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The Journal of Immunology, 2004, 172: 2341-2351.
Copyright © 2004 by The American Association of Immunologists

Immunosuppressive Activity of Endovanilloids: N-Arachidonoyl-Dopamine Inhibits Activation of the NF-{kappa}B, NFAT, and Activator Protein 1 Signaling Pathways 1

Rocío Sancho*, Antonio Macho*, Laureano de La Vega*, Marco A. Calzado*, Bernd L. Fiebich{dagger},{ddagger}, Giovanni Appendino§ and Eduardo Muñoz2,*

* Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba, Facultad de Medicina, Cordoba, Spain; {dagger} Neurochemistry Research Group, Department of Psychiatry, University of Freiburg Medical School, Germany; {ddagger} VivaCell Biotechnology, Denzlingen, Germany; and § Università degli Studi del Piemonte Orientale, Dipartimento de Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Novara, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endogenous N-acyl dopamines such as N-arachidonoyldopamine (NADA) and N-oleoyldopamine have been recently identified as a new class of brain neurotransmitters sharing endocannabinoid and endovanilloid biological activities. As endocannabinoids show immunomodulatory activity, and T cells play a key role in the onset of several diseases that affect the CNS, we have evaluated the immunosuppressive activity of NADA and N-oleoyldopamine in human T cells, discovering that both compounds are potent inhibitors of early and late events in TCR-mediated T cell activation. Moreover, we found that NADA specifically inhibited both IL-2 and TNF-{alpha} gene transcription in stimulated Jurkat T cells. To further characterize the inhibitory mechanisms of NADA at the transcriptional level, we examined the DNA binding and transcriptional activities of NF-{kappa}B, NF-AT, and AP-1 transcription factors in Jurkat cells. We found that NADA inhibited NF-{kappa}B-dependent transcriptional activity without affecting either degradation of the cytoplasmic NF-{kappa}B inhibitory protein, I{kappa}B{alpha}, or DNA binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by NADA in stimulated cells. In addition, NADA inhibited both binding to DNA and the transcriptional activity of NF-AT and AP-1, as expected from the inhibition of NF-AT1 dephosphorylation and c-Jun N-terminal kinase activation in stimulated T cells. Finally, overexpression of a constitutively active form of calcineurin demonstrated that this phosphatase may represent one of the main targets of NADA. These findings provide new mechanistic insights into the anti-inflammatory activities of NADA and highlight their potential to design novel therapeutic strategies to manage inflammatory diseases.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Numerous experimental models (1) have highlighted the key role of T cells in the development of human inflammatory brain diseases, such as multiple sclerosis, viral encephalitis, and possibly post-traumatic processes. Endocannabinoids are a class of lipid mediators found in several tissues and based on a polyunsaturated fatty acid amide or ester motifs (2). Among them, anandamide (arachidonoylethanolamide (AEA)) 3 and 2-arachidonoylglicerol (2-AG) act as mediators in the brain and peripheral tissues, mainly through the activation of brain (CB1) and peripheral (CB2) cannabinoid receptors. Although AEA preferentially binds to CB1, 2-AG is equipotent at both receptor subtypes. AEA can also interact with the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) (also called VR-1) (3, 4). This nonselective cation channel is gated by noxious heat, extracellular protons, and a structurally heterogeneous group of compounds named vanilloids, encompassing also the endogenous ligands N-arachidonoyldopamine (NADA) and N-oleoyldopamine (OLDA) (5, 6, 7). Although NADA binds to both human and rat TRPV1 (6, 8) and CB1 receptor (9), OLDA is a capsaicin-like lipid with full TRPV1 agonist activity, but devoid of affinity for CB receptors (7).

Over the past few years there has been a growing awareness that the endocannabinoid system is exceedingly complex. Indeed, its inherent complexity is further increased by the cross-talk between CB1 and TRPV1 signaling pathways and by the fact that CB- and TRPV1-independent biological activities have been demonstrated for the archetypal endocannabinoids AEA and 2-AG (10, 11, 12). In addition to the effects on peripheral and central nervous systems, endocannabinoids show anti-inflammatory and immunomodulatory activities (13) that, despite their pharmacological relevance, have to date remained mechanistically elusive.

The signal transduction pathways triggered by activation of the TCR/CD3 complex in T cells lead to the immediate activation of transcription factors that regulate a variety of activation-associated genes. Many of them are cytokines and surface receptors that play an important role in coordinating the immune response (14). The signal transduction pathways involved in T cell activation are initiated by the clustering of lipids rafts at the cell surface, with formation of a supramolecular activation complex localized at the Ag-induced immunological synapse (15). Several studies have demonstrated that the presence or absence of specific signaling proteins, such as Cot/Tpl-2, Vav-1, and protein kinase C{theta} (PKC{theta}), within lipids rafts controls lymphocyte signaling (16). Activation of PLC{gamma} by specific tyrosine kinases at the supramolecular activation complex results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the generation of inositol 1,4,5-triphosphate and diacylglycerol. Although inositol 1,4,5-triphosphate mobilizes Ca2+ from intracellular stores, diacylglycerol mediates activation of the PKC family members (17) and the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway (18). As a consequence of an increase of intracellular Ca2+ levels, several signaling pathways are activated (19). For instance, calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is activated and subsequently dephosphorylates NFAT, allowing its nuclear shuttling (20). This transcription factor was first described as an inducible regulatory complex critical for transcriptional induction of IL-2 in activated T cells (21), but was subsequently shown to regulate the transcription of many other genes, including cytokines (IL-4, IFN, TNF, GM-CSF) (22, 23, 24, 25), cell surface receptors such as Fas ligand and CD40 ligand (26, 27), and regulatory enzymes such as cyclooxygenase-2 (28). The NFAT family of transcription factors includes four classical members, NFAT1, NFAT2, NFAT3, and NFAT4 (29), among which NFAT1 and NFAT2 are preferentially expressed in peripheral T cells (30). In the nucleus, NFAT binds to the DNA either alone or in conjunction with AP-1 proteins (31). Nevertheless, the coordinate induction and activation of the transcription factors NFAT, NF-{kappa}B, and AP-1 are required to regulate cytokine gene expression (14).

