Christine Börner

Loss of striatal type 1 cannabinoid receptors is a key pathogenic factor in Huntington’s disease

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntingtons disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntingtons disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntingtons disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntingtons disease, and suggest that activation of these receptors in patients with Huntingtons disease may attenuate disease progression.

Mechanisms of Opioid-Mediated Inhibition of Human T Cell Receptor Signaling

Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid β-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-κB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by μ opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since β-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for β-endorphin in the physiological regulation of T cell activation.

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

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 Δ9‐tetrahydroannabinol (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.

Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to Gi/o proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Δ9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the ζ-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

Comparative analysis of mu-opioid receptor expression in immune and neuronal cells

Morphine modulates neuronal and immune cell functions via mu-opioid receptors. In primary and Jurkat T cells, and Raji B cells mu-opioid receptor transcripts were detected only after stimulation of the cells with IL-4 or TNF-alpha. Moreover, the amount of the induced mu-opioid receptor mRNA in the immune cells was 15 to 200 times less than those in primary cortical and SH SY5Y neuronal cells. Nevertheless, mu-opioid receptor mRNA in immune cells is processed to functional receptors, as demonstrated by morphine-mediated phosphorylation of mitogen activated protein kinase, morphine-mediated up-regulation of IL-4 mRNA and coupling to adenylyl cyclase in Jurkat cells.

STAT6 transcription factor binding sites with mismatches within the canonical 5′‐TTC…GAA‐3′ motif involved in regulation of δ‐ and µ‐opioid receptors

Opioid receptors are expressed in neuronal and immune cells and regulated in response to immunological processes. Herein, we demonstrate up‐regulation of the δ‐opioid receptor gene by interleukin‐4 in immune cells (primary T and polymorphonuclear leukocytes, Jurkat E6 T cells), and in NG 108–15 neuronal cells. We identified an interleukin‐4‐responsive element at nt −671 on the murine gene promoter, to which the transcription factor STAT6 binds, as shown by reporter gene analysis and STAT6/DNA interaction studies in living cells with transcription factor decoy oligonucleotides. STAT6 normally binds to palindromic DNA motifs with a 5′‐TTC…GAA‐3′ core. Notably, the δ‐opioid receptor STAT6 site (5′‐TTC…GGA‐3′) is an imperfect palindrome with a mismatch within this core sequence. A systematic analysis of possible mismatch 5′‐TTC…GAA‐3′ motifs revealed that STAT6 also binds to the sequence 5′‐TTA…GAA‐3′. This motif occurs as a polymorphism in the human µ‐opioid receptor gene ( Kraus et al. 2001  J. Biol. Chem 276, 43901–43908). We show that this mutated element has a significantly reduced STAT6 binding activity which correlates to its reduced interleukin (IL)‐4 inducibility. In contrast, the non‐canonical STAT6 site of the δ‐opioid receptor binds STAT6 with similar high activity as a perfectly palindromic STAT6 site and is strongly inducible by IL‐4.

Analysis of Promoter Regions Regulating Basal and Interleukin-4-Inducible Expression of the Human CB1 Receptor Gene in T Lymphocytes

The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) -3086 to -2490 and nts -1950 to -1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts -648 and -559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt -2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R(+)-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells.

Distinct palindromic extensions of the 5′-TTC…GAA-3′ motif allow STAT6 binding in vivo

