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Human T Cell Receptor SignalingMechanisms of Opioid-Mediated Inhibition of

Höllt and Jürgen KrausHartig, Jonathan A. Lindquist, Burkhart Schraven, Volker Christine Börner, Beate Warnick, Michal Smida, Roland

http://www.jimmunol.org/content/183/2/882doi: 10.4049/jimmunol.08027632009;

2009; 183:882-889; Prepublished online 26 JuneJ Immunol 

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Mechanisms of Opioid-Mediated Inhibition of Human T CellReceptor Signaling1

Christine Borner,* Beate Warnick,* Michal Smida,† Roland Hartig,† Jonathan A. Lindquist,†

Burkhart Schraven,† Volker Hollt,* and Jurgen Kraus2*

Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, causeimmunosuppression. 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 humanT lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid �-endorphininhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1,NFAT, and NF-�B, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited bythe opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. Amore detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase incAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phos-phoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition ofthe leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediatedby � opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since �-en-dorphin 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 �-endorphin in the physiological regulation of T cell activation. The Journal ofImmunology, 2009, 183: 882–889.

O pioids are the most potent analgesics and widely used forthe treatment of severe pain such as cancer pain. How-ever, a great number of studies have convincingly dem-

onstrated that opioids, in particular morphine and derivatives, areimmunosuppressive. This may cause unwanted side effects duringpain treatment, especially in persons with an impaired immunesystem, e.g., patients suffering from cancer or AIDS. It is alsoreasonable to propose that the opioid-induced immunosuppressioncontributes to an increased prevalence of infections like pneumo-nia and HIV among opioid users and addicts (for reviews, seeRefs. 1 and 2). Studies in mouse models have suggested that oneof the mechanisms by which opioids cause immunosuppression isthe inhibition of IL-2 transcription in activated T lymphocytes (3–6). Thus, it was demonstrated that heroin and methadone inhibitthe IL-2 production of activated murine splenocytes (3). Further-more, it was shown that opioid-mediated inhibition of c-fos mRNA(4) and opioid-induced modulation of the cAMP response element-binding transcription factors CREB/CREM/ICER (5, 6) are in-volved in the inhibition of IL-2. However, detailed molecularmechanisms, which link opioids to TCR signaling and lead to itsinhibition, have not been clearly defined. Furthermore, little is

known about opioid-induced immunosuppression in human Tcells.

It is known that the TCR-mediated activation of T cells inducesa defined series of signaling events which culminate in the tran-scription of the gene for the T cell growth factor IL-2 (7–10). In thenonactivated state, i.e., in resting T cells, the cascade is tonicallyrepressed by constitutive phosphorylation of the negative regula-tory site of Lck at Tyr505. This phosphorylation is mediated by thekinase C-terminal Src kinase (Csk),3 which is complexed in restingT cells with the transmembrane adapter protein Cbp/PAG (phos-phoprotein associated with glycosphingolipid-enriched microdo-main/Csk-binding protein). Upon engagement of the TCR, Cbp/PAG is dephosphorylated. This leads to a release of Csk fromCbp/PAG, resulting in a loss of the inhibitory effect of Csk on Lck.These events lead to activation of Lck and initiation of the TCR-induced signaling cascade (11–13). One of the earliest events inthis cascade is the Lck-mediated phosphorylation and thereby ac-tivation of the protein tyrosine kinase Zap70. Activated Zap70 inturn phosphorylates the transmembrane adapter protein linker foractivation of T cells (LAT) on at least four tyrosine residues, lead-ing to the assembly of the calcium-initiation complex (14). Theseevents lead to the induction of calcium flux and also to the acti-vation of the MAPK cascades. In this way, the signal is transducedand finally results in the activation of the transcription factorsAP-1, NF-�B, and NFAT. Together, they transactivate the IL-2

*Department of Pharmacology and Toxicology and †Department of Molecular andClinical Immunology, University of Magdeburg, Magdeburg, Germany

Received for publication August 25, 2008. Accepted for publication May 13, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a research grant from the government of the state ofSachsen-Anhalt (N2/ND: Physiologie und Pathophysiologie signalubertragenderNetzwerke im Immun- und Nervensystem) provided to V.H. and B.S. and a grantfrom the Deutsche Forschungsgemeinschaft (KR 1740/10-1) to J.K.2 Address correspondence and reprint requests to Dr. Jurgen Kraus, Department ofPharmacology and Toxicology, University of Magdeburg, 44 Leipzigerstrasse, 39120Magdeburg, Germany. E-mail address: juergen.kraus@med.ovgu.de

3 Abbreviations used in this paper: Csk, C-terminal Src kinase; cAMPS-Rp, (R)-adenosine, cyclic 3�,5�-hydrogenphosphorothioate; Cbp/PAG, phosphoprotein asso-ciated with glycosphingolipid-enriched microdomain/Csk-binding protein; DPDPE,D-penicillamine2-D-penicillamine5-enkephalin-enkephalin; LAT, linker for activa-tion of T cells; PKA, protein kinase A; siRNA, small interfering RNA.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

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gene, which is almost entirely regulated at the level of transcrip-tion. After being released, IL-2 induces a variety of well-definedsubsequent immune responses.