Stimulation via the TCR-CD3 complex alone is sufficient for NFAT activation (32), but it is insufficient for activation of NF-{kappa}B and AP-1. Thus, a second signal mediated by the CD28 receptors is required for the induction of NF-{kappa}B and AP-1 in Ag-stimulated T cells (33). Appropriate CD28 signaling is provided by the costimulatory molecules B7-1/B7-2 present at the cell surface of APCs (34). The transcription factor NF-{kappa}B is one of the key regulators of genes involved in the immune/inflammatory response as well as in survival from apoptosis (35). NF-{kappa}B is an inducible transcription factor made up of homo- and heterodimers of p50, p65 (RelA), p52, RelB, and c-Rel subunits that interact with a family of inhibitory I{kappa}B proteins, of which I{kappa}B{alpha} is the best characterized (36). In most cell types these proteins sequester NF-{kappa}B in the cytoplasm by masking its nuclear localization sequence. Ag stimulation in T cells triggers a signaling pathway that results in the phosphorylation, ubiquitination, and subsequent degradation of I{kappa}B proteins, resulting in the translocation of NF-{kappa}B from the cytoplasm to the nucleus. Phosphorylation of I{kappa}B{alpha} at serines 32 and 36 is a key step mediated by I{kappa}B kinases (IKKs) (37), and the activation of IKK by different stimuli requires distinct signaling proteins such as PKC{theta}, Cot-2, and AKT/PKB kinases, which are recruited to the Ag-induced immunological synapse (35, 38). In addition to the control of NF-{kappa}B activity exerted at the nuclear translocation level, there is increasing evidence for another complex level of regulation that is mediated by direct phosphorylation of the p65 subunit trans-activation domain (39). This phosphorylation regulates not only the DNA-binding and trans-activation properties of p65, but also the interactions between the transcription factor and the regulatory proteins. To date, several kinases, such as p38 kinase (40), mitogen- and stress-activated protein kinase-1 (41), PKC{zeta} (42), casein kinase II (43), IKK{beta} (44), and the Ca2+/calmodulin-dependent protein kinase IV (45), have been described to phosphorylate the p65 subunit, indicating the complexity of this second level of NF-{kappa}B regulation. In addition, it has been recently shown that IKK{alpha} translocates to the nucleus, where it phosphorylates histone H3, which is required for optimal NF-{kappa}B-dependent gene transcription (46, 47).

The finding that both endocannabinoids and exogenous vanilloids are involved in regulation of the immune response (13, 48) prompted us to investigate the immunosuppressive effects of the endovanilloids NADA and OLDA in the Ag-induced activation events in primary T cells. We disclose in this report that along with NADA-mediated inhibition of IL-2 and TNF-{alpha} gene transcription, there is inhibition of the signaling pathways mediating the activation of transcription factors NF-{kappa}B, NFAT, and AP-1, which are known to play a critical role in the immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

Jurkat cells (American Type Culture Collection, Manassas, VA) were maintained in exponential growth in RPMI 1640 (Life Technologies, Barcelona, Spain) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM HEPES, and antibiotics (Life Technologies). Anti-I{kappa}B{alpha} mAb 10B was a gift from R. T. Hay (Centre of Biomolecular Sciences, St. Andrews, Scotland), the mAb anti-tubulin was purchased from Sigma-Aldrich (St. Louis, MO), the rabbit polyclonal anti-NFAT1 (49) and anti-p65 (1226) were gifts from J. M. Redondo (CBM, Madrid, Spain) and A. Isräel (Institute Pasteur, Paris, France), respectively. The anti-phospho-ERK1/2 (sc-7383) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); the mAbs anti-phospho-p38 (9211S), anti-phospho-c-Jun N-terminal kinase (JNK; 9255S), anti-phospho-p65 (3031S), and anti-phospho-I{kappa}B{alpha} were obtained from New England Biolabs (Hitchin, U.K.). NADA was purchased from Sigma-Aldrich. 2-AG was obtained from Alexis (Bingham, U.K.). OLDA was synthesized as previously described (7). [{gamma}-32P]ATP (3000 Ci/mmol) was purchased from ICN Pharmaceuticals (Costa Mesa, CA). All other reagents were obtained from Sigma-Aldrich.

Plasmids

The AP-1-luciferase (AP-1-Luc) plasmid was constructed by inserting three copies of an SV40 AP-1 binding site into the Xho site of pGL-2 promoter vector (Promega, Madison, WI), the NFAT-Luc plasmid that contains three copies of the NFAT binding site of the IL-2 promoter fused to the luciferase gene (50). The {kappa}B factor (KBF)-Luc contains three copies of the MHC enhancer {kappa}B site upstream of the conalbumin promoter, followed by the luciferase gene (51). The IL-2-Luc (-326 to +45 of the IL-2 promoter) and the TNF-Luc (-1185 pTNF-{alpha}-Luc) plasmids were previously described (50, 52). The plasmid pEF-BOS trunk-Cot, containing the truncated active form of the Cot kinase (53), was obtained from S. Alemany (CSIC, Madrid, Spain). The expression plasmid pSR{alpha}-{Delta}CaM-AI encodes a truncated form of a murine calcineurin catalytic subunit that has Ca2+-independent and constitutive phosphatase (54). The expression vector for IKK{beta} has been described previously (10). The galactosidase 4 (Gal4)-Luc reporter plasmid includes five Gal4 DNA binding sites fused to the luciferase gene (55). The Gal4-hNFAT1 contains the first 1–415 aa of human NFAT1 fused to the DNA binding domain of yeast Gal4 transcription factor and was previously described (28). The Gal4-p65 contains the C-terminal region of the human p65 (aa 286–551) fused to the Gal4 binding domain and was obtained from M. L. Schmitz (University of Bern, Bern, Switzerland). The Gal4-c-jun (wild type) and Gal4-VP16 have been previously described (55, 56).

