH 015 but not with R(+)-methanandamide. The CB2-specific antagonist (AM 630) abolished the THC effect (Fig. 3) . The protein biosynthesis inhibitor cycloheximide does not prevent IL-4 mRNA induction by CBs, indicating that novel protein expression is not needed for this effect (Fig. 3) . The IL-4 antagonist had no effect on the IL-4 mRNA induction by THC, suggesting that there is no positive feedback amplification of IL-4 expression and that IL-4 protein is not involved at this stage (Fig. 3) .


Figure 3
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  		Figure 3. Induction of IL-4 mRNA in Jurkat E6.1 cells, which were stimulated with THC (500 nM), R(+)-methanandamide (500 nM), THC plus AM 281 (500 nM), JWH 015 (250 nM), THC plus AM 630 (500 nM), THC together with cycloheximide (5 µg/ml), and THC together with the IL-4 antagonist IL-4 [R121D,Y124D] (300 nM) for 48 h. RNA and cDNA were prepared and subjected to quantitative real-time RT-PCR monitoring IL-4 transcripts. Results of at least two independent experiments performed in triplicate relative to ß-actin plus SEM are shown. *, P < 0.05; ***, P < 0.001.

 
Time-courses for CB1, IL-4, IL-13, and GATA3 mRNA after stimulation with THC
In the above experiments, transcripts were determined after 48 h of stimulation. To obtain more precise information about gene induction by THC in Jurkat E6.1 cells, time-courses for mRNA for the genes involved were monitored. The induction of CB1 gene mRNA started as late as 40 h after stimulation of Jurkat cells (Fig. 4 ). Next, the mRNA levels for IL-4 and IL-13 during THC stimulation of Jurkat cells were determined. It is known that the IL-4 antagonist binds to the IL-4R{alpha} subunit [20 ]. Consequently, IL-13 signaling, which is also dependent on this receptor subunit, is blocked as well. IL-4 mRNA induction started 32 h after stimulation and peaked at the 40-h time-point (Fig. 4) . In contrast, IL-13 mRNA levels were not influenced significantly by THC (Fig. 4) . It is known that the mRNA of the transcription factor GATA3 is up-regulated after CB and IL-4 stimulation of T cells [11 , 14 ]. Thus, the time-course for THC-mediated GATA3 mRNA was monitored as well. An induction for this mRNA was also observed after 32 h, following the induction of the IL-4 mRNA.


Figure 4
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  		Figure 4. Time-course for mRNA for IL-4, IL-13, GATA3, and CB1 after THC stimulation of Jurkat E6.1 cells, which were stimulated with THC (500 nM) for the indicated times. RNA and cDNA were prepared and subjected to quantitative real-time RT-PCR for transcripts specific for the indicated genes. Quantifications of the results of three independent experiments performed in triplicate normalized to ß-actin are shown. IL-4, IL-13, and GATA3 mRNA levels are shown as fold induction, referring to the left y-axis (at 40 h, P=0.0289 for IL-4, and P=0.0293 for GATA3; at 48 h, P=0.0004 for IL-4, and P=0.0082 for GATA3). CB1 mRNA induction refers to the right y-axis (at 48 h, P=0.0004).

 
Involvement of transcription factors in the THC-mediated induction of IL-4 and CB1
Next, it was investigated which transcription factors mediate induction of the IL-4 and CB1 gene in Jurkat E6.1 cells. Transcription factor decoy oligonucleotides directed against STAT5 completely suppressed transcriptional induction of the CB1 (Fig. 5A ) and IL-4 (Fig. 5B) gene by THC. This indicates that STAT5 acts prior to IL-4 mRNA induction and is involved in the induction of the cytokine, which is in line with earlier observations [11 ]. As controls, mutated (nSTAT5) decoy oligonucleotides were used, in which the binding site for the transcription factor had been destroyed. They had no effect on gene induction. Next, we determined the time-course for CB-mediated phosphorylation of STAT5 in Jurkat E6.1 cells (Fig. 5C) . A weak phosphorylation of STAT5 was observed after ~30 min of stimulation. However, strong phosphorylation of STAT5 appeared after 32 h, which is in line with the delayed increase of IL-4 mRNA beginning 32 h after stimulation of the cells. Next, it was investigated which transcription factors act downstream of the IL-4 induction and contribute to transactivation of the CB1 gene. Typically, IL-4-mediated gene induction in T cells is dependent on the transcription factors STAT6 and GATA3. Up-regulation of GATA3 in response to THC is observed (see Fig. 4 ), and activation of STAT6 by phosphorylation in response to IL-4 and CBs has been demonstrated earlier [11 ]. Thus, transcription factor decoy oligonucleotides directed against both factors were used to investigate their involvement in CB1 transactivation (Fig. 6A ). Decoy oligonucleotides directed against STAT6 significantly inhibited induction of the CB1 gene by THC, whereas decoys for GATA3 did not attenuate CB1 induction. As controls, mutated (nSTAT6 and nGATA3) decoy oligonucleotides had no effect on gene induction. These data indicate that STAT6 but not GATA3 is necessary for transactivation of the CB1 gene. The induction of the IL-4 gene mRNA by THC was not affected by STAT6 and GATA3 decoy oligonucleotides (data not shown), which is in line with our model that they act downstream of IL-4 and are not needed for the IL-4 transactivation. To additionally support our model, activation/phosphorylation of STAT6 by THC and the influence of the IL-4 antagonist thereon were investigated. Indeed, similar to IL-4, THC induced phosphorylation of STAT6, which was blocked by the IL-4 antagonist (Fig. 6B) .