The modulation of the IL-2 production of activated T cells byopioids is pharmacologically of great importance, since it contrib-utes to the immunosuppressive side effects of drugs like morphine.In addition, there may be a physiological regulation of activated Tcells by endogenous opioid peptides like �-endorphin. However,the question of if and how �-endorphin regulates the TCR-medi-ated signaling cascade and whether this is involved in the regula-tion of physiological T cell responses are less well documented.Earlier studies using the opioid receptor antagonist naloxone havesuggested that �-endorphin exerts an inhibitory effect on T cellfunctions like IL-2 production (15) and mitogen-induced spleno-cytes proliferation (16). In line with this observation, it was dem-onstrated more recently that mice lacking �-endorphin showed en-hanced splenocyte proliferation and enhanced levels of IL-2mRNA compared with wild-type mice (17).

Effects of opioids on T lymphocytes require the expression ofspecific opioid receptors (for reviews, see Refs. 18 and 19).Drugs of the morphine type, and thus most of the clinically usedopioids, bind preferentially to � opioid receptors. The expres-sion of functional � opioid receptors on human T lymphocytes isinduced by TNF and IL-4 (20–22). In addition, expression of �opioid receptors is also induced indirectly by drugs and stimuli,which increase the expression of TNF and IL-4, like cannabinoids(23). As demonstrated recently by our group, also anti-CD3/anti-CD28-mediated activation of human T cells induces the expressionof functional � opioid receptors (24). Studies in mice have shownthat morphine is only effective on mitogen-activated T cells, whichmay be explained by an induction of � opioid receptors after T cellactivation (4, 5, 25). Thus, there is reason to believe that the ex-pression of � opioid receptors on human T lymphocytes allows themodulation of T cell responses by ligands for � opioid receptors,such as morphine. The endogenous opioid �-endorphin activatesboth � and � opioid receptors. It was reported that low levels of �opioid receptors are expressed in Jurkat T lymphocytes and thattheir expression is induced in response to IL-4 (26), and anti-CD3/anti-CD28 (our unpublished observations). In murine T cells, itwas shown that anti-CD3 increases the expression of � opioid re-ceptors (27). These observations suggest that also � opioid receptoragonists can modulate T cell functions. In this report, we identifiedmechanisms by which opioids suppress the TCR-triggered signal-ing cascade in activated human T lymphocytes.

Materials and MethodsT cell culture, induction of opioid receptors, T cell activation,and reagents

PBMC were isolated from heparinized blood collected from healthy vol-unteers as previously described (28). Primary human T cells and the humanT cell line Jurkat were cultivated in RPMI 1640 medium (Lonza Verviers)supplemented with 10% FCS and antibiotics (100 U/ml penicillin and 100mg/ml streptomycin; Lonza Verviers). For our experiments, no opioid re-ceptor-overexpressing cells were used, but � and � opioid receptors wereinduced in physiological amounts by IL-4 according to an established pro-cedure, which has been shown to produce functional receptors (21, 22, 26).Briefly, the cells received IL-4 (used at 5 ng/ml; R&D Systems) and wereincubated for 3 days. Then the medium was replaced by fresh mediumwithout IL-4 and the cells were cultured for an additional 24 h. An aliquotof the cells was tested to assure the induction of � and � opioid receptortranscripts. For these cells, the term “opioid receptor-expressing” cells isused throughout this publication. For activation of T cells, 100 �l each ofanti-CD3 and anti-CD28 was used per 106 cells. The sources for anti-CD3(OKT3) and anti-CD28 (248.23.2) were the hybridoma supernatants pro-duced in our laboratories (28). As opioids, morphine (Synopharm), �-en-dorphin (Sigma-Aldrich), and D-penicillamine2-D-penicillamine5-en-kephalin (Sigma-Aldrich) were used at 1 �M. Antagonists were CTAP (�

opioid receptor specific) and naltrindole (� opioid receptor specific), bothused at 250 nM and purchased from Tocris. The protein kinase A (PKA)inhibitor (R)-adenosine cyclic 3�,5�-hydrogenphosphorothioate (cAMPS-Rp; Tocris) was used at 100 �M. The PKA activator 8-Br-cAMP (Tocris)was used at 5 �M. As phosphodiesterase inhibitors, IBMX (500 nM; Sig-ma-Aldrich), rolipram (10 �M; Tocris), BRL50481 (10 �M; Tocris), anddipyridamole (100 �M; Tocris) were used. PMA (Sigma-Aldrich) wasused at 100 nM.

Chronic opioid treatment and IL-2 transcription

Experiments were performed similarly to a described protocol (29).Briefly, opioid receptor-expressing Jurkat cells (106 per sample) were in-cubated for 4 days on anti-CD3/anti-CD28-coated dishes with opioids andopioid antagonists. Then cells were restimulated with anti-CD3/anti-CD28for 15 min to induce IL-2 mRNA and lysed after 4 h for quantitative IL-2RT-PCR. The restimulation and final 4-h incubation of the cells was per-formed in the presence of opioids to exclude “withdrawal”-like effects.

Analysis of transcription factor activity

Oligonucleotides containing binding sequences (core-binding sequencesare underlined) for the transcription factors AP-1 (5�-AAACATATGATTCACCAGGCA-3�), NF-�B (5�-AAAGTTGAGGGGACTTTCCCAGGCCT-3�), and NFAT (5�-GCCCAAAGAGGAAAATTTGTTTCATA-3�)were cloned 5� to the herpes simplex thymidine kinase promoter of thereporter gene plasmid pBLCAT2 (30). These constructs were transfectedinto opioid receptor-expressing Jurkat cells by electroporation (210 V, 950�F) using a Gene Pulser II (Bio-Rad). The day after transfection, cells weretransferred to anti-CD3/anti-CD28- or anti-mouse Ig-coated dishes, with orwithout morphine. After 72 h of transient expression, the reporter gene wasassayed via a chloroamphenol acetyl transferase-ELISA purchased fromRoche.