Isolation of human PBMC and T cell proliferation assays

Human PBMC from healthy adult volunteer donors were isolated by centrifugation of venous blood on Ficoll-Hypaque density gradients (Amersham Pharmacia Biotech, Piscataway, NJ). Cells (105) were cultured in triplicate in 96-well, round-bottom microtiter plates (Nunc, Roskilde, Denmark) in 200 µl of complete medium and stimulated with staphylococcal enterotoxin B (SEB; 1 µg/ml) in the presence or the absence of increasing concentrations of NADA, OLDA, or 2-AG. The SEB activation model was used because this superantigen is presented by B cells and macrophages and activates T cells through TCR and costimulators. The cultures were conducted for 3 days and pulsed with 0.5 µCi [3H]TdR/well (ICN Pharmaceuticals) for the last 12 h of culture. Radioactivity incorporated into DNA was measured by liquid scintillation counting.

Cytofluorometric analyses of cell surface Ag and cell cycle

For cell cycle analyses and measurement of CD25 expression, PBMC (106/ml) were stimulated with SEB (1 µg/ml) in 24-well plates in a total volume of 2 ml of complete medium for 48 h in the presence or the absence of different concentrations of NADA or OLDA. CD25 cell surface fluorescence was measured using a specific mAb and was analyzed by flow cytometry in an EPICS XL flow cytometer (Coulter, Hialeah, FL). For DNA profile analyses, cells were washed in PBS, fixed in ethanol (70%; 24 h at 4°C), followed by RNA digestion (RNase A, 50 U/ml) and propidium iodide (20 µg/ml) staining, and were analyzed by cytofluorometry. Ten thousand gated events were collected per sample, and the percentage of cells in every phase of the cell cycle was determined. The frequency of cells that had undergone chromatinolysis was calculated by determining the sub-G0/G1 fraction.

Transient transfections and Luc assays

Jurkat cells (107/ml) were transiently transfected with the indicated plasmids. The transfections were performed using Lipofectamine reagent (Life Technologies), according to the manufacturer’s recommendations, for 24 h. After incubation with NADA for 30 min, transfected cells were stimulated for 6 h as indicated. Then the cells were lysed in 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol. Luciferase activity was measured using an Autolumat LB 953 (Berthold, Nashua, NJ) following the instructions of the luciferase assay kit (Promega), and the protein concentration was measured by the Bradford method. The background obtained with the lysis buffer was subtracted in each experimental value, and the specific trans-activation was expressed as the fold induction over untreated cells. All experiments were repeated at least four times.

Western blots

Jurkat cells (1 x 106 cells/ml) were stimulated with PMA (20 ng/ml) and ionomycin (0.5 µg/ml) for NFAT1 detection or with PMA and PHA (1 µg/ml) for p65, JNK, ERK1/2, p38, and I{kappa}B{alpha} proteins in the presence or the absence of NADA for the indicated period of time. Cells were then washed with PBS, and proteins were extracted in 50 µl of lysis buffer (20 mM HEPES (pH 8.0), 10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 0.5 mM Na3VO4, 5 mM NaFl, 1 mM DTT, 1 µg/ml leupeptin, 0.5 µg/ml pepstatin, 0.5 µg/ml aprotinin, and 1 mM PMSF) containing 0.5% Nonidet P-40. The protein concentration was determined by the Bradford assay (Bio-Rad, Richmond, CA), and 30 µg of proteins were boiled in Laemmli buffer and electrophoresed in 10% SDS-polyacrylamide gels (or 6% SDS-polyacrylamide for NFAT detection). Separated proteins were transferred to nitrocellulose membranes (0.5 A at 100 V, 4°C) for 1 h. Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C, and immunodetection of specific proteins was conducted with primary Abs using an ECL system (Amersham, Little Chalfont, U.K.).

Isolation of nuclear extracts and mobility shift assays

Jurkat cells (106/ml) were treated with the agonists in complete medium as indicated. Cells were then washed twice with cold PBS, and proteins from total cell extracts (for NF-{kappa}B and AP-1 binding) or nuclear extracts (for NFAT binding) were isolated as previously described (48). The protein concentration was determined by the Bradford method (Bio-Rad). For the EMSA, double-stranded oligonucleotides containing the consensus sites for NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGG-3'; Promega), NFAT (5'-GATCGGAGGAAAAACTGTTTCATACAGAAGGCGT-3'; distalNFAT site of the human IL-2 promoter), and AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3'; Promega) were end-labeled with [{gamma}-32P]ATP. The binding reaction mixture contained 3 µg of nuclear extract (or 15 µg of total extracts), 0.5 µg of poly(dI-dC) (Amersham Pharmacia Biotech), 20 mM HEPES (pH 7), 70 mM NaCl, 2 mM DTT, 0.01% Nonidet P-40, 100 µg/ml BSA, 4% Ficoll, and 100,000 cpm of end-labeled DNA fragments in a total volume of 20 µl. When indicated, 0.5 µl of rabbit anti-NF-AT1 or preimmune serum was added to the standard reaction before addition of the radiolabeled probe. For cold competition, a 100-fold excess of the double-stranded oligonucleotide competitor was added to the binding reaction. After 30-min incubation at 4°C, the mixture was electrophoresed through a native 6% polyacrylamide (4% in the case of NFAT) gel containing 89 mM Tris-base, 89 mM boric acid, and 2 mM EDTA. Gels were pre-electrophoresed for 30 min at 225 V and then for 2 h after loading the samples. These gels were dried and exposed to x-ray film at -80°C.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of T cell proliferation by endovanilloids

We have recently shown that exogenous vanilloids, such as capsaicin and capsiate, affect T cell functions in a TRPV1-independent way (48). Thus, we studied the effects of the endovanilloids NADA and OLDA as well as the endocannabinoid 2-AG on several T cell activation events. In Fig. 1 it is shown that DNA synthesis measured by [3H]TdR uptake in SEB-stimulated T cells was markedly inhibited in a concentration-dependent manner by both OLDA and NADA; the latter was more effective at inhibiting T cell proliferation. In these experiments 2-AG (up to 5 µM) was not found to affect SEB-induced DNA synthesis.