STATs (signal transducers and activators of transcription) are transcription factors downstream of cytokine and growth factor signals. All of the seven different STATs bind to regulatory promoter elements with the common core motif 5’‐TTC(N)2‐4GAA‐3’. A key question is how the different STAT factors recognize “their” response elements, that is, what distinguishes for example STAT1 from STAT6 binding sites. In vivo, binding of the different STATs to DNA elements is highly specific and disruption of the genes for the different STAT factors is accompanied with distinct, non‐overlaping phenotypical effects. As a first step towards discrimination of target sequences for the various STATs, we determined requirements for binding sites for STAT6. In functional assays, six sequences were identified. These have palindromic extensions of the core motif in common (underlined): 5’‐TTTCNNNGAAA‐3’, 5’‐CTTCNNNGAAG‐3’, 5’‐TTTCNNNNGAAA‐3’, 5’‐CTTCNNNNGAAG‐3’, 5’‐TTCCNNGGAA‐3’ and 5’‐TTCANNTGAA‐3’. Different approaches and mutational analysis demonstrated the functionality of these sequences and high specific binding to STAT6. (I) These elements mediate transcriptional induction by interleukin‐(IL)‐4, IL‐13, IL‐15, and platelet‐derived growth factor. (II) When used as “decoy” oligonucleotides, they bind STAT6 and disrupt its function in vivo, attenuating (a) STAT6/IL‐4‐mediated reporter gene transcription and (b) STAT6/IL‐4‐mediated induction of μ‐opioid receptor mRNA of Raji cells.

Activation of human T cells induces upregulation of cannabinoid receptor type 1 transcription.

Objective: Effects of cannabinoids are mediated by CB1 and CB2 receptors. In addition to neuronal effects, cannabinoids are potent modulators of immune functions. In this report, we investigated whether the transcription of these receptors is regulated after activation of T lymphocytes. Methods: CB1- and CB2-specific mRNA of primary human peripheral blood T cells and cells of the human T cell line Jurkat was measured by quantitative real-time RT-PCR in response to CD3/28. Using the decoy oligonucleotide approach, transcription factors involved in the regulation were determined. A promoter analysis was performed using transient transfection of chloramphenicol acetyl transferase reporter gene constructs in Jurkat cells. Results: Activation of human T cells caused an induction of CB1 mRNA expression in primary human T cells (8-fold) and Jurkat cells (29-fold). In contrast, CB2 transcription was not regulated. The CD3/28-mediated upregulation of CB1 involves the transcription factors AP-1, NFĸB and NFAT. Furthermore, 2,490 bp of the CB1 promoter mediated inducibility in response to CD3/28. Conclusions: The upregulation of CB1 in activated T cells, together with the constitutive expression of CB2, enables cellular responses to cannabinoids mediated by both receptor subtypes. It may thus contribute to the understanding of the various modulatory effects of cannabinoids on activated T cells.

T-Cell Receptor/CD28-Mediated Activation of Human T Lymphocytes Induces Expression of Functional μ-Opioid Receptors

Opiates function as immunomodulators, partly by their effects on T cells. Opioids act via μ-, δ-, and κ-opioid receptors, among which the μ-type is of particular interest, because morphine-like opioids preferentially bind to it. Here we report that μ-opioid receptor mRNA was induced after CD3/28-mediated activation of primary human T lymphocytes and Jurkat T cells, neither of which expresses the gene constitutively. Moreover, a reporter gene construct containing 2624 base pairs of the μ-opioid receptor promoter was transactivated by CD3/28 stimulation. Transcriptional induction of the μ-opioid receptor gene was mediated by activator protein-1 (AP-1), nuclear factor-κB, and nuclear factor of activated T cells (NFAT). NFAT was found to bind to three sequences of the μ-opioid receptor promoter, located at nucleotides -1064, -785, and -486. Although the -486 element is in close proximity to a putative AP-1 site, there was no evidence for a combined AP-1/NFAT site. Furthermore, we demonstrated that the induction of inter-leukin-2 mRNA and protein in activated T cells was inhibited by morphine in cells, in which μ-opioid receptors had been induced by CD3/28 monoclonal antibodies (mAbs), and that this effect was blocked by the μ-opioid receptor-specific antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. CD3/28 mAb-induced interleukin-2 transcription was also inhibited by the opioids fentanyl and loperamide. This indicates that the induced μ-opioid receptor mRNA is translated into functional receptor protein. Furthermore, a μ-opioid receptor-enhanced green fluorescent protein-fusion protein was localized in membranes of Jurkat cells and internalized in response to [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin but not morphine. In conclusion, these data emphasize the role of opioids in the modulation of T lymphocyte signaling.