Quantitative real-time RT-PCR

Isolation of RNA and quantitative real-time RT-PCR for �-actin, �, and �opioid receptor transcripts have been described earlier (20, 22, 26). IL-2-specific RT-PCR was performed with 5�-GAAGGCCACAGAACTGAAACATCT-3� and 5�-CTGTTCAGAAATTCTACAATGGTTG-3�primers, with a preincubation for 8 min at 95°C and 50 cycles with 5 s at95°C, 5 s at 65°C, and 10 s at 72°C (specific Tm � 81, 75°C). Quantitativereal-time RT-PCR was done in a total volume of 20 �l on a LightCyclerinstrument using a LightCycler-Fast Start DNA Master SYBR Green I kit(both from Roche) according to the manufacturer’s suggestions.

Surface expression of TCR

After washing in PBS, cells were incubated with murine anti-CD3 for 30min at 4°C. Secondary goat anti-mouse-FITC Abs (Dianova) were incu-bated for 30 min at 4°C and the surface expressions of the TCR weremeasured using a FACSCalibur flow cytometer (BD Biosciences).

Western blots

Western blots were performed as previously described (22, 23, 31). Foractivation, 2.5 � 106 opioid receptor-expressing cells per sample werepelleted, resuspended in anti-CD3/anti-CD28, and incubated at 37°C for 2min. The activation was stopped with 1 ml of ice-cold PBS, cells werepelleted again, and lysed. For protein detection, the following Abs wereused: primary Abs were actin C-11 (Santa Cruz Biotechnology); phospho-p44/42 MAPK Tyr202/Tyr204

; phospho-LAT Tyr191, phospho-Zap70Tyr319, and phospho-Lck Tyr505 (all from Cell Signaling Technology/NewEngland Biolabs). All Abs were from rabbits; the secondary Ab was anti-rabbit IgG (GE Healthcare). Anti-PAG (MEM255) hybridoma was made inthe laboratory of Dr. V. Horejsi (Institute of Molecular Genetics, Prague,Czech Republic) and grown and supernatant was collected in our institute.The secondary Ab anti-mouse-HRP was obtained from Dianova.

cAMP measurement

Opioid receptor-expressing cells were incubated with morphine and �-en-dorphin for 0 h (controls) up to 24 h. Then cells were lysed with 50 mMHCl for 30 min on ice. The competitive cAMP-ELISA was performedaccording to a described procedure (32).

Down-regulation of Cbp/PAG with small interfering RNA(siRNA)

For the construction of silencing plasmids, the sequences targeting humanCbp/PAG (5�-AAGCGATACAGACTCTCAACA-3�), mouse Cbp/PAG

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(5�-AAGCCATACAGACTCTAAACA-3�), and a scrambled sequence(5�-AACGCACGAACTATCAACATA-3�) were cloned into the vectorpCMS3-EGFP. All constructs were sequenced to ensure integrity. Theseconstructs were transfected into Jurkat T cells by electroporation (de-scribed above). The cells were then cultured in RPMI 1640 supplementedwith IL-4 for 4 days to induce opioid receptor expression (described above)and ensure Cbp/PAG protein down-regulation.

Calcium measurement

Cells (three times 105 cells/ml) in RPMI 1640 medium (phenol red free;Invitrogen) containing 10% FCS were loaded with 5 �g/ml Indo-1-AM(MoBiTec) at 37°C for 45 min. After washing, cells were incubated inRPMI 1640 medium (phenol red free) supplemented with 10% FCS at37°C for an additional 45 min. Cells were measured using an LSR I flowcytometer (BD Biosciences) equipped with a 325-nm laser. Cells werestimulated with anti-CD3 and the ratio of the two emission wavelengths(512/20 nm and 400/40 nm) was recorded over time. To confirm properIndo-1 loading and the viability of cells, ionomycin (10 �g/ml; Sigma-Aldrich) was used. The kinetics of the data were analyzed using FlowJosoftware (Tree Star).

Statistical analysis

For statistical evaluations between two groups of samples, Student’s t testswere performed. Multiple comparisons were performed with ANOVA, fol-

lowed by Tukey’s multiple comparison posttest. Asterisks indicate signif-icantly different values (�, p � 0.05; ��, p � 0.01; and ���, p � 0.001).