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  		FIGURE 1. Effects of NADA, OLDA, and 2-AG on T cell proliferation. Human PBMCs were stimulated with SEB (1 µg/ml) in the presence or the absence of increasing concentrations of NADA, OLDA, or 2-AG for 72 h. [3H]thymidine incorporation was measured by liquid scintillation counting and is represented as the mean disintegrations per minute ± SE of three different experiments. (mean ± SE in untreated cells, 12,567 ± 1,548 dpm)

 
Cell cycle analysis in unstimulated T cells showed that the cells remained largely in the G0/G1 phase of the cell cycle. Three days after activation with SEB, T cells were full cycling and progressed through the S, G2, and M phases of the cell cycle (20.7% of the cells), whereas pretreatment with either NADA (2.5 µM) or OLDA (5 µM) almost completely prevented entry of the cells into the S phase of the cell cycle (Fig. 2A). Interestingly, no significant differences were found in the percentage of hypodiploid cells (sub-G0/G1) between SEB-stimulated and SEB- plus endovanilloid-stimulated cells. These results indicate that at the doses used, neither NADA nor OLDA induced cytotoxicity or apoptosis in primary T cells. Next, the effect of endovanilloids on cell surface expression of the activation marker CD25 was studied in SEB-stimulated primary T cells. In Fig. 2B it is shown that both endovanilloids greatly inhibit the percentage of cells expressing the CD25 marker at the cell surface.



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  		FIGURE 2. Effect of NADA on the progression of cell cycle and CD25 expression. A, PBMCs were pretreated with either NADA (2.5 µM) or OLDA (µM) and stimulated with SEB (1 µg/ml) for 72 h. The percentages of cells entering the S and G2/M phases of the cell cycle are indicated. The results are representative of four independent experiments. B, Effects of NADA and OLDA on SEB-induced cell surface expression of CD25. PMBCs were treated as indicated for 48 h, and the expression of CD25 was detected by flow cytometry. The numbers represent the percentage of CD25+ cells and are representative of three different experiments.

 
Effect of NADA on cytokine promoter activity

IL-2 and TNF-{alpha} expression is regulated mainly at the transcriptional level. To evaluate whether the inhibitory effects of NADA on T cell activation and proliferation were mediated by the inhibition of cytokines at the transcriptional level, we investigated the regulation of IL-2 and TNF-{alpha} promoters in Jurkat cells transiently transfected with the promoter reporter plasmids IL-2-Luc and TNF-{alpha}-Luc. After transfection, cells were preincubated with NADA for 30 min, activated with PMA plus PHA for 6 h, and tested for Luc activity. NADA efficiently inhibited PMA- plus PHA-induced luciferase expression driven by both IL-2 and TNF-{alpha} promoters in a dose-dependent manner (Fig. 3, A and B). The inhibitory effects of NADA were not due to an interference with the transcriptional machinery or with the in vitro activity of the luciferase enzyme, as NADA did not affect Luc activity in Jurkat cells transfected with a plasmid encoding the Luc gene under control of the CMV promoter (Fig. 3C). The transcriptional activity of both IL-2 and TNF-{alpha} genes depends on the coordinated activation of several transcription factors, including NF-AT, NF-{kappa}B, and AP-1 families. Thus, we evaluated the effect of NADA on the transcriptional activity of those factors by using luciferase reporter constructs under the control of minimal promoters containing binding sites of each of them. Activation by PMA plus PHA increased the luciferase gene expression driven by these promoters in Jurkat cells, and we found that NADA effectively inhibited each of these promoters in a dose-dependent manner, with NF-AT being the most sensitive transcription factor to the inhibitory activity of NADA (Fig. 4).



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  		FIGURE 3. Effect of NADA on TNF-{alpha} and IL-2 promoter activity. A and B, Jurkat T cells transfected with TNF-{alpha} (A) or IL-2 (B) promoter Luc reporter plasmids were treated for 30 min with increasing concentrations of NADA and then stimulated with PMA (20 ng/ml) plus PHA (1 µg/ml) for 6 h, and Luc activity was measured in the cell lysates. Results are the mean ± SE of three determinations, expressed as the fold induction (observed experimental relative light units/basal relative light units in the absence of any stimuli). C, NADA did not affect Luc activity in Jurkat cells transfected with a CMV-Luc construct.

 


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  		FIGURE 4. Regulation of transcription factor-mediated trans-activation by NADA. A–C, Jurkat T cells were transiently transfected with the Luc reporter plasmid KBF-Luc (A), NFAT-Luc (B), or AP-1-Luc (C), as described in Materials and Methods. Cells were preincubated for 30 min with NADA at the concentrations indicated before stimulation with PMA (20 ng/ml) plus PHA (1 µg/ml) for 6 h. Luc activity was measured, and the results are the mean ± SE of three determinations, expressed as the fold induction (observed experimental relative light units/basal relative light units in the absence of any stimuli).