Figure 5
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  		Figure 5. Involvement of STAT5 in THC-mediated induction of IL-4 and CB1 mRNA in Jurkat E6.1 cells. (A) Inhibition of CB1 mRNA induction by STAT5 decoy oligonucleotides. Cells were treated as indicated with THC (500 nM, 48 h) and decoy oligonucleotides (160 nM) with a binding site for STAT5 or a mutated sequence, which does not bind STAT5 (nSTAT5). Then, specific transcripts relative to ß-actin were determined. Results of at least two independent experiments performed in triplicate plus SEM are shown. (B) Inhibition of IL-4 mRNA induction by STAT5 decoy oligonucleotides (see Panel A description). *, P < 0.05; **/{dagger}{dagger}, P < 0.01; ***/{dagger}{dagger}{dagger}, P < 0.001. (C) Phosphorylation of STAT5 by Western blot analysis. Cells were stimulated with THC (500 nM) for the indicated times. Unstimulated samples (–) were incubated for 48 h in medium containing 1% FCS and vehicle. After gel electrophoresis and blotting, the membrane was hybridized with an antibody against P-STAT5 (upper panel). (Lower panel) The same membrane was reprobed for unphosphorylated STAT5 protein. One representative example out of three experiments is shown.

 

Figure 6
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  		Figure 6. Involvement of STAT6 in THC-mediated induction of CB1 mRNA in Jurkat E6.1 cells. (A) Inhibition of CB1 mRNA induction by STAT6 decoy oligonucleotides. Cells were treated as indicated with THC (500 nM, 48 h) and decoy oligonucleotides (160 nM) with binding sites for STAT6, GATA3, or mutated sequences, which do not bind these factors (nSTAT6, nGATA3). Then, specific transcripts relative to ß-actin were determined. Results of at least two independent experiments performed in triplicate plus SEM are shown. *, P < 0.05; **, P < 0.01; ***/{dagger}{dagger}{dagger}, P < 0.001; n.s., not significant. (B) Phosphorylation of STAT6 by Western blot analysis. Cells were stimulated with THC (500 nM, 48 h), IL-4 (5 ng/ml, 10 min), and/or the IL-4 antagonist (1 h preincubation with 300 nM) as indicated. After gel electrophoresis and blotting, the membrane was hybridized with an antibody against P-STAT6 (upper panel). (Lower panel) The same membrane was reprobed for unphosphorylated STAT6 protein. One representative example out of two experiments is shown.

 
Induction of functional CB1 receptor proteins in Jurkat E6.1 cells by a CB2-specific agonist
Assaying CB1-mediated phosphorylation of MAPK was used to test if the increase in CB1 RNA might be followed by an increase in CB1 protein. To avoid cross-effects on CB2 receptors, which are also present on Jurakt cells, the preferential CB1 agonist R(+)-methanandamide was used for activation of the receptor proteins, and the CB2-specific agonist JWH 015 was used for prestimulation of cells. In unstimulated cells, practically no phosphorylation of MAPK was observed (Fig. 7 ), indicating a low expression level of CB1 receptors, which is in line with the low number of transcripts reported above (see Fig. 1A ). In contrast, in cells pretreated with JWH 015, a remarkable phosphorylation of MAPK was observed after R(+)-methanandamide treatment, suggesting that CB1 receptor expression levels in these cells may be higher (Fig. 7) . The phosphorylation of MAPK was blocked by the CB1-specific antagonist AM 281 (Fig. 7) .