ResultsEffects of morphine and �-endorphin on the transcriptionof IL-2

Activation of the TCR leads to the induction of IL-2. A number ofreports demonstrated that chronic morphine treatment of activatedT cells inhibits this induction in mice (3, 4, 6). Fig. 1A shows theeffects of chronic opioid treatment on IL-2 transcription in opioidreceptor-expressing Jurkat cells (i.e., cells in which opioid recep-tors were induced; see Materials and Methods) after stimulationwith a combination of anti-CD3 and anti-CD28. Similar to murinecells, morphine caused a significant inhibition of anti-CD3/anti-CD28-induced IL-2 mRNA production in the human cells. Thiseffect was mediated by � opioid receptors, since it was blocked byCTAP, a specific � opioid receptor antagonist. Similarly, �-en-dorphin, which is a ligand for � and � opioid receptors, causedinhibition of anti-CD3/anti-CD28-induced IL-2 transcription. Thiseffect was also mediated by � opioid receptors, since it wasblocked by CTAP, but not by naltrindole, a specific � opioid re-ceptor antagonist. To test possible effects on the anti-CD3/anti-CD28-induced IL-2 mRNA production mediated by � opioid re-ceptors more specifically, we additionally tested the specific �opioid receptor agonist D-penicillamine2-D-penicillamine5-en-kephalin (DPDPE). However, treatment of the cells with DPDPEhad no significant effect on IL-2 transcription. Additionally, theeffect of morphine on the activities of the transcription factorsAP-1, NF-�B, and NFAT were investigated. As shown in Fig. 1B,morphine significantly inhibited the anti-CD3/anti-CD28-induced

FIGURE 1. Opioids inhibit TCR-induced IL-2 mRNA formation andtranscription factor activities. A, Effects on IL-2 mRNA. Opioid receptor-expressing Jurkat cells were simultaneously incubated for 4 days on anti-CD3/anti-CD28-coated culture dishes along with opioid receptor agonists(morphine: 1 �M, � opioid receptor specific; �-endorphin (�-End): 1 �M,� and � specific; DPDPE: 1 �M, � specific) and antagonists (CTAP: 250nM, � specific; naltrindole (Naltrin): 250 nM, � specific), as indicatedbelow. Then cells were restimulated in the presence of opioids with anti-CD3/anti-CD28 for 15 min to induce IL-2 mRNA. Before harvesting, cellswere incubated for another 4 h in the presence of opioids. Quantitative IL-2RT-PCR is shown relative to that of �-actin (�-act). The anti-CD3/anti-CD28-induced IL-2 mRNA amount of samples that contained no opioids(controls) was set to 100%. The means of at least three independent ex-periments performed in triplicate � SEM are shown. Secondary compar-isons are indicated by brackets (�, p � 0.05; ��, p � 0.01; and ���, p �0.001). B, Effects on transcription factor activities. Opioid receptor-ex-pressing Jurkat cells were transfected with thymidine kinase (tk)-chloro-amphenicol acetyl transferase (CAT) reporter genes with binding sites forthe transcription factors AP-1, NF-�B, and NFAT. Cells were incubatedeither on anti-CD3/anti-CD28- or Ig-coated dishes in the presence andabsence of morphine (Mo; 1 �M) for 72 h as indicated. The means of atleast three independent experiments performed in triplicate � SEM aredisplayed. Secondary comparisons are indicated by brackets (�, p � 0.05;��, p � 0.01; and ���, p � 0.001).

FIGURE 2. Morphine decreases the TCR-mediated calcium flux. A andB, Morphine decreases the TCR-mediated calcium flux in opioid receptor-expressing Jurkat T cells. The cells received a single dose of morphine (1�M; MO) and were incubated with the drug for 1 or 2 h or left untreated(controls, CO). Then the anti-CD3-induced calcium flux was measured bythe fluorescence emission ratio of Indo-1 (‚: time of addition of anti-CD3).In addition, the ionomycin (10 �g/ml)-evoked calcium flux was measuredin the control cells (Œ: time of addition of ionomycin) A, Example of atypical experiment. B, Quantification of the morphine effect. The means ofat least three independent experiments � SEM are shown. Samples werecompared with morphine-untreated controls (�, p � 0.05). C, The effect ofmorphine on the TCR-mediated calcium flux is absent in naive Jurkat Tcells, which do not express � opioid receptors. An example of a typicalexperiment is shown.

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activities of all three transcription factors in opioid receptor-ex-pressing Jurkat cells.

Effects of morphine and �-endorphin on the anti-CD3/anti-CD28-induced signaling cascade of T cells

One of the early events in T cell activation is mobilization ofintracellular calcium. As shown in Fig. 2A, incubation of opioidreceptor-expressing Jurkat cells with morphine for up to 2 h grad-ually inhibited the TCR-induced calcium flux by �60% (Fig. 2B).In contrast, morphine had no effect on the calcium flux in naive Tcells, which do not express � opioid receptors (Fig. 2C).

To obtain further insight into the mechanism(s), by which opi-oids inhibit anti-CD3/anti-CD28-induced IL-2 transcription, wenext investigated their effects on the signaling cascade downstreamof the TCR. The activation states of MAPK, LAT, and Zap70 weredetermined in Western blot experiments using phospho-specificAbs (Fig. 3). Activation of opioid receptor-expressing primaryhuman T cells with anti-CD3/anti-CD28 for 2 min induced arobust phosphorylation of MAPK, LAT, and Zap70. However,this phosphorylation was markedly reduced when the cells wereincubated with morphine for 2– 8 h before their activation (Fig.3A). Simultaneous incubation of the T cells with morphine andthe � opioid receptor-specific antagonist CTAP abolished theeffect, indicating that it is mediated via � opioid receptors (Fig.3B). Similar effects were observed when opioid receptor-ex-pressing primary T cells were incubated with �-endorphin (Fig.3C). The inhibitory effect of �-endorphin on anti-CD3/anti-CD28-induced MAPK, LAT, and Zap70 phosphorylation wasabolished after coincubation of the cells with �-endorphin and

CTAP, but not after coincubation with �-endorphin and naltrin-dole, a specific antagonist for the � opioid receptor (Fig. 3D).This indicates that the inhibitory effect of �-endorphin is alsomediated via � opioid receptors. As shown in Fig. 3, E and F,the inhibitory effects of morphine and �-endorphin on the TCR-triggered activation of MAPK are clearly visible at time pointslater than 80 min of incubation of the cells with the drugs. Theeffects of the opioids were not observed in naive T cells, whichdid not express � opioid receptors (Fig. 3G).