 
NADA inhibits phosphorylation and transcriptional activity of the NF-{kappa}B p65 subunit in stimulated Jurkat cells

The signaling pathways that activate NF-{kappa}B include a complex activation of regulatory kinases resulting in the phosphorylation and degradation of the I{kappa}B proteins and nuclear translocation of NF-{kappa}B (35). In addition to this pathway, a second level of NF-{kappa}B activation involves the phosphorylation of p65 and subsequent stimulation of NF-{kappa}B trans-activation (41, 57). Thus, to investigate the level at which NADA exerted its inhibitory effect on NF-{kappa}B activation, we stimulated Jurkat cells with PHA and PMA for various times in the presence or the absence of NADA (2.5 µM). Proteins from total cell extracts were analyzed for NF-{kappa}B DNA binding activity by EMSA, and steady state levels of I{kappa}B{alpha} were determined by Western blot. The kinetic experiments revealed a clear increase in NF-{kappa}B binding to DNA after 15 min of stimulation, which was maintained through the time of stimulation (Fig. 5A). The increased DNA binding was concomitant with degradation of I{kappa}B{alpha}, which was clear after 15 min of PHA plus PMA stimulation and recovered after 45 min of stimulation. However, in the presence of NADA, I{kappa}B{alpha} phosphorylation and degradation were not affected in the stimulated cells. Even the recovery of I{kappa}B{alpha} protein to the basal levels, which also depends on NF-{kappa}B activation (58), was delayed in the presence of NADA, which did not affect the steady state level of {alpha}-tubulin (Fig. 5B). Moreover, NF-{kappa}B activation induced by overexpression of IKK{beta} was inhibited by NADA in a concentration-dependent manner (data not shown). The DNA binding specificity was studied by supershift experiments with specific anti-p50 and anti-p65 (RelA) Abs and by cold competition experiments with unlabeled competitors, and the heterodimer p50/p65 was identified as the main complex (data not shown) (10, 48). To determine whether NADA affected the phosphorylation status of the p65 trans-activation domain in Jurkat cells, we stimulated the cells with PMA plus PHA for 30 min in the presence or the absence of increasing concentrations of NADA. As expected, the nuclear NF-{kappa}B binding activity to DNA was not modified by the presence of NADA at concentrations between 1 and 5 µM (Fig. 5C). However, Western blot determination of p65 phosphorylation revealed that NADA clearly inhibited PMA- plus PHA-dependent p65 phosphorylation (serine 536) in a concentration-dependent manner (Fig. 5D). To further analyze whether NADA inhibits directly p65 transcriptional activity, we performed cotransfection experiments using Gal4-p65, a fusion protein between the trans-activation domain of p65 (aa 286–551) and the DNA binding domain of the yeast Gal4 trans-activator, together with a reporter plasmid containing the Luc gene under the control of a Gal4-responsive element (Gal4-Luc). This system has the advantage that the Gal4 trans-activator fusion protein is exclusively nuclear and thus is regulated independently of I{kappa}B (59). The results presented in Fig. 5E revealed that transcriptional activity of Gal4-p65 was increased (~1.6-fold) upon treatment of the cells with PMA plus PHA, and this induction was inhibited by the presence of NADA in a concentration-dependent manner. Pretreatment with NADA did not affect the Luc activity induced by the fusion protein Gal4-VP16. In addition, we found that NADA inhibited the transcriptional activity of Gal4-p65 in unstimulated cells, suggesting that the basal phosphorylation activity of p65 could also be inhibited by NADA (data not shown).



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  		FIGURE 5. Effect of NADA on the NF-{kappa}B activation pathway. A and B, Jurkat T cells were incubated with NADA for 30 min, treated with PHA and PMA for the indicated times, and then tested for NF-{kappa}B binding (A) and for I{kappa}B{alpha} phosphorylation and degradation and {alpha}-tubulin expression (B). C and D, Effect of NADA on p65 phosphorylation and transcriptional activity. Jurkat T cells were incubated for 30 min with increasing concentrations of NADA and then stimulated with PMA and PHA for 45 min, and total protein was extracted. NF-{kappa}B DNA binding was determined by EMSA (C), and the steady state of p65 and p65-phosphorylated (Ser536) forms of the protein was analyzed by Western blot (D). E, Jurkat T cells were transiently cotransfected with the plasmids Gal4-p65 and pGal4-Luc or with the plasmid VP-16-Gal4 and pGal4-Luc. Twenty-four hours after transfection, the cells were incubated for 30 with increasing concentrations of NADA and stimulated with PMA plus PHA for 6 h, and Luc activity was measured. Results are the mean ± SE of three different experiments and are presented as relative light units.

 
NADA inhibits dephosphorylation and transcriptional activity of NFAT

The regulation of IL-2 gene transcription is cell type specific and requires the coordinate activation of transcription factors such as NF-{kappa}B, NFAT, and AP-1. Transcriptional activation of NFAT requires its translocation to the nucleus, where it binds to specific consensus sites in the promoter region of the IL-2 gene (60). TCR signaling that activates NFAT can be mimicked by a combination of PMA plus the calcium ionophore, ionomycin. To study whether NADA inhibits NFAT activation, we first performed EMSAs with nuclear extracts of Jurkat cells stimulated with PMA plus ionomycin in the presence or the absence of increasing concentrations of NADA. Using the distal NFAT site of the IL-2 promoter (61), we found a major complex that was retarded in PMA- plus ionomycin-treated cells, and the binding to DNA of this complex was clearly inhibited in the presence of increasing concentrations of NADA. This complex was characterized as NFAT1 by supershift experiments with an anti-NFAT1 antiserum (49) and by cold competition experiments (Fig. 6A). To dissect the mechanism responsible for NFAT inhibition by NADA, we studied the dephosphorylation of NFAT1 by Western blot using a specific antiserum against NFAT1. In Fig. 6B it is shown that upon treatment with PMA plus ionomycin, NFAT1 was dephosphorylated in Jurkat cells, and NADA inhibited this dephosphorylation in a concentration-dependent manner. To further demonstrate the inhibitory effects of this endovanilloid in the NFAT trans-activation activity, we cotransfected Jurkat cells with a Gal4-Luc reporter plasmid along with the construct Gal4-NFAT1 that encodes the N-terminal region of the NFAT1 (aa 1–415), which contains both trans-activation and regulatory domains, fused to the Gal4 DNA binding domain. As shown in Fig. 6C, trans-activation mediated by Gal4-NFAT1 was enhanced in the presence of PMA plus ionomycin, and as expected, increasing doses of NADA progressively inhibited this trans-activation.