Figure 7
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  		Figure 7. Induction of functional CB1 receptor proteins. Jurkat E6.1 cells were preincubated with JWH 015 (250 nM) for 5 days to allow sufficient protein expression or were left untreated (–). Then, medium was removed and replaced by PBS for 1 h. Then, methanandamide-induced phosphorylation of MAPK was assayed by Western blot analysis [R(+)-methanandamide, 500 nM]. The CB1 antagonist AM 281 (500 nM) was added 1 h prior to methanandamide. After gel electrophoresis, proteins were blotted, and membranes were hybridized with antibody against phosphorylated MAPK (P-MAPK). The same membranes were rehybridized with antibody against unphosphorylated ERK2. One representative example out of four experiments is shown. {dagger}{dagger}, P < 0.01; ***, P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we addressed the question of whether CBs regulate the expression of their cognate receptor genes in T cells. It was found that the CB1 gene is up-regulated in response to CBs, that induction and release of IL-4 are necessary for the up-regulation, and that the transcription factor STAT5 is required for the IL-4 mRNA induction. Previously, we reported that the µ-opioid receptor gene is induced by CBs in Jurkat cells, which involves STAT5 activation and IL-4 induction as well [11 ]. To understand mechanisms more precisely, we herein present detailed time-kinetics of mRNA levels and the activation of STAT5 by phosphorylation. It is surprising that phosphorylation of STAT5 in response to CBs is characterized by a relatively long lag phase. Often, phosphorylation of STAT factors is achieved within minutes after stimulation. Recently, it was shown that STAT5 is phosphorylated in response to opioids mediated by µ-opioid receptors [21 ]. This is an interesting finding, as CBs often have qualitatively similar effects on neuronal and immune functions as opioids [22 , 23 ]. Furthermore, the receptors for both classes of substances are structurally similar. Phosphorylation of STAT5 by opioids was monitored within a period of 15 min [21 ]. However, the authors used COS-7 cells, which express neither µ-opioid receptors nor STAT5 but overexpressed transfected proteins. The Jurkat cells that were used in our experiments physiologically express CB2 receptors and STAT5. Physiological levels, however, are likely to be much lower than those in overexpressing cells, in which effects therefore may be observed at earlier time-points. Another reason, which would explain the relatively long time until STAT5 is activated, could be that CBs first induce the release of a certain factor, which is already present in the cells and then again, activates the cells prior to STAT5 activation. This would also explain why no new protein synthesis is needed for IL-4 induction, which follows STAT5 activation. The exact mechanisms underlying phosphorylation of STAT5 in Jurkat cells after CB stimulation remain to be elucidated. The up-regulation of IL-4 transcription is followed by the induction of CB1 transcripts with a gap of ~8 h. During this time, STAT6 is activated in response to IL-4, which is required for CB1 transactivation. The mechanisms by which the CB1 gene is transactivated by STAT6 will be investigated in the future, studying the recently identified promoter sequences of the CB1 gene [24 ]. Finally, to complement RNA data on the induction of the CB1 gene, Western blot experiments were performed, which suggest an increase in CB1 receptor protein in cells that have been pretreated with CB2 agonists. In these cells, CB1-mediated phosphorylation of MAPK as a functional marker for CB1 receptor protein is increased significantly. Generally, the majority of immunomodulatory effects of CBs is mediated via CB2 receptors. It is also interesting that up-regulation of CB1 gene expression in Jurkat cells by CBs is mediated by CB2 receptors. Their activation and the following induction of IL-4 expression are probably the reason why CB1 genes are regulated oppositely in neuronal and immune cells. Although there is increasing evidence that CB2 receptors are also expressed in neuronal cells in the brain, at least at low levels [25 , 26 ], it is possible that these cells do not release IL-4. However, according to our results, only if CB2 expression and the ability of the cells to respond with IL-4 induction are guaranteed could one expect up-regulation of the CB1 gene by CBs. Additional mechanisms may even cause down-regulation of CB1 transcripts in neuronal cells in the brain, as reported earlier [9 , 10 ]. It is interesting that microglia cells are known to express CB2 receptors and potentially respond with cytokine expression [27 , 28 ], which might thus lead to CB2-mediated CB1 induction in the brain. However, to our knowledge, such a mechanism has not been reported so far. In other cells of immune tissue, where the mechanism via CB2 and IL-4 is possible, the CB1 gene may be up-regulated by CBs. This has been suggested earlier in humans [2 ] and confirmed for T cells on a cellular level in this study. Although CB2 receptors are the classical peripheral CB receptors, some immunomodulatory effects of CBs are mediated by CB1 receptors. In particular, endogenous as well as exogenous CBs have been shown to reduce inflammation and inflammatory pain, and it was demonstrated that beside CB2 receptors, also CB1 receptors participate in these neuroimmune interactions [29 30 31 32 ]. Furthermore, it was shown that the CB1 gene is up-regulated in inflammation [29 ]. On a molecular level, it has been shown, e.g., that CBs activate the anti-inflammatory IL-1R{alpha} [33 ] and inhibit proinflammatory mediators such as IL-6 and IL-8 [34 ] and that CB1 receptors are involved therein. This study suggests that anti-inflammatory actions of CBs may be amplified by up-regulation of CB1 receptors in response to CBs themselves and the anti-inflammatory cytokine IL-4, which may help to maintain the physiological balance between pro- and anti-inflammatory processes.


   ACKNOWLEDGEMENTS
 
The study was supported by grants from the Doktor-Robert-Pfleger-Stiftung, Bamberg, Germany (to J. K.), and the German Bundesministerium für Bildung und Forschung, Förderkennzeichen 01ZZ0407 (to C. B.). The authors thank Uwe Lendeckel (Institute of Experimental Internal Medicine, University of Magdeburg, Germany) for generously providing primary human T cells.

Received March 28, 2006; revised September 6, 2006; accepted September 11, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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