To more precisely identify the level at which � opioid receptorsand the TCR communicate, we examined Lck, the kinase respon-sible for initiating TCR signaling and the activation of Zap70. Lckis phosphorylated in unstimulated T cells at Tyr505, by which sig-naling downstream of the TCR is prevented. We observed that inopioid receptor-expressing primary T cells (Fig. 4) and Jurkat cells(data not shown) the dephosphorylation of Lck at Tyr505, whichoccurs after TCR engagement, is inhibited by morphine, as well asby �-endorphin. In contrast, the opioids did not inhibit the TCR-induced dephosphorylation of Lck at Tyr505 in naive cells, whichdo not express � opioid receptors (Fig. 4). As a control, and to ruleout the possibility that the effect of the opioids might be due todown-regulation or internalization of the TCR, the presence ofTCR on the cell surface was determined by flow cytometry. It wasshown that 98% of the opioid receptor-expressing Jurkat cells werepositive for the TCR. After 2 h of morphine treatment, the numberof TCR-positive cells did not change significantly, indicating thatmorphine does not influence the surface expression of the TCR(data not shown).

FIGURE 3. Opioids inhibit the TCR-induced phos-phorylation of Zap70, LAT, and MAPK. Primary hu-man T cells received a single dose of opioids (morphineor �-endorphin (�-End; both 1 �M) and were incubatedwith the drugs for the indicated time periods. Then thecells received fresh medium and were either left un-treated or activated with anti-CD3/anti-CD28 for 2 minand lysed. Blots were probed for phospho-specific pro-teins (P-) and actin as a control. Examples of represen-tative experiments are depicted, which were performedat least two times in duplicate. A, Effects of morphine inopioid receptor-expressing cells. B, The effect of mor-phine in opioid receptor-expressing cells is blocked bysimultaneous addition of the � opioid receptor antago-nist CTAP (250 nM). C, Effects of �-endorphin (�-End)in opioid receptor-expressing cells. D, The effect of�-endorphin in opioid receptor-expressing cells isblocked by simultaneous addition of the � opioid re-ceptor-specific antagonist CTAP (250 nM), but not bythe � opioid receptor-specific antagonist naltrindole(Naltrin; 250 nM). E and F, Temporal dissection of theeffects of morphine (E) and �-endorphin (F) in opioidreceptor-expressing cells. A quantification of two indi-vidual experiments performed in duplicate is depictedon top (asterisks indicate samples significantly differentfrom opioid-untreated, anti-CD3/anti-CD28-activatedsamples; secondary comparisons are indicated by brack-ets; ��, p � 0.01 and ���, p � 0.001). G: The effect ofthe opioids on the TCR-mediated phosphorylation ofZap70, LAT, and MAPK is absent in naive T cells,which do not express � opioid receptors.

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Agonists for the � opioid receptor inhibit TCR signaling via thePKA-Cbp/PAG/Csk-Lck pathway

It is known that the phosphorylation of the inhibitory Tyr505 of Lckis mediated by Csk (33). Csk is recruited to the membrane bybinding to Cbp/PAG (11), which, in turn, enhances its activity(34). The activity of Csk can be further enhanced by PKA-medi-ated phosphorylation of Csk (35). Thus, activation of PKA viaelevated levels of cAMP exhibits an inhibitory effect upon TCRsignaling by stabilizing the inhibited state of Lck (36). Therefore,we monitored the intracellular cAMP concentration in Jurkat cellsafter morphine and �-endorphin treatment (Fig. 5). In opioid re-ceptor-expressing cells, both substances caused an initial decreasein the cAMP formation at the 15-min time point. However, longerincubation of the cells with the opioids caused a significant ele-vation of cAMP, which reached a maximum of �550% of thecontrols (Fig. 5A). In contrast, the opioids did not affect the cAMPconcentration in naive T cells, which do not express � opioidreceptors (Fig. 5B). In the opioid receptor-expressing cells, the �opioid receptor-specific antagonist CTAP blocked both the mor-phine- and �-endorphin-induced increase in cAMP levels, whereasthe � opioid receptor-specific antagonist naltrindole had no effect(Fig. 5C). Furthermore, pertussis toxin inhibited the morphine- and�-endorphin-induced increase in cAMP levels, indicating that theeffect is mediated by Gi/o proteins (Fig. 5C). Next, we aimed tofind a possible explanation for the terminal decrease in cAMP thatoccurs between the 4- and 24-h time points and that brings cAMPlevels back to control levels. Speculating that phosphodiesterasesmight be involved, we found that coincubation of cells with mor-phine and the unspecific phosphodiesterase inhibitor IBMX inhib-ited this decrease. Thus, cAMP levels remained at maximal levelsat the 8- and 16-h time points (data not shown) and the 24-h timepoint (Fig. 5D). To more specifically determine the types of phos-phodiesterases, which are involved in this cAMP decrease, we nexttested rolipram (inhibitor of phosphodiesterase 4), BRL50481 (in-hibitor of phosphodiesterase 7), and dipyridamole (inhibitor ofphosphodiesterases 5, 6, 8, 10, and 11) (Fig. 5D). Our results in-dicate that the phosphodiesterases 4 and 7 are involved in the ter-minal decrease in cAMP, since rolipram and BRL50481 signifi-cantly reduced the cAMP decrease. Applied together, bothsubstances inhibited the cAMP decrease to a similar extent as