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  		FIGURE 6. Effect of NADA on the NFAT activation pathway. A, NFAT DNA binding activity was analyzed for nuclear extracts from Jurkat T cells stimulated for 2 h with PMA plus ionomycin (Io; 1 µM) in the absence or the presence of increasing concentrations of NADA. The specificity of NFAT binding to DNA was demonstrated by supershift and cold competition assays (A, right panel). B, NADA inhibits PMA- plus ionomycin-induced NFAT dephosphorylation. Jurkat T cells were treated as described in A, and the phosphorylation status of NFAT1 was identified by Western blot analysis. C, Jurkat cells were cotransfected by lipofection with 0.7 µg of a Gal4-Luc reporter plasmid/ml together with 0.3 µg of the expression vector coding for the fusion protein Gal4-human NFAT11–415. After 24 h, cells were pretreated, or not, with increasing doses of NADA and further stimulated with PMA (20 ng/ml) plus ionomycin (1 µM) for 6 h. Results are the mean ± SE of three determinations, expressed as the fold induction (observed experimental relative light units/basal relative light units in absence of any stimuli).

 
Effects of NADA on AP-1 transcriptional activation and mitogen-activated protein kinase (MAPK) phosphorylation

It has been shown that in the nucleus activated NFAT binds to the AP-1 family of transcription factors to increase the rate of transcription of target genes such as IL-2 or TNF-{alpha} (62). Moreover, a cross-talk between signaling pathways that activates both NFAT and AP-1 has been demonstrated in different experimental systems (14, 31). To study the effects of NADA on AP-1 activation, we stimulated Jurkat cells with PMA plus PHA in the presence of NADA and total cell extracts obtained for EMSA and Western blot analyses. Fig. 7A shows that NADA also effectively inhibited in a concentration-dependent manner PMA- plus PHA-induced AP-1 DNA binding. In addition, by using specific Abs that recognize the phosphorylated and activated forms of three major MAPKs, we demonstrated that NADA, at the same AP-1 inhibitory concentrations, was able to inhibit the phosphorylation of JNK and p38, but not mitogen-induced activation of both ERK isoforms (ERK1 and -2; Fig. 7B). NADA pretreatment did not affect the steady state levels of {alpha}-tubulin and the expression of the three nonphosphorylated MAPKs (data not shown). To further study the effects of NADA on c-Jun transcriptional activity, we cotransfected Jurkat cells with the Gal4-Luc reporter plasmid along with the construct Gal4-c-Jun that encodes the trans-activation domain of c-Jun (aa 1–111), and 24 h later the cells were pretreated with increasing concentrations of NADA before stimulation with PMA plus PHA for 6 h. Similar to the results obtained with the AP-1 reporter construct (Fig. 4C), NADA, at the same concentrations that were effective at inhibiting JNK activation and AP-1 binding to DNA, strongly inhibited the transcriptional activity of the Gal4-c-Jun construct (Fig. 7C).



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  		FIGURE 7. Effects of NADA on the MAPK and AP-1 activation pathways. A, Jurkat T cells were preincubated with NADA at the indicated concentrations for 30 min, followed by stimulation with PMA and PHA for 1 h. AP-1 DNA binding activity in total cell extracts from stimulated cells was studied by EMSA. B, NADA inhibits the phosphorylation of p38 and JNK, but not the phosphorylation of ERK1 and -2. Jurkat T cells were incubated with increasing concentrations of NADA and were further stimulated with PMA plus PHA for 30 min. The phosphorylation status of MAPKs and the steady state levels of {alpha}-tubulin were analyzed by Western blot using specific anti-phospho mAbs. C, Jurkat cells were cotransfected with 0.7 µg of a Gal4-Luc reporter plasmid/ml together with 0.3 µg of the expression vector coding for the fusion protein Gal4-c-Jun. After 24 h, cells were pretreated, or not, with increasing doses of NADA and further stimulated with PMA (20 ng/ml) plus PHA (1 µg/ml) for 6 h. Luc activity was measured, and the results are the mean ± SE of three determinations, expressed as the fold induction (observed experimental relative light units/basal relative light units in absence of any stimuli).

 
Effects of NADA on Cot kinase- and calcineurin-induced NFAT and NF-{kappa}B activation in Jurkat cells

Cot kinase is a protein serine/threonine kinase, classified as a MAPK kinase kinase, implicated in T cell activation. This kinase is involved in the signal transduction pathways leading to the activation of NF-{kappa}B, NFAT, and AP-1 transcription factors (28, 63, 64). To analyze the effects of NADA in Cot kinase-induced transcriptional activity of NF-{kappa}B and NFAT, we cotransfected Jurkat cells with a construct encoding an active form of this kinase (pEF-BOS trunk-Cot) and either the Gal4-NFAT1 or the KBF-Luc plasmids; 24 h after transfection, cells were incubated with increasing concentrations of NADA. In Fig. 8, A and B, it is shown that NADA strongly inhibited the trans-activation activity of both transcription factors. As Cot kinase can also activate NFAT through calcineurin-dependent and -independent pathways (28), we were interested to investigate whether this endovanilloid was also able to inhibit both NFAT and NF-{kappa}B-dependent trans-activation induced by overexpression of an active form of the phosphatase calcineurin (54). Cotransfection of the catalytic subunit of calcineurin increased NFAT and NF-{kappa}B transcriptional activities that were abrogated by NADA in a concentration-dependent manner (Fig. 8, C and D). Our results suggest that NADA and perhaps other endovanilloids may inhibit IL-2 gene expression and T cell activation by targeting a common step in the signaling pathway that regulates the activation of NFAT, NF-{kappa}B, and AP-1 transcription factors.