IBMX did alone. To test whether the ability of opioids to inhibitTCR signaling is dependent on cAMP-activated PKA, we appliedthe PKA antagonist cAMPS-Rp. Indeed, preincubation of opioidreceptor-expressing primary T cells (Fig. 6) and Jurkat cells (datanot shown) with cAMPS-Rp for 1 h before opioid incubation com-pletely abolished the inhibitory effects of morphine and �-endor-phin on the anti-CD3/anti-CD28-induced phosphorylation ofMAPK and Zap70 and dephosphorylation of Lck. To investigatewhether Cbp/PAG/Csk is involved in the inhibition of TCR sig-naling by � opioid receptor agonists, we down-regulated the ex-pression of Cbp/PAG using siRNA (Fig. 7). As shown in Fig. 7A,transient transfection of siRNA directed against human Cbp/PAGresulted in an �90% down-regulation of the protein comparedwith a control (scrambled) siRNA. Transfection of siRNA specificfor the mouse protein, which has two nucleotide exchanges com-pared with the human gene, resulted in an inhibition of about one-third of Cbp/PAG expression compared with the control siRNA.Transfection of control siRNA had no detectable effect on Cbp/PAG expression compared with untransfected, naive cells (data notshown). In opioid receptor-expressing Jurkat cells transfected withsiRNA directed against human Cbp/PAG, the inhibitory effect of

FIGURE 4. Opioids inhibit the TCR-induced de-phosphorylation ofLck at Tyr505. Opioid receptor-expressing primary human T cells (on top)and naive T cells, which do not express � opioid receptors (below) re-ceived a single dose of opioids (morphine or �-endorphin (�-End), both 1�M) according to the scheme below the gels and were incubated with thedrugs for 4 h. Then the cells received fresh medium and were either leftuntreated or activated with anti-CD3/anti-CD28 for 2 min and lysed. Blotswere probed for phospho-specific proteins (P-) and actin as shown. Exam-ples of typical experiments are depicted, which were performed at least twotimes in duplicate.

FIGURE 5. Modulation of the cAMP formation in T cells by opioids. Aand B, Time-dependent modulation of cAMP formation in opioid re-ceptor-expressing (A) and naive (B) Jurkat cells. The cells received asingle dose of opioids (morphine (Mo) and �-endorphin (End), both 1�M) and were incubated with the drugs for up to 24 h. Then cells wereharvested and the amounts of intracellular cAMP were determined. Thedata represent the means � SEM of at least three independent experimentsperformed in triplicate. C, The opioid-induced increase in cAMP formationin Jurkat cells is mediated by � opioid receptors and Gi/o proteins. The cellsreceived simultaneously a single dose of opioids (morphine and �-endor-phin, both 1 �M) and opioid receptor antagonists (CTAP: 250 nM, �specific; naltrindole (Naltrin): 250 nM, � specific) as indicated and wereincubated for 4 h. For other samples, cells were treated with pertussis toxin(PTX; 10 ng/ml) for 16 h, then opioids were added and cells were incu-bated for an additional 4 h. After the 4-h opioid incubation, all cells wereharvested and cAMP was measured. The data represent the means � SEMof at least three independent experiments performed in triplicate (��, p �0.01). D, The phosphodiesterases 4 and 7 are involved in the opioid-in-duced terminal decrease of cAMP at the 24-h time point. Jurkat cells re-ceived simultaneously a single dose of morphine (1 �M) and the phos-phodiesterase inhibitors IBMX (unspecific, 500 nM), rolipram (specific forphosphodiesterase 4; 10 �M), BRL50481 (specific for phosphodiesterase7; 10 �M), or dipyridamole (DPDM, inhibitor of phosphodiesterases 5, 6,8, 10, and 11; 100 �M) as indicated and were incubated for 24 h. Then cellswere harvested and cAMP was measured. The data represent the means �SEM of at least three independent experiments performed in triplicate (�,p � 0.05 and ��, p � 0.01).

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morphine and �-endorphin and 8-Br-cAMP, which was used as acontrol that directly activates PKA, on TCR signaling was almostcompletely blocked as demonstrated by monitoring the phosphor-ylation of Zap70 (Fig. 7B). Applying siRNA specific for the mouseprotein resulted in a weak blockade of the inhibitory opioid effect.In contrast, applying control siRNA had no influence on the in-

hibitory effect of the opioids. Together, these data suggest that �opioid receptor agonists inhibit TCR signaling via the PKA-Cbp/PAG/Csk-Lck pathway. If this was indeed the case, then the PMA/ionomycin-induced activation of MAPK, in which Lck-regulatedTCR activation is bypassed, should not be influenced by opioids.Indeed, although the incubation of opioid receptor-expressing Ju-rkat cells with morphine and �-endorphin for 4 h produced ele-vated cAMP levels, which are required for the activation of PKA,the PMA/ionomycin-induced activation of MAPK was not influ-enced (Fig. 8).

DiscussionIn this report, we addressed the question how opioids inhibit theTCR-mediated production of IL-2 in human T lymphocytes. Weinvestigated the effects of morphine, as the pharmacologicallymost important opioid, and �-endorphin, as an endogenous opioid.The mechanisms are summarized in a schematic diagram pre-sented in Fig. 9.

One of the first steps in these opioid-T cell interactions was anincrease in cAMP, which was evoked by both opioids and medi-ated by � opioid receptors. Thus far, detailed mechanisms for theregulation of cAMP by � opioid receptor ligands are poorly un-derstood. It has been suggested from studies in other cells that theactivation of � opioid receptors leads to an initial decease incAMP levels, which is due to their coupling to the inhibitory Gi/o

proteins. Under certain experimental conditions, e.g., removal ofthe ligand, a compensatory, sometimes overshooting increase incAMP levels occurring thereafter was observed (37, 38). However,most studies were performed in � opioid receptor-overexpressingcells of various tissues that normally do not express the receptorand, therefore, are of limited relevance to the physiological regu-lation of cAMP levels by opioids in human T cells. In activatedmurine T cells, it was found that morphine treatment for 18 h leadsto a weak increase in cAMP (6). However, the determination ofcAMP levels at a single, fixed time point only may be insufficientto establish a role for this molecule in the opioid-mediated mod-ulation of TCR signaling, because of the biphasic, time-dependentmodulation of the cAMP levels caused by opioids. For the firsttime, we demonstrated that simple incubation of human T cellswith opioids, without any further manipulation like removal of the

FIGURE 6. The PKA inhibitor cAMPS-Rp suppresses the inhibitory effectof opioids on anti-CD3/anti-CD28-induced TCR signaling. Opioid receptor-expressing primary human T cells were treated with cAMPS-Rp (100 �M) for1 h as indicated in the scheme below the gel. Then cells received a single doseof opioids (morphine or �-endorphin (�-End), both 1 �M) according to thescheme and were incubated with the drugs for 4 h. Then the cells receivedfresh medium and were either left untreated or activated with anti-CD3/anti-CD28 for 2 min and lysed. Blots were probed for phospho-specific proteins(P-) and actin as shown. Examples of typical experiments are depicted, whichwere performed at least two times in duplicate.

FIGURE 7. Down-regulation of Cbp/PAG suppresses the inhibitory ef-fect of opioids on anti-CD3/anti-CD28-induced TCR signaling. A, Down-regulation of Cbp/PAG. Western blot experiment showing the average ex-pression (Av. Exp.) of Cbp/PAG 4 days after transfection of opioidreceptor-expressing Jurkat cells with siRNA directed against human (h), ormouse (m) Cbp/PAG, and a nonmatching, scrambled (sc) siRNA. B, Effectof the siRNA on the opioid-inhibited TCR signaling. Opioid receptor-ex-pressing Jurkat cells were transfected with siRNA directed against humanand mouse Cbp/PAG and a scrambled siRNA as indicated. Nucleotidesshown in white on black mark mutations in the sequences compared withthe human Cbp/PAG siRNA. Four days later, cells received a single doseof opioids (morphine or �-endorphin (�-End), both 1 �M) or the cAMPanalogon 8-Br-cAMP (5 �M) according to the scheme and were incubatedfor 4 h. Then the cells received fresh medium and were either left untreatedor activated with anti-CD3/anti-CD28 for 2 min and lysed. Blots wereprobed for phospho-specific (P-) Zap70 and actin as shown. Examples ofrepresentative experiments are depicted, which were performed at least twotimes in duplicate.

FIGURE 8. Opioids have no effect on the PMA/inonomycin (Io)-in-duced activation of MAPK. Opioid receptor-expressing Jurkat T cells (top)and naive Jurkat cells, which do not express � opioid receptors (below),received a single dose of opioids (morphine or �-endorphin (�-End), both1 �M) according to the scheme below the gels and were incubated with thedrugs for 4 h. Then the cells received fresh medium, were activated withPMA/inonmycin (100 nM/1 �M) for 2 min according to the scheme, andlysed. Blots were probed for phospho- (P-) MAPK and actin as shown.Representative experiments are shown. After the 4-h opioid incubation,aliquots of the cells were taken and amounts of intracellular cAMP weredetermined (shown on the right-hand side). The data represent themeans � SEM of two independent experiments performed in duplicate (��,p � 0.01).

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opioid, induced a short, initial decrease in cAMP levels, which wasfollowed by a marked increase in cAMP. This increase was sen-sitive to pertussis toxin and thus mediated by Gi/o proteins. Theelevated cAMP levels were most pronounced �2–4 h after opioidaddition. Thereafter, they decreased again, reaching control levelsat the 24-h time point. This final decrease in cAMP levels was dueto phosphodiesterase activity, catalyzed by the phosphodiesterases4 and 7.