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  		FIGURE 8. NADA inhibits both Cot and calcineurin-induced NF-{kappa}B and NFAT transcriptional activities. A and B, Jurkat T cells were cotransfected with the plasmid pEF-BOS trunk-Cot containing the truncated active form of the Cot kinase together either with a combination of the Gal4-Luc reporter plasmid and the expression vector Gal4-human NFAT11–415 (A) or with the plasmid KBF-Luc (B). C and D, Effect of NADA on calcineurin-induced NF-{kappa}B and NFAT transcriptional activities. Jurkat T cells were transiently cotransfected with an expression vector encoding for the catalytic subunit of calcineurin ({Delta}CAM-AI) together with either a combination of the Gal4-Luc reporter plasmid and the expression vector Gal4-human NFAT11–415 (C) or with the plasmid KBF-Luc (D). In all the cases and after 24 h of transfection, the cells were incubated with the indicated concentration of NADA for 6 h, and Luc activity was measured as described above. Results are the means of three determinations, expressed as the fold induction.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Invasion of lymphocytes into brain parenchyma is a common feature in different brain pathologies, including viral encephalitis, multiple sclerosis, stroke, and other post-traumatic processes (1). Thus, the role of the immune system in neurodegenerative diseases is an important area of investigation, and new therapeutic strategies based on immune modulation are under consideration, especially for autoimmune disorders such as multiple sclerosis (65). In this sense, in the past few years the endocannabinoid system has emerged as a potential and interesting target for the development of new lead compounds with therapeutic activity in several brain diseases. Recently, a functional interplay between the endocannabinoid and endovanilloid systems has been suggested (66, 67). Furthermore, we and others have shown that both plant-derived vanilloids and AEA may exert biological functions through CB receptors and TRPV1-independent pathways (10, 48, 68). In this report we extended our studies to analyze the immunosuppressive effects of the recently discovered endovanilloids, NADA and OLDA, and we describe for the first time that both compounds are potent inhibitors of early and late activation events in Ag-stimulated human peripheral T cells.

In our experiments we found that in mitogen-activated cells, NADA could completely suppress both IL-2 and TNF-{alpha} gene transcription at concentrations ranging between 1–2.5 µM. As the physiological concentrations of NADA are unknown, it is difficult to speculate about the physiological relevance of this finding. However, the relatively high concentrations of NADA and OLDA required for in vitro T cell inhibition should not be seen as evidence against a physiological role for these lipid mediators. For instance, the effect of the endovanilloid/endocannabinoid AEA on TRPV1 was also shown to occur at concentrations >1 µM, but additional experiments have shown that the threshold for TRPV1 activation by AEA can be sensibly lowered by several regulatory factors (69).

NADA and 2-AG share an arachidonoyl moiety, and both are inhibitors of T cell activation. Nevertheless, different mechanisms might underlie their activity. Thus Ouyang et al. (70) have shown that 2-AG inhibits IL-2 production in mitogen-activated mouse splenocytes with an ED50 value ranging between 2 and 4 µM. However, in T cell lines, much higher concentrations (up to 50 µM) were required to inhibit 50% of the NFAT- and AP-1-mediated transcriptional activities (70). Moreover, and in contrast to the findings for NADA, both 2-AG and cannabinol are very weak NF-{kappa}B inhibitors (70, 71). Thus, a likely explanation for these results is that 2-AG, like cannabinol, mediates its immunosuppressive activity in T cells by targeting the CB2 receptor. Nevertheless, studies are in progress to determine whether the immunosuppressive effects of 2-AG in serum-free medium are mediated exclusively by targeting the CB2 receptor or by additional CB-independent pathways. Also the possible synergistic effects of NADA and 2-AG could explain the differences between the effects of the two endocannabinoids on T cell activation.

As human peripheral T cells and Jurkat T cells do not express the TRPV1 receptor (data not shown), and neither NADA nor OLDA binds the CB2 receptor, the only one expressed in Jurkat T cells (10, 72), it is likely that the inhibitory activity of these endovanilloids on T cell activation could be mediated by a novel pathway, which seems to be independent of the vanilloid and cannabinoid receptors identified to date. One possibility is that NADA may enter the cells by a process of simple diffusion, as has been recently suggested for AEA (73). Once inside the cells, NADA or a metabolite could interact with a specific component of the signaling cascade, leading to inhibition of IL-2 gene transcription. Alternatively, NADA may target other ionic channels differently from the TRPV1, such as T-type calcium channels (74) and the K+ channel TASK-1 (75), which have been shown to be inhibited by endocannabinoids.

Ag-specific T cell proliferation requires TCR/CD3 signaling and a second signal provided by CD28. Signaling pathways activated by both CD3 and CD28 couple the signals from the plasma membrane with the activation of transcription factors that regulate the expression of a large number of genes during T cell activation program. We show in this report that NADA inhibits the activation of NF-{kappa}B, NFAT, and AP-1 transcription factors in Jurkat T cells, suggesting that NADA can target a common element upstream of these transcription factors. Such an upstream element could be the Cot/Tpl2 kinase, which has been demonstrated to participate in the signaling pathway that activates NF-{kappa}B, AP-1, and NFAT transcription factors (28, 63, 64). However, the finding that NADA does not affect either I{kappa}B{alpha} degradation and NF-{kappa}B binding to DNA or ERK activation strongly suggests that the molecular target for NADA may be downstream of Cot kinase. Interestingly, NADA inhibits catalytic subunit of calcineurin-induced NFAT1-dependent gene transcription, and the activation of the MAPKs, p38, and JNK, resembling the effects described for the calcineurin inhibitors cyclosporine A (CsA) and FK506 (76).

Although the cross-talk between calcineurin and NF-{kappa}B signaling has been described (77), the mechanism by which CsA inhibits NF-{kappa}B is not well understood. It has been described that CsA inhibits the inducible degradation of both I{kappa}B{alpha} and I{kappa}B{beta} upon T cell activation (78). Moreover, CsA inhibits both the binding to DNA and the transcriptional activity of NF-{kappa}B in primary cerebellar neurons (79). However, CsA can also affect the NF-{kappa}B signaling pathway through calcineurin-independent mechanisms (80), which may not be shared by NADA. As a working hypothesis, it is possible that NADA inhibits a calcineurin-dependent pathway that results in the activation of nuclear kinases that may phosphorylate p65 in the nucleus, thus increasing its transcriptional activity. As p65-serine 536 is a target for IKK{beta} (44), and this kinase may form nuclear complexes with IKK{alpha}, p65, and the cAMP response element binding protein (46), it is possible that NADA, by targeting calcineurin, could disrupt this complex, then preventing the phosphorylation and acetylation of histone H3 by IKK{alpha} and the phosphorylation of p65 by IKK{beta}, two key signals that may be required for optimal NF-{kappa}B-dependent gene transcription. Interestingly, the functional interaction between calcineurin and the CREB signaling pathway has been demonstrated (81) and supports our hypothesis about the possible inhibitory mechanism of action of NADA in the NF-{kappa}B pathway, which requires further experimentation (Fig. 9).



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  		FIGURE 9. Proposed mechanism for NF-{kappa}B, NFAT, and AP-1 inhibition by the endocannabinoid NADA. Calcineurin targeting by NADA may result in inhibition of nuclear p65 phosphorylation, inhibition of JNK activity, and inhibition of direct dephosphorylation of cytoplasmic NFAT.

 
The endogenous occurrence of long chain fatty acids functionalized with aromatic bioactive amines in mammals has long been postulated (82). Then arachidonoyl-serotonin and NADA were synthesized in vitro and were found to be potent inhibitors of the enzyme fatty acid amide hydrolase, the enzyme responsible for AEA metabolism (9, 83). The recent discovery that NADA and other lipids mediators, such as OLDA, exist in several brain areas is of outstanding interest not only for their direct mechanisms of action, but also for their possible interplay with other components of the endocannabinoid system (6, 7). Thus, in addition to the CB and fatty acid amide hydrolase-independent effects described in this report for NADA and OLDA in T cells, it is also possible that, under proinflammatory conditions, these compounds may inhibit the hydrolysis and/or the uptake of AEA, with an overall increase in its bioavailability. In this sense, it has been suggested that lipid mediators simultaneously synthesized and released can have synergistic effects not only by heightening the potency of the major bioactive compound, but also by targeting distinct signaling pathways involved in the inflammatory processes.

Biosynthesis and inactivation of NADA and OLDA are both unknown. Formation from the acylation of dopamine with arachidonic acid seems possible (6), as does their inactivation by catechol-O-methyltransferase (6). The identification of mechanisms regulating the endogenous levels of NADA and OLDA could be a new target worth pursuing to modulate the levels of endovanilloids in inflammatory brain conditions.


   Acknowledgments
 
We thank Dr. Alain Israël (Institute Pasteur, Paris, France) for the anti-p65 antisera, Dr. Juan M. Redondo (Centro de Biologia Molecular-Universidad Autónoma de Madrid, Madrid, Spain) for the anti-NFAT1 antisera, Dr. R. T. Hay (Centre for Biomolecular Sciences, St. Andrews, Scotland) for the mAb 10B, and colleagues Dr. L. Schmitz (Bern, Switzerland), Dr. Manuel López-Cabrera (Hospital de la Princesa, Madrid, Spain), Dr. Manuel Fresno (Centro de Biologia Molecular-Universidad Autónoma de Madrid, Madrid, Spain), and Dr. Susana Alemany (Instituto de Investigaciones Biomédicas-Centro Superior de Investigaciones Cientificas, Madrid, Spain) for providing plasmids. Finally, we thank Carmen Cabrero-Doncel for her assistance with the manuscript.


   Footnotes
 
1 This work was supported by Ministerio de Ciencia y Tecnologiá Grant SAF2001-0037-C04-02 and European Union Grant QLK3-CT-2000-00463 (to E.M., B.L.F., and G.A.). A.M. was supported by Ministerio de Ciencia y Tecnologiá Grant SAF2002-01157. Back

2 Address correspondence and reprint requests to Dr. Eduardo Muñoz, Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Medicina, Avenida de Menendez Pidal s/n, 14004 Cordoba, Spain. E-mail address: fi1muble{at}uco.es Back

3 Abbreviations used in this paper: AEA, arachidonoylethanolamide; 2-AG, 2-arachidonoylglicerol; CB, cannabinoid receptor; CsA, cyclosporine A; ERK, extracellular signal-regulated kinase; Gal4, galactosidase 4; IKK, I{kappa}B kinase; I{kappa}B, {kappa}B inhibitor; JNK, c-Jun N-terminal kinase; Luc, luciferase; MAPK, mitogen-activated kinase; NADA, N-arachidonoyldopamine; OLDA, N-oleoyldopamine; PKC, protein kinase C; SEB, staphylococcal enterotoxin B; TRPV-1, transient receptor potential channel, vanilloid subfamily member 1; KBF, {kappa}B factor. Back

Received for publication August 12, 2003. Accepted for publication December 4, 2003.


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