It is known that agents, which lead to increased cAMP levels,may inhibit the TCR-activated signaling cascade of T lymphocytesvia the PKA-Cbp/PAG/Csk-Lck pathway (8). In line with thismodel, our experiments using a PKA inhibitor strongly suggestthat the effect is mediated via this enzyme. The data using siRNAdemonstrated the involvement of Cbp/PAG. Since interactions be-tween Cbp/PAG and Csk are crucial for the function of the “masterregulator” Lck, these data suggest that � opioid receptor ligandsinhibit TCR signaling by superactivating Csk and thereby inhibit-ing Lck. The stable phosphorylation of the inhibitory Tyr505 withinthe C terminus of Lck produced by the opioids is likely to lead tointramolecular binding between the Src homology 2 domain andthe inhibitory phospho-tyrosine of Lck resulting in the formationof a closed conformation (discussed in Ref. 8). As a consequence,this may prevent interaction of Lck with its substrates, e.g., theTCR complex, resulting in a blockade of TCR-mediated signalingand IL-2 induction. In line with this model, we demonstrated thatdownstream activation of the TCR pathway with PMA/ionomycinwas not inhibited by opioids. The opioid-inhibited signaling cas-cade then results in decreased activities of the transcription factorsAP-1 and NF-�B, which contribute to the induction of IL-2. Inaddition, we demonstrated inhibition of the TCR-activated calciumflux by morphine. This is likely to result in the inhibition of thecalcium/calmodulin-dependent transcription factor NFAT, whichalso contributes to the induction of the IL-2 gene (5). In our cel-lular model, the time course for the inhibition of TCR signaling by� opioid receptor ligands is in good accordance with the kineticsof cAMP accumulation. Noteworthy, it is reasonable to assumethat an application of morphine will cause inhibition of the func-tion of activated T cells also in vivo in humans. Since the biolog-ical half-life of morphine is �2–4 h, considerable concentrationsof morphine will be present in an organism for several hours,which may cause the inhibition of IL-2 production. One should

recall that orally applied morphine has an even longer half-life of�8–12 h. Furthermore, also the metabolite morphine-6-glucuro-nide is still biologically active. Together, this might markedly in-crease the time window within which inhibitory effects on T cellsmight be observed in vivo after morphine treatment, comparedwith the cellular model shown in this study. To sum up, our dataare likely to be directly relevant for pharmacological opioid ap-plications. Similar to morphine, the endogenous opioid �-endor-phin had comparable effects on anti-CD3/anti-CD28-induced IL-2mRNA and the TCR-activated signaling cascade. Interestingly,these effects were mediated by � opioid receptors only, although�-endorphin binds to � as well as � opioid receptors. In line withthese findings, the � opioid receptor-specific agonist DPDPE hadno effect on the TCR-mediated induction of IL-2. This suggeststhat TCR-induced IL-2 production is not influenced by activationof � opioid receptors. Nevertheless, it was reported by others thatspecific � opioid receptor agonists caused robust activation ofAP-1 and NFAT transcription factors and induction of IL-2 in �opioid receptor-overexpressing Jurkat cells (39). However, suchstrong effects may be due to an unphysiologically high amount ofreceptors present in the overexpressing cells.

It is becoming increasingly clear that �-endorphin is expressedin T cells and up-regulated in response to several conditions andstimuli (40, 41). Most notably, up-regulation of �-endorphin in Tcells was demonstrated in response to T cell activation (40, 42),which may cause an inhibitory feedback regulation within the pro-cesses of T cell activation. Furthermore, it was shown that �-en-dorphin itself induces up-regulation of its own expression in Tcells (43). In addition, the peptide is up-regulated in various im-mune effector cells in response to inflammation (41, 44, 45). Takentogether, it is very likely that under certain conditions and, at leastlocally, a remarkable amount of �-endorphin is present. Our datasuggest that �-endorphin has a physiological regulatory function inthe immune system by inhibiting T cell activation. They thus sup-port earlier findings obtained in mice, which demonstrated en-hanced splenocyte proliferation and enhanced levels of IL-2mRNA after application of the opioid receptor antagonist naloxone(15, 16) and in knockout mice lacking �-endorphin (17). At themoment, the physiological relevance of the �-endorphin inhibitionin TCR signaling is unclear. Since high amounts of the opioid arereleased from the pituitary during stress, it might contribute tostress-induced immunosuppression. It is interesting to speculateabout effects of �-endorphin on activation of T cells in the brain.Being a classic neuromodulator, it can be assumed that remarkableamounts of the opioid are present in the brain. Therefore, activa-tion of T cells infiltrating the brain, e.g., after inflammation may bereduced by �-endorphin, which would establish a neuroprotectivefunction of the opioid within the neuroimmune cross-talk.

AcknowledgmentsWe thank Camilla Merten and Helga Tischmeyer for excellent technicalassistance. Plates and reagents for the cAMP-ELISA were a gift from Her-mann Ammer (Institute of Veterinary Medicine, University of Munich,Munich, Germany).

DisclosuresThe authors have no financial conflict of interest.

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FIGURE 9. Schematic diagram summarizing mechanisms of the opi-oid-induced inhibition of TCR signaling. Lck is essential for the initiationof TCR signaling (1). Signals transmitted via either the TCR or the IL-4R(2) induce the expression of the � opioid receptor gene (MOR) (3). Bindingof ligands to the � opioid receptors results in a Gi/o protein-mediated ac-cumulation of cAMP. This in turn activates the cAMP-dependent PKA.PKA phosphorylates Csk, which is bound to the transmembrane adaptorprotein PAG. Together, PKA and PAG enhance Csk activity, generating astable phosphorylation of the inhibitory Tyr505 within the C terminus ofLck (4). This is likely to lead to intramolecular binding between the Srchomology 2 domain and the inhibitory phospho-tyrosine of Lck resulting inthe formation of a closed conformation. This prevents interaction of Lckwith its substrates, e.g., the TCR complex, resulting in a blockade of TCR-mediated signaling and IL-2 induction.

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